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The role of interleukin-12 in the pathogenesis of Sendai virus-induced airway disease

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The role of interleukin-12 in the pathogenesis of Sendai virus-induced airway disease
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Stone, Amy Elizabeth Seymour, 1973-
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Cytokines ( jstor )
Dendritic cells ( jstor )
Fibrosis ( jstor )
Infections ( jstor )
Inflammation ( jstor )
Inoculation ( jstor )
Lungs ( jstor )
Messenger RNA ( jstor )
Rats ( jstor )
Sendai virus ( jstor )
City of Gainesville ( local )

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THE ROLE OF INTERLEUKIN-12 IN THE PATHOGENESIS OF SENDAI VIRUS-
INDUCED AIRWAY DISEASE















BY

AMY ELIZABETH SEYMOUR STONE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2002


























This work is dedicated to my mother, Dr. Sandra Fields Seymour, who instilled me with

the courage and desire to become an educated woman.














ACKNOWLEDGMENTS

I would like to start by thanking my family; my parents, Andy, and Sydne were all

so supportive through this whole thing. I really appreciate their never wavering support.

I thank all of my friends for never letting me take myself too seriously when things

were tough. I appreciate my coworkers/fellow graduate students Diane Hulse and Tracy

Jack for sticking with me through the highs (happy hour) and lows (molecular biology) of

the last few years.

I would not have been able to complete this work without the support of my life

partner, Andy. I cannot thank him enough for his love, patience, and ability to make me

laugh at the times when I needed it.

Special thanks go to William Castleman for giving me the opportunity to embark on

this dual degree program, for giving me guidance through the system, and for setting a

quality example as a scientist and a leader.

I thank the members of my committee, Mary Brown, Thomas Brown, Steeve

Giguere, Elizabeth Uhl, and former committee members Ammon Peck, Trenton Schoeb,

and Rosalia Simmen for their ideas and assistance.

Additionally, I appreciate all of the editorial and motivational help that Carol

Detrisac provided in the completion of this document. I would also like to acknowledge

the other graduate student in my lab, Xuezhong Cai, who provided expertise,

encouragement, and humor whenever needed.








Many thanks go to Karen Dukes, Heather Sorenson, Jeff Mans, Kristen Ruest, Mark

Hickey, Timothy Holloway, and Christy Voakes, who provided help with laboratory

work as well as much friendship.

Special appreciation and admiration go to Tracy Jack, who was my listening ear,

my shoulder, my colleague, and my friend through this whole process.















TABLE OF CONTENTS

page

ACK NO W LED G M EN TS ................................................................................................. iii

TA BLE OF CON TEN TS..................................................................................................... v

LIST O F TAB LES ........................................................................................................... viii

LIST O F FIG UR ES ........................................................................................................... ix

ABSTRA CT....................................................................................................................... xi

CHAPTER

1 LITERA TU RE REV IEW ................................................................................................ I

Interleukin-12 ................................................................................................................. 1
IL-12 in Bacterial Infections................................................................................. 12
IL- 12 in Protozoal Infections................................................................................ 13
IL-12 in Fungal Infections .................................................................................... 15
IL- 12 in V iral Infections ....................................................................................... 16
The Role of IL-12 in A llergy and A sthm a................................................................... 18
Rodent M odel for V irus-Induced Pulm onary D isease................................................. 21

2 RESEAR CH PLA N A N D PRO TO CO L ....................................................................... 24

Hypothesis and Specific A im s ..................................................................................... 24
Background/Significance ............................................................................................. 24
Gaps in Knowledge to Be Addressed by This Research.............................................. 26
Research and D esign M ethods ..................................................................................... 26
Overview of Experim ents and Schedule...................................................................... 26
Experiment 1: Pulmonary Expression of IL- 12 in Sendai Virus-infected BN and F344
Rats ....................................................................................................................... 26
Objectives ............................................................................................................. 26
Rationale ............................................................................................................... 27
D esign and M ethods ............................................................................................. 28
Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats ................... 31
O objectives ............................................................................................................. 31









Rationale ............................................................................................................... 32
Design and M ethods ................................................................... ..................... 33

3 INCREASED EXPRESSION OF PULMONARY INTERLEUKIN-12 (IL-12) IN
SENDAI VIRUS-RESISTANT F344 RATS .......................................................... 38

Summary ...................................................................................................................... 38
Introduction ....................................................................................... ........................... 39
M materials and M ethods ............................................................................................. 42
A n im als ................................................................................................................. 42
Viral Procedures and Sample Collection.............................................................. 42
Cytokine mRNA ............................................................................................... 43
Protein Analysis.................................................................................................... 44
Enzyme-linked immunosorbent assay (ELISA) .............................................. 44
Immunohistochem istry.................................................... ............... .... ...... 45
Data Analysis ..................................................... ................................................... 46
Results................... .................................................................................... ................... 46
Virus-Resistant F344 Rats Have Increased Expression of Pulmonary IL-12
m R N A ............................................................................................................. 46
F344 Rats Have Increased Pulmonary IL-12 Protein After Sendai Virus Infection
........................... ............................... ............. .... ........ ....... 49
F344 Rats Have Increased Numbers of IL-12 p40 Expressing Cells in the
Bronchioles After Sendai Virus Inoculation................................................... 50
F344 and BN Rats Do Not Have Detectable Differences in the Expression of IL-
18 mRNA ........................................................................................................ 53
D discussion ................................................................. ................................................... 54

4 EXOGENOUS INTERLEUKIN-12 (IL-12) ADMINISTRATION REDUCES THE
SEVERITY OF SENDAI VIRUS-INDUCED CHRONIC AIRWAY FIBROSIS
AND REM ODELING IN BN RATS................................. ..................................... 59

S um m ary ..................................................................... ................ ................................ 59
In production .............................................................................. .................................... 60
M materials and M ethods ............................................................................... ........... 62
A n im als ................................................................................ .................................62
Viral Procedures and Sample Collection .............................................................. 62
IL-12 Treatment Protocol ..................................................................................... 63
Cytokine mRNA ................................................................................................... 63
Enzyme-Linked Immunosorbent Assay (ELISA)..................... ............................ 65
BrdU Immunohistochemistry...................................... ..........................................65
Analysis of Bronchiolar Inflammation and Fibrosis ............................................. 66
Data Analysis ........................................................................................................ 67
Results ........................................ .................... ..................... 68
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Amount of Bronchiolar Inflammation and Fibrosis .................................... 68
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Bronchiolar W all Thickness ........................................................................... 69









IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Number of BrdU Labeled Fibroblasts in the Bronchiolar Walls.................... 70
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Increases the
Pulmonary Expression of IFN-y ..................................................................... 70
IL-12 Treatment of BN Rats on Day 2 After Sendai Virus Inoculation Alters the
Levels of IL-18 or IL-4 mRNA ...................................................................... 73
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Does Not
Alter the Respiratory Clearance of Sendai Virus............................................ 76
D iscu ssio n .................................................................................................................... 76

5 GENERAL SUMMARY AND FUTURE DIRECTIONS ............................................ 80

O bjectiv e 1 ................................................................................................................... 80
O objective 2 ................................................................................................................... 81
O objective 3 ................................................................................................................... 8 1
O objective 4 ................................................................................................................... 82
C conclusions ...................................................................................................... .. .......... 82
Future Studies .......................................... .................................................................... 83

APPENDIX

A PRELIMINARY DATA ............................................................................................86

Experiment 1: Pulmonary Expression of IL-12 in Sendai Virus-Infected BN and F344
R ats ............... .. ...................................................................................................... 86
D ilutional R T -PC R ........................... ....................... ............................................. 86
Lung Lavage Fluid ELISA.................................................................................... 86
Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats ................... 88

B IL-12 DOSAGE FOR TREATMENT TRIAL.............................................................. 89

Experim ental D esign .................................................................................................... 89
R results and C onclusions............................................................................................... 90

C IN SITU HYBRIDIZATION FOR INTERLEUKIN-12 ......................................... 93

Protocol For In Situ Hybridization......................................................................... 93
Problems Solving For Difficulties With In Situ Hybridization.................................... 94
Observations of In Situ Hybridization Experiments ........................ ............................ 95

LIST OF REFERENCES ................................................................................................... 98

BIOGRAPHICAL SKETCH ...................................................................... ...... ...... 111I















LIST OF TABLES


Table page

2-1 Table of Experimental Design: Experiment 1 ............................................................28

2-2 Table of Experimental Design: Experiment 2...................................................................34

3-1 Comparative CT Method ofcDNA Relative Quantitation......................................... 44

B-I Experimental Design for IL-12 Dosage Trial ............................................................89

B-2 Percentages of Inflammatory Cells in the IL-1 2 Treated BN Rats...........................91















LIST OF FIGURES


Figure page

2-1 Experimental design diagram for experiment 2 ......................................................... 34

3-1 Real-time PCR analysis of IL-12 p 40 mRNA in whole lung samples of BN and
F344 rats after Sendai virus infection .................................................................... 47

3-2 Real-time PCR analysis of IL-12 p35 mRNA in whole lung samples of BN and F344
rats after Sendai virus infection ....................................................................... 48

3-3 Real-time PCR analysis of IL-12 p40 mRNA in trachea samples of BN and F344 rats
after Sendai virus infection ............................................................................... 48

3-4 Real-time PCR analysis of IL-12 p35 mRNA in trachea samples of BN and F344 rats
after Sendai virus infection ................................................................................. 49

3-5 ELISA analysis of IL-12 total protein in F344 and BN rats strains after Sendai virus
inoculation................................................. .......................................... ... ...... 50

3-6 ELISA analysis of IL-I 2 p40 protein in F344 and BN rats strains after Sendai virus
in ocu latio n ................................................................ .............................................. 50

3-7 OX-6 immunohistochemistry in the bronchiole of a F344 rat three days after
inocu lation .............................................................................................................. 5 1

3-8 Density of OX-6 positive dendritic cells in the bronchioles of F344 and BN rats.....51

3-9 Density of IL-12 p40 positive dendritic .............................. ....................................52

3-10 IL-12 p40 immunohistochemistry in the wall of bronchiole of a F344 rat at two
days after Sendai inoculation ............................... ........................ .................. ........ 52

3.11 Density of IL-12 p40 positive macrophages in the bronchioles of BN and F344 rats.
.............. ........................................................... ...................................................... 5 3

3-12 Real-time PCR analysis of IL-18 mRNA expression in lung samples of BN and
F344 rats after Sendai virus infection .............................. ...................................... 54

4-1 The percent of bronchioles containing evidence of inflammation and/or fibrosis at 14
days after inoculation............................................................................................. 68









4-2 Airway morphometric analysis of bronchiolar wall thickness at 14 days after virus
in ocu lation .................................................................................. .... ..................... 69

4-4 Immunohistochemical analysis of fibroblast BrdU labeling at 10 days after Sendai
inocu lation ....................................................................................... .................... 72

4-5 Immunohistochemical analysis of fibroblast BrdU labeling at 14 days after Sendai
inoculation............................................................................................... ............ 72

4-6 Competitive PCR analysis of IFN-y mRNA in BN rats 3 days after inoculation
treated at day 0 or day 2 w ith 1L- 12....................................................................... 73

4-7 ELISA analysis of whole lung homogenates from BN rats at 7 and 10 days after
virus inoculation ......................................... ........................................................ ... 74

4-8 Analysis of real-time PCR for the detection ofIL-I 8 mRNA .................................... 75

4-9 Analysis of real-time PCR for the detection of IL-4 mRNA......................................75

4-10 Viral titer results from plaque assays at 7 days after inoculation ............................. 76

5-1 RT-PCR of IL-12RP2 in BN and F344 rats at non-infected control levels ............ 84

A-1 Dilutional RT-PCR analysis of mRNA from virus-infected BN and F344 rats ........ 86

A-2. ELISA analysis of IL-12 p70 protein in concentrated lavage samples from small
numbers virus-infected and control rats ................................................................. 87

A-3 ELISA analysis of IL-12 p40 protein (homodimers and monomers) in concentrated
lavage samples from small numbers virus-infected and control rats .................. 87

A-4 ELISA analysis of IFN-y in concentrated lavage samples ................ ........................ 88

B-1 The percentage of cells identified as macrophages, neutrophils, lymphocytes, and
epithelial cells in all treatment groups ............................................................... 91

B-2 Pneumonia indices of BN rats in all treatment groups .............................................. 92

C-I Anti-Sense IL-12 p40 mRNA In Situ Hybridization ofbronchiole wall in a F344 rat
at 5 days after Sendai inoculation .......................................................................... 96

C-2 Anti-Sense IL-12 p40 mRNA In Situ Hybridization of inflammatory aggregate in the
bronchiole wall of an F344 rat at 3 days after inoculation ....................................96

C-3 Sense IL-12 p40 mRNA In Situ Hybridization in the airway of a F344 rat 5 days
after inoculation ..................................................................................................... 97















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE ROLE OF INTERLEUKIN-12 IN THE PATHOGENESIS OF SENDAI VIRUS-
INDUCED AIRWAY DISEASE


By

Amy Elizabeth Seymour Stone

December 2002


Chair: William L. Castleman
Major Department: Veterinary Medicine


Brown Norway (BN) rats are susceptible to Sendai virus-induced chronic airway

inflammation that results in fibrosis and functional abnormalities resembling asthma.

Fischer (F344) rats are resistant to these virus-induced changes and have earlier viral

clearance, increased expression of Th-1 cytokines (e.g., interferon-y, IFN-y), and do not

develop pulmonary function abnormalities. In contrast, BN rats are Th-2 type cytokine

responders (e.g., interleukin-4, IL-4) and have delayed viral clearance. Due to the critical

role of interleukin-12 (IL-12) in regulating the IFN-y cytokine response in intracellular

infections, I hypothesized that virus-resistant F344 rats are higher IL-12 gene responders

than BN rats. Levels of IL-12 p40 messenger (mRNA) were measured by real-time

polymerase chain reaction (RT-PCR) and IL-12 protein was detected by lung

homogenate enzyme-linked immunosorbent assay (ELISA) at several time points after








viral inoculation. Although virus infection resulted in increased IL-12 production in both

strains, F344 rats had significantly more IL-12 p40 mRNA than BN rats at 0-3 days

(early) after virus inoculation (p<0.05). Furthermore, IL-12 total protein levels were

elevated in F344 rats as early as 2 days following viral challenge, and the numbers of IL-

12 p40 protein expressing cells were also significantly increased in their bronchioles at 2

and 3 days following Sendai inoculation (p<0.05). To evaluate the potential protective

role of IL-12 in virus-induced airway injury, BN rats were given IL-12 intraperitoneally

at either the time of (day 0) or two days after viral inoculation (day 2). In contrast to

infected rats given saline, infected rats treated with IL- 12 at day 0 had 22.1% lower levels

of chronic airway inflammation, 23.8% lower levels of airway fibrosis, and 42% and

62.5% decrease in bromodeoxyuridine (BrdU)-labeled fibroblasts at 10 and 14 days after

inoculation respectively (p<0.05). Day 0 treated BN rats had a 4-fold increase in the

pulmonary IFN-y mRNA and a 77% increase in IFN-y protein as compared to saline-

treated, virus-inoculated controls. In contrast, day 2 IL-12 treatment induced a 20%

increase in bronchiolar airway wall thickness, a 12.5% increase in BrdU-labeled

fibroblasts at 14 days after inoculation, and an increase in pulmonary IL-4 mRNA

(p<0.05). The results are consistent with the hypothesis that resistance to virus-induced

airway damage in F344 rats is due, at least in part, to their high virus-induced IL-12 gene

expression.













CHAPTER 1
LITERATURE REVIEW

Interleukin-12

Interleukin- 12 (IL- 12) is a heterodimeric cytokine, which plays a role in the

induction of cell-mediated and T helper type-1 (Th-1, CD4 T cells) immune responses

(1). This cytokine is produced primarily by antigen presenting cells in response to

intracellular bacterial, viral, protozoal, and fungal infections (1). The main function of

IL-12 is to direct the "cross talk" between the phagocytic antigen presenting cells and

effector lymphocytes by inducing the production of cytokines, particularly interferon-y

(IFN-y), and by enhancing lymphocyte cytotoxic activity (2). Directly or indirectly, IL-

12 is involved in the activation of macrophages, the generation and survival of Th-1 cells,

the generation of cytotoxic T lymphocytes (CTL, CD8 T cells), and the suppression of

IgG1 and IgE production (2). IL-12, synergistically with interleukin-2 (IL-2), tumor

necrosis factor-a (TNF-ct), and interleukin-18 (IL-18) induces the maturation of Th-1

cells from Th-0 precursors by inducing the production of IFN-y from resting and

activated natural killer (NK) and T cells (1). IL-12 maintains high levels of IFN- y once

the CD4 and CD8+ T cell types are established to resolve the infection (2). Additionally

IL-12 acts to prevent the outgrowth and development of Th-2 cells and their production

ofTh-2 cytokines (3).

The structure of IL-12 is a unique heterodimer composed of two disulfide-linked

subunits, p35 and p40 (representing the approximate molecular weights) (4). These two









subunits are encoded on two unrelated genes residing at independent loci in the mouse,

human, and rat genomes (5-7). No sequence homology exists between the two subunits,

but the p35 subunit shares homology with interleukin-6 (IL-6), granulocyte colony

stimulating factor (G-CSF), and chicken myelomonocytic growth factor (8). The p40

subunit is not homologous to other cytokines, but is a part of the hemopoietin receptor

family most closely resembling the IL-6 receptor a-subunit and the ciliary neutrotrophic

factor receptor (5,9). Coexpression and covalent linkage of both chains of this cytokine

in the same cell is required for the generation of the functionally active heterodimer (10).

Expression of the p35 chain is ubiquitously constitutive. The secretion of free p35

subunit has not been demonstrated (11-12). Both subunits are induced by intracellular

infections via the subsequent activation of phagocytic cells (13). The p40 gene is located

only in IL-12 producing cells, and is produced at much higher levels than the p35 chain

or p70 heterodimer (11-12). The rate of p70 production is limited by p35 expression

because the p40 subunit is produced by phagocytic cells at a few-fold to 1000-fold higher

levels than the active heterodimer (14-15). Secretion of IL-12 p40 monomers and

homodimers can have an inhibitory effect on the expression and production of the p70

heterodimer in murine T and NK cells (10). The p40 homodimer may be acting as a

physiologic antagonistic regulator in the mouse system or may just be competing for the

IL-12 cellular receptor (2). In humans, the p40 homodimer has modest ability compete

with the heterodimer for IL-12 biological activity (16). Recently, human airway

epithelial cells have been shown to produce high levels of p40 subunit, the possible role

being to attract macrophages to the site of airway inflammation during mucosal defense

(17).








The predominately transcriptionally regulated p40 subunit gene spans 13 Kbp

containing eight exons preceded by a classical promoter (18,1). The murine p40 promoter

contains three essential transcription factor-binding sites, a nuclear factor kappa B (NF-

KB) site, a CCAAT enhancer-binding protein site (C/EBP), and an adaptor protein-

binding site (AP-1), that are involved in lipopolysaccharide (LPS) and IFN-y promoter

activation (19-20). The human p40 promoter also has three essential cis-acting elements

in its promoter, including a NF-KB site, an Ets (erythroblastosis virus oncogene

homologue) core element (Ets-2, interferon regulatory factor-1 [IRF-1], interferon

consensus sequence binding protein, and c-Rel), and a C/EBP site (18,21-22). The

regulatory mechanisms of this promoter appear to involve functional synergy between the

Ets and NF-KB transcription factors in the human gene, and the C/EBP factor also

interacts functionally with the NF-KB factor in the promoters of both species (21-22).

The trans-acting proteins interacting with the binding sites mediating the activation of the

IL-12 p40 gene have not been well elucidated. However, a novel repressor element, GA-

12 binding protein (GAP-12), has been shown to reduce inducible IL-12 p40 gene

transcription in response to interleukin-4 (IL-4) and prostaglandin-E2 (PGE2) in human

monocytes (23).

Due to the constitutive expression of the IL-12 p35 gene in many tissue types, many

studies have focused on the p40 gene expression. However, p35 subunit production is

more tightly regulated as it appears to be controlled translationally and transcriptionally

(24). The murine p35 gene has multiple transcription start sites at either of two 5' exons

resulting in mRNA isoforms with different untranslated regions (5'UTR) (25). The

human p35 gene also has multiple forms initiating from two separate exons, suggesting








that the p35 genes in both species are similarly regulated (14). Under non-stimulated

conditions, p35 transcripts contain an additional upstream ATG from a region whose

presence inhibits translation (24). Stimulated cells produce transcripts that lack this

upstream ATG that can be translated into p35 protein; the proportion of each set of

transcripts in vivo depends on the stimulus (14,24-25). The multiple transcription start

sites suggest the existence of two promoters, and the switch between the two may depend

on the initiating transcription factor (14). There are multiple NF-KB sites, IRF-1 sites,

and an AP-2 site within both promoter regions (14). Only the NF-KB, particularly c-Rel,

and the proximal most IRF-1 sites have proven critical for efficient transcription (13,14).

There are several other putative elements that may contribute to regulation, including

gamma-associated elements (GAS), IFN-stimulated response elements (ISRE), and

interferon consensus sequence binding sites (ICSBP) (14). Confusion regarding the gene

expression of IL-12 p35 remains, due to the inability of conventional methods, such as

northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), to

accurately distinguish between the at least four different mRNA isoforms.

The IL-12 receptor is composed of two subunits (IL-12RI31 and IL-12Rp32) that are

members of the cytokine receptor super family most closely related to glycoprotein 130

(gpl130) (26). Coexpression of these two receptors is required for the formation of a

high-affinity IL-12 binding site in human cells; however in mouse cells the IL-12Rpl 1

subunit confers high and low-affinity binding (26-27). The IL-12Rp32 subunit contains

three tyrosine residues and is essential for signal transduction in both species (26-28).

There is a 68% homology between the human and mouse receptors, and both are thought

to be regulated and expressed in a similar manner (26). Expression of IL-12Rp2 protein








may be limited to Thi cells and this expression may correlate with IL-12 responsiveness

(29-30). The IL-12RP31 subunit is present on resting NK and T cells and is up regulated

during activation (30). The IL-12Rp2 subunit is not present on resting CD4+ T cells but

is up regulated upon antigen activation of the T cell receptor (TCR) (30). Upon T cell

activation, the cytokines interleukin-4 (IL-4) and IFN-y can modify this subunit

expression. During T cell maturation, IL-4 can inhibit IL-12Rp32 subunit expression, thus

losing the ability to respond to IL-12 after TCR binding (30). When low levels of IL-4

are present, IFN-y is required for IL-12Rp2 expression (30). However, if there is no

detectable IL-4, the presence of IFN-y is not necessary (30). The IL-12Rp32 chain

appears also to be more highly regulated than the IL- 12 RP3 1 by cytokines such as IL- 10

and TGF-p1 (31).

Once IL-12 is bound to its receptor, the complex induces the rapid tyrosine

phosphorylation of both Janus (JAK) and Tyrosine kinases (Tyk) (32). Following the

activation of the JAK and Tyk, three members of the signal transducers and activators of

transcription (STAT) family are phosphorylated and activated [STAT-1, STAT-3, and

STAT-4] (33-34). STAT-4 is not activated by any other cytokine except IFN-oc in human

cells, so it is unique to the IL-12 signaling pathway (35). STAT-4 is directly involved in

the transcription of IFN-y, primarily through two binding sites in the first intron (36-37).

STAT-4 has been shown to be present and activated with translocation to the nucleus in T

cells, NK cells, and dendritic cells (38). STAT-4 knock out mice have an almost

identical phenotype to IL-12 or IL-12R knock out mice and produce no IFN-y in response

to IL-12 stimulation confirming this pathway in the IL-12 biological response (36,39).

Additionally, there is evidence that IL-12 can induce transcription of an Ets transcription








factor called ERM in differentiated Th-1 cells through the binding of the TCR (40). The

induction of this factor, although it does not activate STAT-4, is dependent on the

presence of STAT-4 within the cell (40). This pathway may regulate another aspect of

Thi behavior or may require cooperation to regulate IFN-y production (40).

The Role of Interleukin-12 in Regulating the Immune Response

The production of IL- 12 in the immune response is complex and can be initiated by

several different pathways. Phagocytic cells, including dendritic cells, produce IL-12 by

T cell-independent and T cell-dependent means. Infectious agents, including bacteria,

bacterial products, both metazoan and protozoan parasites, fungi and viruses, induce the

production of IL-12 by phagocytic cells initiating the inflammatory process independent

ofT lymphocytes (15, 41). Additionally during inflammation, but independent of

infectious agents or T cells, IL- 12 production is induced by the interaction of adhesion

molecules with substrates of the inflammatory cascade, such as the interaction of the

CD44 adhesion molecule with low molecular weight hyalumrnonan (42). This mechanism

may contribute to macrophage activation due to the proinflammatory roles of IL-12 and

IFN-y (2,42). The T cell-dependent mechanism of IL-12 production by antigen

presenting cells depends on the CD40/CD40 ligand interaction with activated T cells

(43). The antigen presenting cells induce IL-12 and up regulate receptors, such as B7 on

monocytes and dendritic cells, thus activating antigen specific T cells (43). During this

interaction, the p70 heterodimer is produced more or as efficiently as the p40 chain,

indicating the effective up regulation of both p35 and p40 peptide chains (14,24,43). The

T cells then produce IFN-y and the cytokine granulocyte/monocyte-colony stimulatory

factor (GM-CSF), which enhance the ability of the antigen presenting cells to produce IL-








12 (45-46). Two signals are required for both the T cells (CD40L and IFN-y) to produce

IFN-y and for most of the antigen presenting cells (innate signal from infection and

CD40L) to produce IL-12 in response to CD40 ligation (47). This bi-directional, two-

signal interaction functions to maintain the T cell-independent mechanisms of IL-12

production that initiated the inflammatory process (48).

The ability of these various pathways to induce IL-12 production is regulated by

both positive and negative feedback mechanisms. IL-12 induces the production of IFN-y

by T and NK cells; IFN-y then enhances the expression of IL-12 by phagocytic cells and

neutrophils (14,21,49). There are several other cytokines produced in response to IL-12

expression, such as TNF-a, GM-CSF, Interleukin-8 (IL-8), and interleukin-1I3 (IL-1P)

(43,50). TNFac and GM-CSF are also involved in positive feedback loops with IL-12.

Specifically, TNF-a enhances the ability of IFN-y to prime phagocytic cells for IL-12

production, and GM-CSF has a priming effect on IL-12 production from phagocytic cells,

primarily at the level of the p40 gene (51,52). Other Th-1 cytokines, such as IL-18, IL-2,

and IL-15, are costimulatory with IL-12 in the production of IFN-y. IL-2 or IL-12 alone

can stimulate the production of IFN-y from NK and T cells, but when acting together the

half-life of the IFN-y mRNA is doubled and IL-2 increases IL-12RP2 expression on

activated NK cells, thus enhancing the production of IFN-y (53, 54). IL-15 shares the

same biological functions with EL-2 and seems to interact with IL-12 through similar

pathways (55). IL-18 also acts with IL-12 to induce IFN-y in T cells and NK cells, but

this combination also is capable in vitro of stimulating IFN-y enhancement in mouse

dendritic cells and macrophages, suggesting the existence of an autocrine feedback loop

in these professional antigen presenting cells (55-57).








The counter mechanisms that down regulate the positive amplification of IL-12

production are mediated by IFN-y and they prevent uncontrolled cytokine production.

Interleukin-10 (IL- 10), potent inhibitor of IFN-y production directly inhibits the

production of IL-12 by antigen presenting cells (58). IL-10 is able to block the

proliferation of Th-1 cells through the inhibition of IL-12 transcripts, other soluble

cytokines, and costimulatory surface molecules by antigen presenting cells (58). In both

mice and humans, IL-10 also prevents the development of mature, differentiated dendritic

cells (59-60). However, IL-12 induces the production of IL-10 in T cells and primes T

cell clones for high IL-10 production, thus inducing a negative feedback loop to reduce

its own expression (61). Other inhibitors of IL-12 include transforming growth factor-P

(TGF-0), PGE2, and partial inhibitors IL-4, TNF-a and IL-13 (62). Like IL-10, TGF-P1i

suppresses IL-12 at the transcriptional level; however it also appears to reduce the

stability of the IL-12 p40 mRNA (63). IL-4 and interleukin-13 (IL-13) can suppress IL-

12 expression when added simultaneously with a stimulus to cell cultures. However,

monocytes primed with IL-4 or IL-13 prior to stimulation can significantly enhance IL-12

expression (62, 64). These overlapping cytokine regulation pathways are postulated to be

extra backup mechanisms to prevent uncontrolled production ofproinflammatory

cytokines.

Although B cells, neutrophils, microglial nerve cells, and macrophages produce

some IL-12, dendritic cells have been identified as extremely efficient producers of the

IL-12 that can act in inducing Th-1 responses upon antigen presentation (65-67). Due to

the heterogeneity of both human and mouse dendritic cells, it is not clear which type or

maturational state of dendritic cell is the major producer of IL-12. Human monocyte-








derived dendritic cells or myeloid dendritic cells (DC1) obtained from cultures treated

with GM-CSF and IL-4 produce high levels of the bioactive IL-12 p70 in response to

various stimuli (65). Alternatively, the plasmacytoid dendritic cells (lymphoid origin)

have been reported to produce lower levels of IL-12 (68). In the mouse, the CD8a-

positive dendritic cells, which may be comparable to the human plasmacytoid dendritic

cells, are more efficient producers of IL-12 when compared to myeloid dendritic cells

after intracellular infection in vivo (69). Dendritic cells from both species are unique

antigen presenting cells because 1) they produce bioactive IL-12 upon specific interaction

with T cells without additional stimuli; 2) their production of IL-12 is critical for optimal

proliferation and IFN-y production by activated Th-1 cells; 3) they prime resting, naYve T

cells that, once restimulated, produce Th-1 cytokines (66). The ability of dendritic cells

to propagate Th-1 differentiation is due to their levels of STAT-4 (70). IL-4 can reduce

the amount of STAT-4 in maturing dendritic cells, reducing the amount of IL-12

dependent IFN-y produced as well as the Th-1 signaling capacity of these cells (70).

Once dendritic cells are mature, IL-4 can no longer inhibit the production of STAT-4

(70). Dendritic cells, depending on type and maturational status, are therefore capable of

initiating the antigen-activated immune response in the innate branch of the immune

system and then directing the Th-1 I response to steer the humoral immune response.

Many parameters direct the development of Th-1 or Th-2 cells from a naive

precursor. These include the antigen presenting cells used for priming, the dose of

antigen encountered, the costimulatory cell-surface molecules, the genetic background of

the cells, and the cytokine milieu present in the environment (71). Changing one of these

factors can alter the resulting T cell phenotype. Ultimately the cytokines IL-4 and IL-12








act directly on T cells through STAT-6 and STAT-4 respectively to deliver final

differentiation signals (38,71). Naive CD4+ T cells are activated by interaction with

antigen presenting cells, primarily dendritic cells, through the TCR (30,66). This initiates

expression of the IL-12RP2 subunit and up-regulates the IL-12RP1 subunit already

present on the surface ofT cells (30). The presence of even minute quantities of IL-4

will inhibit IL-12RP2 expression; however, the presence of IFN-y enhances this subunit's

expression and can even reverse IL-4 inhibition (30, 71). The source of IFN-y is likely to

be NK cells, T cells, and IL-12 stimulated dendritic cells and macrophages (1, 38,57).

Once the capacity of T cells to respond or not to respond to IL-12 is established, IL-12

acts through STAT-4 via the IL-12R to determine the Th-1 or IFN-y producing phenotype

(39). This process is comparable between the human and mouse species except that

human Th-2 cells maintain their IL-12 responsiveness through low levels of IL-12p2

receptor subunit. In addition, IFN-a is as effective, and in some instances more effective,

an inducer of lL-12RP32 chain in human T cells (72). Generally, the choice between a

Th-1 and Th-2 phenotypic T cell response is dependent on the balance between the levels

of IlL-4 and IL-12 during the maturation of naYve T cells.

As previously noted, one of the main actions of lL-12 in the inflammatory response

to pathogens is to direct the development of Th-1 cells; however IL-12 has several other

very important functions. EL-12 is a potent inducer of IFN-y from CD4+ and CD8+ T

cells, NK cells, and y/5 T cells (2). IL-12 also directly enhances the cytotoxicity of NK

cells and CTLs by inducing the expression of genes encoding cytotoxic granules (e.g.,

performin) and by endowing the CTLs with the ability to mediate antibody-redirected lysis

of target cells (2,73). These effects can be additive when synergistic cytokines such as








IL-2 and IFN-y are present, but IL-12 alone is capable of inducing these effects (2).

Additionally, IL-12 is the major factor required for the differentiation of CD8 T cells

and 7/8 T cells, priming them to be polarized to produce Th-lcyokines, similar to its

effect on their CD4+ counterparts (2). IL-12 can also function as an inhibitor of Fas-

mediated, non- B cell lymphoma oncogene-2- (BCL-2) -dependent T-cell apoptosis (2).

IL-12 is critical in the activation of macrophages through the Th-1 differentiation,

enabling them to produce bactericidal and anti-viral cytokines (67). Recent evidence

using infection models suggests that IL-12 is not only required to initiate these responses

but also maintains antimicrobial functions, such as the ability of memory T cells to

produce IFN-7 at later stages of infection (3).

Investigations of the humoral side of the inflammatory response demonstrated that

IL-12 could suppress the production of Th-2 type antibodies (IgGI and IgE) and increase

the production (10- to 1000- fold) of Th-1 type antibodies IgG2a, IgG2b, and IgG3 (74).

This effect is mediated predominantly by the increased expression of IFN-y; however

removal of IL-12 alone also affects the type of antibodies produced (74). This regulation

is similar to the interaction of IL-12 with IL-4, in that if cells are primed or boosted, IL-

12 can modestly enhance the production of IgG1 and IgE (74). Recent studies have also

shown that IL-12 increases the production of complement fixing antibodies. In addition

certain aspects of the complement cascade may directly modulate the production of IL-12

in various infections and delayed-type hypersensitivity reactions (75). IL-12 is at the

interface of all aspects of the immune response to intracellular infection with interacting

links between the innate and immune responses.








The Role of IL-12 in Infectious Diseases

IL-12 in Bacterial Infections

Intracellular infection of mice with Listeria monocytogenes has been used

extensively as a model to study the role of Th-1-dependent, cell-mediated immunity,

including the role of IL-12 in intracellular infections (76). L. monocytogenes resistant

mice, such as C57BL/6, have macrophages and dendritic cells with higher IL-12 (52%

more total IL- 12 p70) producing capacities that activate NK and y/8 T cells to secrete

IFN-y, and promote the development of Th-1 immunity early during acute infection (77,

78). Additionally, administration of recombinant IL-12 (rIL-12) increases resistance to

this bacterium, and its antibody neutralization leads to increased bacterial susceptibility

(79). The role for IL-12 in sustaining the response in murine listeriosis is unclear (3).

The evidence for the critical need for early IL-12 production in these models is proven

whereas the evidence for late production is still controversial.

IL-12 is induced at high levels in response to Salmonella, mycobacteria, and other

bacterial components. Mice infected with attenuated S. dublin bacteria had increased IL-

12 production in the lymph nodes and Peyer's patches and were protected against

subsequent infection with the virulent agent (79, 80). The emergence of a protective Th-

1 immune response is dependent on IL-12 in murine Mycoplasma tuberculosis infections

(2,79). As in Listeriosis, the addition ofrIL-12 to susceptible BALB/c mice increased

survival and delayed lung pathology (81). Blocking IL-12 with monoclonal antibodies

increases susceptibility in normally M. tuberculosis resistant mice, similar to the

phenotype observed in IL-12 p40 deficient mice (2, 81). The addition ofrIL-12 to human

cell cultures increased the cytolytic activities of NK and CD4+ T cells against monocytes








infected with M. tuberculosis (82). Recombinant IL-12 also increases the proliferative

responses of peripheral blood lymphocytes and stimulates the antibacterial properties of

macrophages in patients with M avium infection (83). Even the addition of a synthetic

oligonucleotide containing a palindromic sequence from mycobacteria, a sequence of

DNA from Escherichia coli, or the B subunit ofE. coli can induce the expression of IL-

12 mRNA p40 and p35 in mouse splenocytes in cell culture and intestinal lymphoid

tissue in vivo (79). IL-12 has significant roles in other bacterial infections such as

Brucella abortus and Klebsiella pneumoniae, but research has not been as extensive in

these infections (79).

IL-12 in Protozoal Infections

The production of IL-12 is important in the initiation and the maintenance of the

Th-1 response in cutaneous and visceral leishmaniasis. The susceptibility of BALB/c

mice to the cutaneous, intracellular protozoan, Leishmania major, is in part due to the

genetic background of the T lymphocytes (84). Though the expression of IL-12 mRNA

and protein levels is similar between resistant and susceptible mouse strains, susceptible

BALB/c mice T cells lose the ability to generate IL-12-induced Th-1 responses and

instead form an IL-4-induced Th-2 response that is ineffective in clearing the pathogen

(84). The important role of IL-12 in resistant mouse strains, such as C57BL/6 and C3H,

seems to be its ability to function as a growth factor for Th-1 cells by the intensification

of IFN-y production and the suppression of IL-4 and IL-10 (79). Treatment of the

susceptible BALB/c mouse strain with IL- 12 during the first week of infection with L.

major induces resistance to the infection with a shift in the immune system from Th-2 to

a Th-1 response and the cured animals are resistant to subsequent rechallenge (2). In









addition, during L. major infections resistant C3H mice upregulate the mRNA expression

oflL-12Rp31 and -P2 subunits on CD4+and CD8+ T cells (85). In contrast, susceptible

BALB/c mice show no increase in IL-12 receptor subunits upon infection with L. major

(85). The antibody neutralization of IL-12 in Leishmania-resistant strains converts these

mice to susceptibility (79).

The treatment of BALB/c mice with rIL-12 two weeks after L. major infection

abrogates its protective effects and can even enhance the expression of IL-4 (79). Thus,

it is difficult to reverse an already established Th-2 immune response. Alternatively, IL-

12 may be needed to maintain a Th-1 response in animals where a Th-1 response has

been initiated. Despite the development of a Th-1 response in IL-12 p40 knockout (IL-12

KO) mice transiently treated with rIL-12, the animals were unable to sustain a Th-1

response in the absence of IL-12 past the acute phase of infection (79, 86). These IL-12

KO mice in the absence of 1L-12 treatment developed evidence of a Th-2 response (86).

IL-12 may also play an important role in visceral leishmaniasis in mice and humans. In

L. donovani mouse infections, in which susceptibility is associated with a failing Th-1

immune response, treatment with rIL-12 has been effective in reversing the disease

process (79). The addition of rL-12 to cultures of human peripheral blood mononuclear

cells from patients with visceral leishmaniasis restores the proliferative and IFN-y

producing capacities of these cells (79). Therefore, in mice and in humans, IL-12 appears

to initiate and maintain cell-mediated immunity, as well as suppress the Th-2 response to

Leishmania infection.

The role of IL-12 in other protozoal infections appears to be quite similar to that in

leishmaniasis, but the differential expression of EL-12 is much greater. C3H mice have









twice the total IL-12 protein in serum and splenic tissues as compared to BALB/c mice in

response to Trypanosoma cruzi infection (87). This difference has been associated with

increased infection resistance due, most likely, to observed increases in NK cell

cytotoxicity and the levels of IL-12 dependent IFN-y protein (87). Additionally, rIL-12

treatment of the susceptible BALB/c mice led to resistance during the acute phase of the

disease, but was ineffective during the chronic infection (87). Cells from Toxoplasma

gondii infected, IL-12 KO mice, transiently treated with rIL- 12, were unable to produce

IFN-y upon antigenic stimulation without the addition of IL-12 (3). There is evidence

that the Th-1 response was initiated and that Th-1 cells were developed, but the T cell

memory was not functional without IL-12 (3). These mechanisms, along with IL-12's

ability to upregulate TNF-a, cell surface molecules, and to increase the phagocytic ability

of antigen presenting cells, are also important in resistance to the mouse protozoal

diseases Plasmodium chabaudi and Cryptosporidium parvum (79).

IL-12 in Fungal Infections

In many fungal infections, the establishment of a Th-1 type reaction is critical to

development of phagocyte dependent protection and the production of inhibitory

cytokines such as IL-4, IL-10, and the IgE antibody is associated with disease

progression (79). DBA/2 mice are genetically resistant to Coccidioides immitis and are

induced to produce five times more IL-12 p40 mRNA in their lungs as compared to C.

immitis-susceptible C57BL/6 mice (79, 88). Neutralization of IL-12 in the DBA/2 strain

by monoclonal antibodies to IL-12 leads to severe disease, and conversely administration

ofrIL-12 to the fungus-susceptible strain (BALB/c) decreases susceptibility to clinical

disease progression (79).








Recombinant IL-12 administered at the time of murine Cryptococcus neoformans

infection results in protection from disseminated infection including pneumonia and then

meningitis (89). This effect seems to be mediated by an increase in the numbers of

pulmonary inflammatory cells, a decrease in the number of neural yeast cells, and

detectable IFN-y mRNA in the lungs of treated mice (89). Later administration ofrIL-12

fails to protect these mice against dissemination of the infection with C. neoformans, with

no detectable pulmonary IFN-y mRNA (89). The role of lL-12 in human fungal

infections continues to be investigated due to the importance of human

immunodeficiency virus (HIV) related susceptibilities to opportunistic fungal infections.

IL-12 in Viral Infections

IL-12 plays an important role in viral defense; however its role is more complex

than in other types of intracellular infections. In murine viral infections such as murine

cytomegalovirus (MCMV), respiratory syncytial virus (RSV), influenza, and herpes

simplex virus (HSV), IL-12 is critical in the early activation of NK cells and the

establishment of a Th-1 antiviral immune response (2, 79, 90). During MCMV and RSV

infection, IL-12 p70 levels increase in serum (50% and four-fold respectively) at early

time points after infection (90, 91). However lymphocytic choriomeningitis virus

(LCMV) infection does not induce detectable IL-12 levels, but instead activates the T-

cell IFN-y responses through the IL-12 inhibitory cytokines IFN-a3 (92). During LCMV

in the absence of IEFN-op the IL-12 response is inducible and indicates an alternative

pathway to NK cell activation and IFN-y production (92).

Low dose IL-12 administration has some protective effects in LMCV infection;

however higher doses can lead to decreases in T cell activity and increases in T cell








necrosis (92). Low doses of IL-12 increased NK cell cytotoxicity, CD4+ and CD8+ T

lymphocyte numbers, IFN-y production, and antiviral status (92). In other murine viral

infections, such as mouse hepatitis virus, encephalomyocarditis virus, and mouse

adenovirus infection rIL-12 administration induced protection (79). In mice transgenic

for the hepatitis B antigen, IL-12 suppressed autoantibody production (Th2 to Thi shift),

inhibited virus replication in the liver and kidneys, and increased IFN-y production (79).

In many of these infections peak IL-12 levels are noted 1-3 days (i.e., early) after

infection and are usually transient (2,79).

IL-12 may also play opposing roles in the outcome and/or associated pathology in

the same infection (79). In corneal HSV infection, local over-production of IL-12 leads

to a virus-specific Th-1 reactivity and immunopathologic disease (93). However, in

thermally injured mice, IL-12 promotes resistance to HSV infection (79). In humans, the

measles virus actually down regulates the expression of L-12 in vivo. In human

monocyte cell cultures measles infection selectively impairs the expression of IL-12

without affecting other cytokines (94). This decreased IL-12 production is dependent on

the activation of the measles virus cellular receptor CD46, a regulator of the complement

gene cluster (94). Thus, it appears that there is some plasticity in the immune response to

viral infections depending on the genetics of the host, the cytokine environment, and the

type of viral pathogen.

IL-12 has also been significant in the pathology and possible treatment of HIV

infection in humans. During in vitro studies with peripheral blood mononuclear cells

(PBMC) and T cells from HIV-infected individuals, it was demonstrated that these cells

produce less IL-12 than non-infected controls (79). Although macrophages from these








individuals express low levels of IL-12, these cells do not respond to normal stimulation

(2, 79). IL-12 treatment in vitro has been shown to enhance NK cytotoxicity in HIV

infected cells, and is able to increase the cytotoxic activity of lymphocytes in non-

infected donors against HIV-infected target cells (2). IL-12 treatment has also improved

the ability for immune cells to recall antigens and to prevent T-cell-receptor-induced

apoptosis (79). The protective ability of IL-12 against the HIV virus may be to help

maintain the CD4+ T cell population from apoptotic destruction by shifting the T cells to

a Th-1 profile that is less permissive to HIV than the Th-2 cell type (2,79).

The Role of IL-12 in Allergy and Asthma

Asthmatic disease involves intermittent airway obstruction, bronchial smooth

muscle cell hyperreactivity to bronchoconstrictors, and chronic bronchial inflammation

(95). This persistent airway inflammation ultimately leads to remodeling of the airway

epithelial cells and the deposition of collagen by proliferating fibroblasts (95). The

primary lesion of asthma consists of the accumulation of CD4+ Th-2 cells and in some

cases eosinophils in the airway mucosa (95). The Th-2 cells direct the persistent

inflammation through the cytokines IL-4, IL-13, IL-5, and IL-9 (96). Although IL-4 is

the main cytokine responsible for Th-2 differentiation and the high IgE levels observed in

many asthmatics, IL-13 and IL-5 are involved in bronchoconstriction and eosinophilia

respectively (96). Chemokines such as RANTES (regulated upon activation, normal T-

cell expressed and secreted) eotaxin, and macrophage inflammatory protein 1 c (MIP-1 a)

also act on eosinophils and T cells to enhance their recruitment and activation (95-96).

These inflammatory mediators have become proposed sites for therapeutic modulation

and have been studied in both human and animal models of allergic disease.








IL-12 appears to have an immunomodulatory effect on the predominantly Th-2

driven pulmonary inflammation in rodent (rat and mouse) models of allergic airway

disease and human asthma. IL-12 KO mice sensitized and challenged with ovalbumin

(OVA) have pronounced eosinophilic airway inflammation with enhanced IL-4 and TNF-

a levels in the bronchoalveolar lavage fluid (4). Recombinant IL-12 given

intraperitoneally in a murine model of ovalbumin-induced, allergic, airway inflammation

suppresses antigen-induced airway eosinophilia, circulating IgE levels, and airway

hyperresponsiveness in a dose dependent manner (97). The administration ofrlL-12 was

timed during either allergic sensitization (early dosage) or the hypersensitivity of

inflammation in the lung (late dosage) (97-98). Early dosages or early and late dosages

combined were effective in C57BL/6 suppressing all signs of the asthma-like phenotype,

however the late doses alone were not as effective, especially in reducing IgE levels (97-

98). In addition, rlL-12 administration to rats by intraperitoneal dosing also inhibits

allergen-induced inflammation and the sensitization to allergens (99). Brusselle et al.

examined the mechanism of the inhibitory effects of EL-12 on airway inflammation using

IFN-y receptor deficient (IFN-yR-KO) mice (100). Recombinant IL-12 given by aerosol

to IFN-yR-KO and wild-type mice during sensitization inhibited airway eosinophilia and

specific IgE production in the wild-type mice and increased these parameters in IFN-yR-

KO mice possibly due to a more established Th-2 response (100). Similar to the previous

studies, rIL-12 given only during the hypersensitivity phase (late) inhibited the airway

eosinophilia, but not the circulating IgE in the wild-type mice (100). The inhibition of

eosinophil influx into the airways by IL-12 appears IFN-y dependent during initial

sensitization and IFN-y independent during the secondary allergic response. These









results suggest that endogenous and exogenous IL-12 play important roles in curtailing

the allergic response in the airways, and the timing of the expression is critical to

suppress the Th-2 response.

In humans, bronchial biopsies from asthma patients compared to normal controls

show decreases in the numbers of IL-12 producing cells, reduced airway IL-12 mRNA,

and a reduction in the ability of the PBMCs in vitro to produce IL-12 during stimulation

as compared to normal non-atopic controls (4). The expression of IL-12Rp2 on T cells of

asthmatics is also reduced, partly due to diminished production of IL-12 and enhanced

secretion of IL-4 by their PBMCs (101). Furthermore, there are intrinsic defects of the

CD4+ T cells, which reduce their ability to respond to IL-12 with IL-12R32 expression

(101). Problematically, systemic administration of rIL-12 to human asthmatics has

several toxic effects such as general malaise/flu-like symptoms and cardiac arrhythmias

(99). Mucosal administration by airway aerosolization of rlL-12 in mouse and non-

human primate models abrogates airway eosinophilia and airway hyperresponsiveness, as

well augmenting the expression of pulmonary IFN-y (4, 102). In addition, lung mucosal

IL-12 gene delivery via viral vectors prevents the development of allergic disease, airway

hyperresponsiveness, and suppressed established allergic responses (4,103). This form of

gene therapy also reversed the suppression of local antiviral cell-mediated immunity

resulting in rapid resolution of viral infection in previously susceptible mice (103). These

therapies may enable exogenous administration of rIL-12 to asthmatic individuals

without the side effects associated with systemic treatment.








Rodent Model for Virus-Induced Pulmonary Disease

Viral bronchiolitis during infancy (less than 1 year of age) in humans has been

associated with chronic airway dysfunction and may be a factor in the development of the

asthmatic phenotype (104-105). These pulmonary function abnormalities include

increased airway resistance and airway hyperresponsiveness to airway smooth muscle

agonists such as methacholine (104,106). Viral infections may be inducing persistent

structural abnormalities through direct inflammatory injury and repair mechanisms that

may lead to permanent structural abnormalities (107). Another factor in the development

of asthma is the association between elevated IgE levels and a predominating Th-2 type

cytokine response in some infants that develop asthma (108). Diminished IFN-y

production can be demonstrated in the cord blood mononuclear cells from infants with

increased risk of developing atopic diseases, such as asthma, and in the PBMCs of infants

that develop virus-associated airway function abnormalities (108). The asthmatic

phenotype then appears to be a combination of inheritable factors (cytokine dysregulation

and/or atopy) and environmental components (viral infections, allergens, and other lower

airway antigens).

Parainfluenza virus type 1 (Sendai) infection in weanling rats produces pulmonary

structural and functional abnormalities, such as bronchiolar hypoplasia and alveolar

dysplasia leading to increased airway resistance and hyperresponsiveness (109-110).

These abnormalities have been developed as an animal model of virus induced airway

disease with many features that resemble human asthma including; episodic, reversible

airway obstruction, airway hyperresponsiveness to methacholine, chronic airway wall

inflammation, and airway wall remodeling (111-113). Young rats infected with Sendai









virus develop severe bronchiolitis followed by pulmonary growth abnormalities,

including bronchiolar hypoplasia, alveolar dysplasia, and increases in bronchiolar airway

wall thickness (113). Sendai virus-induced increases in bronchiolar wall thickness are

due to increases in inflammatory cells (macrophages, mast cells, eosinophils and

lymphocytes), airway wall edema, fibroblast proliferation, and collagen and extracellular

matrix deposition (113). Previously, it has been determined that Brown Norway (BN)

rats are susceptible to virus-induced chronic inflammation and remodeling leading to

airway function abnormalities, whereas Fischer 344 (F344) rats are highly resistant to

these virus-induced effects (111).

The development of the Sendai virus-induced abnormalities may be related to the

initial immune response early after viral infection. The virus-susceptible BN rat strain

differs from the F344 in response to viral infection as it has a greater pulmonary

expression of the Th-2 cytokines IL-4 and IL-5, less IFN-y production, and fewer CD8+ T

lymphocytes at early time points after viral inoculation (112, 114-116). In addition, BN

rats have higher serum IgE levels with enhanced recruitment of airway mast cells,

eosinophils, and prolonged viral replication within the airways when compared to the

F344 rat strain (115-116). The immune effector cells from BN rats are also less

responsive to IL-12 stimulation. Splenocytes and NK cells from uninfected BN rats

secrete significantly less IFN-y upon stimulation with IL-12 or Sendai virus than

splenocytes and NK cells from uninfected F344 rats (117). In the viral repair process,

BN rats have increased and prolonged expression of the fibrosis-inducing cytokines TGF-

P31 and TNF-ax in the macrophages surrounding the airways at 10 and 14 days after viral

infection (113, 118). Modulation of the immune response by the exogenous








administration of recombinant IFN-y, given 4 days before and during the first week of

viral infection, prevents the development of persistent airway inflammation, fibrosis, and

the associated chronic airway dysfunction in the BN rats (119). Thus, differences in the

genetic immune response (i.e., the cytokine response) to parainfluenza virus are critical in

determining whether chronic airway dysfunction and asthma-like disease will develop.

The above findings indicate that the cytokine response is an important component

of the Sendai virus-induced pulmonary damage associated with chronic airway

dysfunction. These results combined with the properties of IL-12 outlined in the previous

sections, indicate that F344 rats may be more resistant to Sendai virus-induced

bronchiolar damage and fibrosis because they produce higher levels of IL-12 early in

response to viral infection. The studies outlined here are designed 1) to investigate the

pulmonary expression of IL-12 in the F344 and BN rat strains in response to viral

infection and 2) determine if systemic treatment with IL-12 could abrogate or lessen the

severity of Sendai virus-induced bronchiolar inflammation and fibrosis that is associated

with airway dysfunction in BN rats.













CHAPTER 2
RESEARCH PLAN AND PROTOCOL

Hypothesis and Specific Aims

The goal of this research was to determine the role ofinterleukin-12 (IL-12) in the

development of resistance to chronic airway disease induced by parainfluenza (Sendai)

virus during early life. The hypothesis to be tested was that F344 rats are more resistant

to virus-induced airway damage and fibrosis because they produce high levels of IL-12

early in response to virus that up-regulates Th-1 I cytokine responses, antiviral immunity,

and reduces airway fibrosis. There were four specific aims:

1) To compare the pulmonary IL-12 mRNA and protein responses of virus- resistant
F344 and virus-susceptible BN rats following Sendai virus infection.

2) To determine if F344 rats have greater numbers of pulmonary cells and differing
cell types that express IL-12 in response to Sendai virus infection than BN rats.

3) To determine if Sendai virus-induced airway damage in BN rats can be reduced by
IL-12 treatment early in the virus infection.

4) To compare the airway IL-12 p35 and p40 mRNA responses of virus-resistant F344
and virus-susceptible BN rats following Sendai virus infection (This specific aim is
contingent on the results from the second specific aim. If there is differential
expression of IL-12 in the dendritic cell or other cells types in the large airways
based on the results of the in situ hybridization and immunohistochemistry, then
this specific aim will be explored).

Background/Significance

Inbred rats differ in susceptibility to Sendai virus-induced chronic airway disease

that resembles human asthma. The BN rat strain is highly susceptible to this virus-

induced damage and produces a predominantly Th-2-type acute cytokine response to

virus with increased levels of IL-4 and IL-5 seven days after inoculation (115). In









contrast, the F344 rat strain is resistant and produces an earlier Th-l-type cytokine

response with high IFN-y mRNA levels three days after virus inoculation and higher IFN-

y protein levels in the bronchoalveolar lavage fluid seven days after inoculation (115).

BN rats have high serum IgE levels and persistent inflammation characterized by the

enhanced recruitment of mast cells and eosinophils in response to Sendai viral infection

(114,116,120). F344 rats have a higher CD8 T cell response to Sendai infection and

have viral titers comparable to non-infected controls seven days after virus inoculation

(115-116). BN rats continue to have persistent airway inflammation with increased

numbers of macrophages expressing the pro-fibrotic cytokines TNF-a and TGF-01 in

their airways at 10,14, and 30 days after inoculation (113,118). Additionally, BN rats

have virus-induced proliferation of fibroblasts in the bronchiolar walls chronically after

virus inoculation (118). These changes remodeling the airways are associated with virus-

induced increases in pulmonary resistance and hyperresponsiveness that persists for 28 to

65 days after inoculation.

IL-12 is a heterodimeric cytokine in the Th-1 cytokine group that is produced in

response to many intracellular infections including viral, protozoal, fungal, and bacterial

(1-3). This cytokine, produced mostly by antigen presenting cells dendriticc cells and

macrophages), promotes antiviral immune responses, in part by inducing IFN-y

production by immune effector cells (2). Differential L-12 expression has been shown

to be important in susceptibility to intracellular pathogens in several rodent models (79).

Administration of recombinant IL-12 confers resistance in rodent and primate infectious

and allergic disease models (79,97-103). F344 rats have higher virus-induced pulmonary

IFN-y expression and increased NK and T lymphocyte responsiveness to IL-12 or Sendai








virus (117). Therefore, expression of lL-12 may play a major role in controlling the

difference in susceptibility to virus-induced airway inflammation, fibrosis, and

hyperresponsiveness in these two rat strains.

Gaps in Knowledge to Be Addressed by This Research

1) Is early IL-12 gene expression increased in the virus-resistant F344 rats as
compared to virus-susceptible BN rats in this model?

2) Can exogenous administration of IL-12 reduce the effects of virus-induced damage
and fibrosis in the pulmonary tissues?

3) Which cell types are responsible for producing IL-12, post-viral infection, in the
lungs?

4) Is the dendritic cell IL-12 response greater in the F344 rats early after Sendai virus
inoculation?

Research and Design Methods

Overview of Experiments and Schedule

Experiment 1: Comparison of IL-12 response in virus-susceptible BN rats and virus-

resistant F344 rats. (Specific Aims 1, 2, and 4) (Year 1 and 3)

Experiment 2: Can Sendai virus-induced airway damage be reduced in virus-susceptible

BN rats by exogenous administration of IL-12 early in viral infection? (Specific Aim 3)

(Year 2)

Experiment 1: Pulmonary Expression of IL-12 in Sendai Virus-infected BN and
F344 Rats

Objectives

1) To determine if virus-resistant F344 rats have higher levels of IL-12 pulmonary
expression in response to Sendai viral inoculation as compared to virus-susceptible
BN rats.

2) To determine the location and amounts of p70 protein in the pulmonary tissues in
the two rat strains.

3) To determine the level of the IL-12 synergistic cytokine, IL-18, mRNA in the
pulmonary tissues of both rat strains.








4) To compare the accumulation of dendritic cells, a primary IL-12 producing cell,
into the airways of both rat strains.

Rationale

Levels of IL-12 have not been compared in this rat model consisting of BN and

F344 inbred rat strains. It has been determined that F344 rats have higher IFN-y

expression than BN rats in response to viral infection and that IL-12 is a potent inducer of

IFN-y in many intracellular infections (119, 1-4). It is of interest to determine if the

levels in pulmonary tissue post Sendai viral infection differ between the two strains.

Previous studies have determined the protein expression of the IL-12 p70 heterodimer

locally in areas of intracellular infection (2, 79). Given that F344 rats have a higher

expression of IFN-y post viral infection, the IL-12 signaling pathway may be an

important factor in the antiviral response. This study is designed to determine the levels

and location (i.e., cell types) that are expressing IL-12.

IL-18 can act synergistically with IL-12 to induce and/or augment the production of

IFN-y. It has been shown to act specifically at the IFN-y promoter to augment production

from Thi cells (57). The combination of IL-12 and IL-18 together has been shown to

induce extremely high amounts of IFN-y protein (643-fold increase) and IFN-y gene

expression in NK cells above resting or non-activated human NK cells (55). It may be

possible that there is not differential expression of IL-12 in this model, but that an

augmenting cytokine like IL-18 is expressed at greater levels in the virus-resistant F344

rats.

Dendritic cells have been shown to be one of the earliest cells responders in the rat

airways after Sendai virus infection (121). Sendai virus initially replicates in airway

epithelial cells; the earliest IL-12 responses should be from intramural and epithelial








dendritic cells (121). Measurement of total lung mRNA may mute any differences at the

airway level by averaging all of the lung tissues together. Focused examination of

airway-specific expression may be required to elucidate these potentially important

differences in IL-12 (122). Previously most of the studies examining regulation of EL-12

have focused on the p40 subunit and its regulation at the transcriptional level in IL-12

producing cells. Recent studies have shown that in CD8+ dendritic cells the production

of IL-12 p70 heterodimer requires the induction of the p35 subunit (13). Since IL-12 p40

homodimers can bind to the IL-12Rp2 subunit and antagonize IL-12 function, the

biological activity may be, at least in part, determined by the ratio of p70 to p40

homodimer. This indicates that p35 levels may be an important limiting factor in IL-12

production by dendritic cells (13). In this model it may important to examine the p40

subunit at the lung and airway level and the p35 subunit production to elucidate any

critical differences that may exist in IL-12 regulation between these two rat strains.

Design and Methods

Twenty-two day old weanling male BN and F344 rats were aerosol-inoculated with

Sendai virus or remained unexposed to Sendai virus (lung changes same as

chorioallantoic fluid exposed) in separate but equal housing, and studied at 0, 1, 2, 3, 5, 7,

10, and 14 days post inoculation.

Necropsy and tissue processing. The rats were anesthetized with sodium

pentobarbital (approximately 2001ag/g body weight) or urethane (1.5g/kg body weight)

and killed by exsanguination via cardiac puncture. Lung lavages for ELISA were

performed through intratracheal cannulation with phosphate buffered saline (PBS). For

RT-PCR and ELISA, the lungs and tracheas were removed, immediately frozen in liquid









nitrogen, and then stored at -80C until processed. Lungs and tracheas were removed and

fixed for 2 hours by tracheal perfusion with 4% paraformaldehyde-PBS (pH = 7.4) and

embedded in paraffin, for in situ hybridization. For immunohistochemistry lungs were

removed, inflated with O.C.T. embedding medium compound (Tissue-Tek, Torrance,

CA), sectioned, and placed into molds. These lungs were then frozen immediately in

liquid nitrogen and then stored at -80C until processing.

Table 2-1. Table of Experimental Design: Experiment 1
Assay Sample Purpose
Reverse-Transcriptase RNA from BN and F344 rat Detection of differences in p35
Polymerase Chain lungs and tracheas (frozen)/virus and p40 IL-12 mRNA levels post
Reaction (RT-PCR) exposed and non-infected viral inoculation
controls
Immunohistochemistry Frozen lung sections from BN Detection and location of cells
and F344 rats/virus exposed and expressing IL-12 p40 protein and
non-infected controls the OX-6 dendritic cell marker
In situ Hybridization Paraffin lung and trachea sections Location of the IL-12 p40
from BN and F344 rats/virus mRNA message within the
exposed and controls pulmonary tissues
Enzyme-Linked Lung homogenates and lung Determination of the level of IL-
Immunosorbent Assay lavage fluid from BN and F344 12 p70 and p40 in the total lung
(ELISA) rats/virus exposed and controls tissue and secreted into the
airways

Dilutional and real-time RT-PCR. Frozen lungs and tracheas were weighed and

total RNA was extracted by phenol/chloroform extraction or using the RNeasy midi kit

(Qiagen, Inc, Valencia, CA) (123,124). Preliminary pulmonary mRNA levels were

measured in a small number of rats using by 10-fold dilutional RT-PCR method with

RNA dilutions ranging from neat to 1:10,000 (125). The primers for IL-12 p40 and the

housekeeping gene product, hypoxanthine-guanine phosphoribosyltransferase (HPRT)

were designed from previously published sequences and all reactions were optimized for

temperature, Mg2 concentration, and primers (125,126) (Appendix A, Figure A- 1).









Reverse transcriptase reactions and real-time PCR for IL-12 p35, IL-12 p40, IL-18

mRNA, and the housekeeping gene rat glyceraldehyde-3-phosphate dehydrogenase

(GADPH) were performed on total RNA extracted from the lungs and tracheas of

individual rats. The primers for each of these cytokines were TaqMan pre-developed

assay reagents for gene expression quantification (Applied Biosystems, Foster City, CA).

Every time cDNA was synthesized, parallel TaqMan assays were run for GADPH and

the target cytokine in separate wells (124).

Immunohistochemistry. Antibodies against IL-12 p40 and OX-6 major

histocompatibility complex (MHC), dendritic cell marker were used to detect bronchiolar

cell cytokine expression or to identify the cell type, respectively. The labeled cells were

quantitated and the numbers of positive cells were compared between treatment groups.

In situ hybridization. A portion of the dilutional IL-12 p40 PCR product was

subcloned and used as a template for the production of RNA probes (125, Appendix C).

The digoxigenin- IL-12 p40 labeled antisense riboprobe was constructed and used to

qualitatively determine which cell types were expressing p40 mRNA in lung and trachea

sections. Control sections were incubated with a labeled sense riboprobe. Northern

analysis was done to verify the success of the IL-12 p40 probe binding to rat RNA

samples with high levels of IL-12 p40 mRNA expression (Appendix C).

Enzyme-linked immunosorbent assay. The IL-12 p 70 protein levels were

determined preliminarily in concentrated lung lavage fluid samples (Millipore

Ultrafree-4 centrifugal filter and tube Millipore Corporation, Bedford, MA) from

individual rats using an ELISA kit according to manufacturers instructions (murine IL-12

p70, Biosource International, Camarillo, CA) (Appendix A, Figure A-2). Due to the








ability of IL-12 p40 monomers and dimers to antagonize the effects of biologically active

IL-12 p70, the IL-12 p40 protein was measured in the lavage fluid using an ELISA kit

according to the manufacturers instructions (Quantikine M Murine Mouse IL-12 p40

ELISA, R&D Systems, Minneapolis, MN) (17,125) (Appendix A, Figure A-3). Based on

the low levels of IL-12 p70 and p40 detected in concentrated lung lavage fluid ELISA

results, the same analyses were done using the supernatants from whole lung

homogenates from control and virus-infected rats of both strains.

Data analysis. The final quantitation of cytokine mRNA levels detected by real-

time PCR was done using the comparative CT (cycle threshold) method and was reported

as relative transcription of the n-fold difference relative to a calibrator cDNA (LPS-

stimulated rat lung) (124). The density of protein labeling was assessed by counting and

identifying based on morphology the number of labeled cells per mm ofbronchiolar

basement membrane. Group means for all assays were compared by one-way analysis of

variance (ANOVA) using a computer-based statistical program (Sigma-Stat, Jandel Corp.

San Rafael, CA). Kruskal-Wallis analysis of variance was used on ranks if normality test

of group means failed. In addition, t-tests, Wilcoxin-Mann-Whitney test, and Student-

Newman-Keuls method of pair wise multiple comparison procedures were used

depending on numbers of groups being compared as well as the variance within each of

the individual groups (113).

Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats

Objectives

1) To determine if administration of exogenous IL-12 early in viral infection increases
resistance to virus-induced chronic airway disease.








2) To determine the mechanismss, if IL-12 administration does increase resistance,
by which IL-12 may be functioning to promote protection from virus-induced disease.

Rationale

The exogenous administration of recombinant IL-12 in numerous murine and

primate infectious disease models increases resistance to infection and/or to the

pathogen-associated, disease phenotype. Intraperitoneal (IP) administration ofrIL-12 has

been the method used in many of these studies, additionally local and mucosal rIL-12

treatment has also been effective at the sites ofpathogenesis (79,102-104). In most

disease models the administration of IL-12 directly, by the production of IFN-y, drives

the development of the Th-1 type immune response with simultaneous suppression of the

Th-2 immune response (2, 79).

IL-12 is elevated most often early in the disease process, particularly in viral

infections where peak IL-12 levels are noted 1-3 days after infection (2,79). The timing

of the cytokine treatment has been shown to be important in both the infectious models

and the allergic disease models (79, 97-98). If the Th-2 response or allergic sensitization

has already occurred, then IL-12 may actually serve to accentuate or reverse these

responses (79, 97-98). Once the T lymphocytes have lost the ability to respond to IL-12,

possibly due to the absence of the IL-12Rp2 subunit, altering the immune response

appears to be very difficult (29, 30).

In the murine model of Leishmaniasis IL-12 the administration of neutralizing

antibody converts normally Leishmania-resistant C57BL/6 and CH3 mice to

susceptibility, and rIL-12 treatment confers resistance to the Leishmania-resistant

BALB/c strain (79,127). The important role for IL-12 in resistant strains seems to be its

ability to function as a growth factor for Thi cells by the intensification of IFN-y








production and to suppress the production ofIL-4 to undetectable levels (79,127). IL-12

treatment within our model could act in a similar manner increasing IFN-y levels and

reducing IL-4 levels after Sendai virus infection.

Preliminary results have shown that IL-12 p40 mRNA levels are elevated in the

early days post viral infection. This is consistent with previous studies in this model, in

which IFN-y mRNA and protein levels also peak at early time points (119). IFN-y has

been shown to be a potent mediator of the Th-1 response and treatment of BN rats before

and early after Sendai infection prevents post viral chronic bronchiolitis (119). This

experiment is designed to determine if early exogenous administrations of IL-12 to BN

rats will up regulate Th-1 type and reduce chronic airway fibrosis and remodeling.

Design and Methods

Twenty-two day old weanling male BN were aerosol-inoculated with Sendai virus

or remained unexposed to Sendai virus (lung changes same as chorioallantoic fluid

exposed) in separate but equal housing, and studied at 0, 3, 7, 10, and 14 days post

inoculation (DPI). L-12 treatment groups were injected IP with 3p.g of mouse

recombinant IL-12 or a comparable volume of saline at the time of virus inoculation (0

DPI) or two days after inoculation (2 DPI) (Biosource International, Camarillo, CA) (See

Figure 2-1). If rats were in the 10 or 14 days post inoculation, they were injected IP at

200 mg/g of rat with 5-bromo-2-deoxyuridine (BrdU)(Sigmna, St. Louis, MO) twelve

hours prior to necropsy.









Table 2-2. Table of Experimental Design: Experiment 2
Assay Sample Purpose
Reverse-Transcriptase RNA from BN rat lungs Detection of differences in IFN-
Polymerase Chain (frozen)/virus-exposed and non- y, IL-18, and IL-4 mRNA levels
Reaction (RT-PCR) infected controls with and after IL-12 treatment
without rIL-12 treatment
Immnunohistochemistry Paraffin lung sections from BN Detection and evaluation of
rats /virus exposed and non- fibroblast proliferation in the
infected controls with and bronchiolar walls
without IL-12 treatment
Differential Cell Counts Lung lavage fluid from BN rats Determination of total numbers
/virus-exposed and non-infected of cells and types in BAL
controls with and without rIL-12
treatment
Histology and Paraffin lung sections from BN Evaluate the extent of
Bronchiole Wall /virus exposed and non-infected inflammation and fibrosis in the
Morphometry controls with and without IL-12 bronchioles
treatment
Enzyme-Linked Lung homogenates and lung Determination of the level of IL-
Immunosorbent Assay lavage fluid from BN rats/virus 12 p70 and p40 in the total lung
(ELISA) exposed and controls with and tissue and secreted into the
without rIL-12 treatment airways
Viral Plaque Assays Lung BN rats (frozen)/ virus Determination of the levels of
exposed with and without rIL-12 virus remaining in the treatment
treatment groups at various time points


IL-12 or Saline Treatment of
BN Rats (3gg IP)
0 or2
Days After Virus inoculation
or Non-infected Controls
Injected
oon Same Day
BrdU Histology to i
Identify I
Fibrotic Cells in
the Airways '
10 and 14 Days DPI


Airway Morphometry |
to Compare the
thickness of Airways Histology to
10 and 14 Days DPI Characterize Airway


RT-PCR to Measure
IFN-y mRNA
0, 3, 7 Days DPI


Figure 2-1. Experimental design diagram for experiment 2









Necropsy and tissue processing. The rats were anesthetized with sodium

pentobarbital (approximately 200gg/g body weight) or urethane (1.5g/kg body weight)

and killed by exsanguination via cardiac puncture. Lung lavages for ELISA and cells

counts (differential and total) were performed through intratracheal cannulation with

phosphate buffered saline (PBS). For RT-PCR and ELISA, the right lungs were

removed, immediately frozen in liquid nitrogen, and then stored at -80C until processed.

The left lungs were tied off, removed and fixed for 2 hours by tracheal perfusion with 4%

paraformaldehyde-PBS (pH = 7.4) and embedded in paraffin for immunohistochemistry

and histology.

Real-time and competitive RT-PCR. Reverse transcriptase reactions and real-

time PCR for IL- 18 mRNA and GADPH were performed on total RNA extracted from

the lungs of individual rats. The primers these cytokines were TaqMan pre-developed

assay reagents for gene expression quantification (Applied Biosystems, Foster City, CA).

The primers and TaqMan probe for BN rat IL-4 were designed using the Primer Express

software (Applied Biosystems, Foster City, CA). The sense and antisense primers were

made in the mRNA sequence to ensure discrimination between cDNA and genomic

DNA. Dilutional curves were evaluated to assure that the efficiency of the IL-4 primers

compare to the GADPH primers. Every time cDNA was synthesized, parallel TaqMan

assays were run for GADPH and the target cytokine in separate wells (124).

IFN-y was detected in lung tissue by a competitive RT-PCR method (126). Primers

for IFN-y and HPRT were constructed from published sequences and each assay was

optimized for temperature, Mg concentration, and primers (126). For each cDNA

sample reactions comparisons were made to 0, 0.5, 5, and 50 femptograms (fg) of a








competitive fragment (126). PCR products were stained with ethidium bromide and

separated electrophoretically on 1.5-% agarose gels.

BrdU immunohistochemistry. Transverse sections were taken from the fixed left

lungs of rats treated with BrdU: at the hilus, between the hilus and the cranial lobe

margin, and at the same distance from the hilus to the caudal lobe margin.

Immunohistochemical staining using the antibody to BrdU was preformed and the

numbers of BrdU-labeled fibroblasts per mm ofbronchiolar basement membrane were

counted.

Histology and bronchiolar wall morphometry. Serial hematoxylin and eosin

(H&E) stained, paraffin sections were scored for airway wall inflammation and fibrosis.

Bronchioles were scored as positive for inflammation if the wall had five or more

inflammatory cell types eosinophilss, lymphocytes, or macrophages). Bronchioles were

scored as positive for fibrosis/remodeling if the walls were thickened with increased

fibroblasts and deposition of collagen. Collagen was identified using Masson's

Trichrome and Manuel's Reticulin stains. Additionally, the area of the bronchiolar wall

from the bronchiolar epithelial basement membrane to basement membrane of the

surrounding alveolar walls was measured. Bronchiolar wall area was divided by the

perimeter ofbronchiolar basement membrane to calculate the thickness of the wall

(square micrometers of bronchiolar wall per micrometer of bronchiolar basement

membrane) (113).

Enzyme-linked immunosorbent assay. The IFN-y protein levels were determined

in concentrated lung lavage fluid (Millipore Ultrafree-4 centrifugal filter and tube

Millipore Corporation, Bedford, MA) samples from individual rats using an ELISA kit








according to manufacturers instructions (rat IFN-y, Biosource International, Camarillo,

CA). Preliminary analysis using concentrated lung lavage fluid samples detected an

increase in levels of IFN-y after viral infected, but no significant differences in the IL-12

treatment groups (Appendix A) (Figure A-4). Based on the minimal effects detected in

concentrated lung lavage fluid, the same analyses were done using the supernatants from

whole lung homogenates from individual animals in the same treatment groups.

Virology. Viral titers, to determine viral clearance, were measured in homogenates

of frozen lung, using a standard plaque assay, and expressed as plague forming units/g

lung tissue (128).

Data analysis. The final quantitation ofcytokine mRNA levels detected by real-

time PCR was done using the comparative CT (cycle threshold) method and was reported

as relative transcription of the n-fold difference relative to a calibrator cDNA (LPS-

stimulated rat lung) (124). The competitive RT-PCR data are reported as non-normalized

mRNA abundance in competitive fragment units (119). Fibroblast proliferation was

assessed by counting the number ofBrdU-labeled fibroblasts (identified due to spindled

shape) per mm ofbronchiolar basement membrane. Group means for all assays were

compared by one-way analysis of variance (ANOVA) using a computer-based statistical

program (Sigma-Stat, Jandel Corp. San Rafael, CA). Kruskal-Wallis analysis of variance

was used on ranks if normality test of group means failed. In addition, several pair wise

multiple comparison procedures were used depending on numbers of groups being

compared as well as the variance within each of the individual groups (113).













CHAPTER 3
INCREASED EXPRESSION OF PULMONARY INTERLEUKIN-12 (IL-12) IN
SENDAI VIRUS-RESISTANT F344 RATS

Summary

Brown Norway (BN) and Fischer (F344) rats differ in their susceptibility in early

life to Sendai virus-induced persistent airway inflammation, chronic airway remodeling,

and airway hyperresponsiveness. These characteristics, as well as other phenotypic

characteristics serve as an experimental model of virus-induced asthma. Virus-

susceptible BN rats mount a predominantly Th-2 cytokine response (IL-4-dominated) to

Sendai virus, whereas virus-resistant F344 rats respond to infection with a Th-1 cytokine

pattern (IFN-y-dominated). F344 rats are more efficient in clearing the virus and in

resisting the induction of chronic airway lesions. We hypothesized that an earlier and

more robust IL-12 response was responsible for the differing IFN-y expression and viral

resistance of F344 rats. IL-12 mRNA and protein expression were evaluated by real-time

PCR, ELISA, and quantitative immunohistochemistry for IL-12 positive dendritic cells in

the lungs and tracheas of BN and F344 rats. F344 non-infected control rats had higher

pulmonary EL-12 p40 mRNA levels than the non-infected control BN rats. Virus-induced

increases in EL-12 p40 mRNA were detected as early as 2 days after inoculation, while

virus-induced increases in IL-12 p40 mRNA were not detected in BN rats until 3 days

after inoculation. F344 rats had higher concentrations of IL-12 total in the lung than BN

rats at 2 days after inoculation. Virus-induced increases in bronchiolar OX-6 positive and

IL-12 p40 dendritic cells were observed as early as 2 days following inoculation in F344








rats. No dendritic cell response was detected in BN rats. These results indicate that

resistance to the sequelae of Sendai virus infection by F344 rats is associated with their

earlier and higher production of IL-12.

Introduction

Viral bronchiolitis in infant children can be associated with chronic bronchiolar

dysfunction and is implicated as an important risk factor for the development of asthma

and other airway abnormalities (129,130). A rat model of virus-induced bronchiolar

damage has been developed in weanling and neonatal rats that has characteristics similar

to human asthma. These characteristics include episodic and reversible bronchiolar

obstruction, bronchiolar hyperresponsiveness to methacholine, chronic bronchiolar

inflammation lymphocytess, macrophages, eosinophils, and mast cells) and bronchiolar

wall remodeling (111-113). This model can be used to examine the possible direct roles

that viral infections have in pulmonary function abnormalities and what genetic factors

may be contributing to the susceptibility to viral sequelae during early life (119).

Interleukin-12 (IL-12) has been associated with resistance to intracellular infections

(2). IL-12 is produced by antigen presenting cells, particularly dendritic cells, during

infection and induces IFN-y production by natural killer (NK) and cytotoxic T cells

(CD8) (2,57). Through this direct up-regulation of IFN-y, IL-12 drives the development

of T helper- 1 (Th-1) type immune response with simultaneous suppression of the T

helper-2 (Th-2) immune response without the requirement for IFN-y (1-3). IL-12 directly

increases the cytotoxic killing capacities of NK and cytotoxic T cells (2). The IL-12 p35

and p40 subunits covalently link to form the biologically active p70 heterodimer (2).

Many cells constitutively express the p35 chain, however the p40 gene is expressed only








in IL-12 producing cells (10). The expression of both chains is induced upon

intracellular infection and the subsequent CD40 ligand binding of antigen presenting cells

(14,47).

Differential expression of IL-12 has been shown to be important in intracellular

infections in several rodent models. DBA/2 mice are genetically resistant to C. immitis

and produce five times more IL-12p40 mRNA in their lungs as compared to C. immitis-

susceptible C57BL/6 mice (79,88). Neutralization of IL-12 in the DBA/2 strain leads to

severe disease. Administration of rIL-12 to another fungus-susceptible mouse strain

(BALB/c) decreases susceptibility to clinical disease (79). Similarly, C3H mice that are

resistant to the protozoan T. cruzi have twice the IL-12 total protein in serum and splenic

tissue as compared to susceptible BALB/c mice. This differential IL-12 response has

been associated with increased resistance and an increased IFN-,y protein response (87).

Additionally, resistant C57BL/6 mice have 52% more IL-12 total protein in splenic

dendritic cells early in murine listeriosis than susceptible BALB/c mice (78). Finally,

C57BL/6 mice have a four-fold greater increase in pulmonary IL-12 expression in

response to respiratory syncytial virus and mild disease as compared to DBA/2 and

BALB/c mice which have lower IL-12 levels and develop increased bronchiolar

hyperactivity and mucus production leading to a more severe disease process (91).

In viral infections such as mouse choriomeningitis (MCMV), corneal herpes

simplex virus and murine influenza, EL-12 is critical in the early activation and

maintenance of the Th-1 immune response (2,90,93). In many of these responses, IL-12

acts through up-regulation of IFN-y, which has been shown to control viral infections and

tissue alterations associated with exaggerated repair mechanisms, such as those








associated with bleomycin and immune complex-induced lung injury (90,131-132).

Recently it has been determined that the p40 chain of IL-12 may also play a role in

mouse Sendai viral infection systemically as a macrophage chemoattractant, as well as at

a local level where it is produced in by bronchiolar epithelial cells (17). IL-12 also has

been shown to inhibit bronchiolar hyperresponsiveness and bronchiolar eosinophil

recruitment in several rodent and primate models of allergic sensitization (79,97-98).

As previously noted, it has been determined that Brown Norway (BN) rats are

susceptible to Sendai virus-induced inflammation and remodeling, whereas Fischer 344

(F344) are highly resistant to the viral effects (111). The development of the Sendai

virus-induced abnormalities may be related to the early immune response after viral

infection. BN rats have greater expression of interleukin-4 (IL-4) and decreased levels of

IFN-y production, high mast cell and eosinophil response, fewer CD8+ T lymphocytes,

and prolonged levels of viral replication in comparison to F344 rats (119). BN rats also

have increased mRNA expression of profibrogenic cytokines TGF-p1 and TNF-ct at 10

and 14 days after Sendai virus infection (113,118). Treatment of Sendai virus-inoculated

BN rats with IFN-y results in a reduction of virus-induced bronchiolar inflammation,

bronchiolar fibrosis, and ultimately results in less severe pulmonary dysfunction (119).

Given the critical role that IL-12 plays in inducing IFN-y in the regulation of the Th-1

immune response and its differential expression in several models of intracellular

infection, we hypothesized that F344 rats are more resistant to Parainfluenza type-1

(Sendai) virus-induced bronchiolar damage and fibrosis because they produce higher

levels of IL-12 early in the response to viral infection. The objectives of this study were








to determine the quantity of pulmonary IL-12 mRNA and protein expression and to

determine the structural and cellular location of this expression in the airways of both rat

strains after Sendai virus infection.

Materials and Methods

Animals


Weanling (22 days old), male, pathogen-free BN/RijHsd (24 rats) and F344/NHsd

(24 rats) rats were purchased from Harlan Sprague Dawley, Inc. Madison, WI and

Indianapolis, IN respectively. The control and virally infected animals were housed

separately in adjacent, identical Micro-Isolator VCL-HDTM individually HEPA

filtered/ventilated cages (#10419ZTGA ZytemTM plastic Micro-IsolatorTM system, Lab

Products, Inc. Seaford, DE). The University of Florida Animal Care and Use Committee

approved all procedures.

Viral Procedures and Sample Collection

The rats were inoculated with aerosolized parainfluenza (Sendai) virus type 1 strain

P3193 (117). The numbers of rats used for each experimental technique are indicated in

the figures located within the results section. Briefly, rats in the virus-inoculation group

were exposed to an aerosol of virus at a concentration of 1-3 plaque-forming units (PFU)

per ml of gas via a Glas-Col Aerosol Exposure Apparatus for 15 minutes (Tri-R) (Glas-

Col, Terre Haute, IN). At 0, 1, 2, 3, 5, 7, 10, and 14 days after inoculation, rats from each

group were immobilized via deep anesthetization with sodium pentobarbital

(approximately 200,g/g body weight) or urethane (1.5g/kg body weight) and killed by

exsanguination via cardiac puncture. Lung lavages were performed through intratracheal

cannulation with phosphate buffered saline (PBS). The lungs taken for homogenization








and RT-PCR were frozen in liquid nitrogen and stored at -80C. The lungs and tracheas

used for in situ hybridization were tied off, perfused with 4% paraformaldehyde PBS

(pH 7.4) (30 cm H20 pressure for 2 hours) and embedded in paraffin. Finally, the lungs

used for immunohistochemistry tied off, inflated with O.C.T. embedding medium

compound (Tissue-Tek, Torrance, CA), sectioned, and placed into molds. These

tissues were stored at -80C until processing.

Cytokine mRNA


Frozen lungs were weighed and RNA was extracted by a phenol/chloroform

method or by using the RNeasy midi kit (Qiagen Inc, Valencia, Ca) (121,122). The

RNA samples were pre-treated with DNase I using the Deoxyribonuclease I,

amplification grade kit (Invitrogen, Carlsbad, CA) to remove genomic DNA. The

Reverse Transcriptase (RT) reactions were preformed using the AdvantageTM RT-for-

PCR Kit (Clontech Laboratories, Inc, Palo Alto, CA). Polymerase chain reaction (PCR)

primers and probes for rat IL-12 p40, rat IL-12 p35, rat interleukin (IL-18), and for the

housekeeping gene rat glyceraldehyde-3-phosphate dehydrogenase (GADPH) were

TaqMan pre-developed assay reagents for gene expression quantification (Applied

Biosystems, Foster City, CA). Every time cDNA was synthesized, parallel TaqMan

assays were run for GADPH and the target cytokine in separate wells. The PCR

reactions contained 900 nM of each primer, 250 nM of the TaqMan probe, PCR

Mastermix (TaqMan Universal PCR Mastermix, Applied Biosystems) containing 10 mM

Tris-HCl, 50 mM KC1, 5 mM MgCl2, 2.5 mM deoxynucleotide triphosphates, 0.625 U

AmpliTaq Gold DNA polymerase per reaction, 0.25 U AmpErase UNG per reaction, and

2 j.1 of the cDNA sample in a final volume of 25 pl. The samples were amplified in an








automated fluorometer (ABI Prism 7700 Sequence Detection System, Applied

Biosystems). Amplification conditions were 2 min at 50C, 10 min at 95C, 40 cycles of

15 s at 95C and 60 s at 60C. Final quantitation was done using the comparative CT

(cycle threshold) method and was reported as relative transcription of the n-fold

difference relative to a calibrator cDNA (LPS-stimulated rat lung) (Table 3-1) (124).

Table 3-1 Com parative CT Method of cDNA Relative Quantitation A
Average CT Average ACT AACT 2"T
GADPH CT____
Target GADPH CT Target CT- ACT ACT Calibrator Relative
cytokine CT average from 3 GADPH CT difference to
average from consecutive wells Calibrator = LPS the calibrator
3 consecutive (Same sample as Stimulated rat lung
wells target cytokine) cDNA

Protein Analysis

Enzyme-linked immunosorbent assay (ELISA)

Lungs were harvested as previously described and homogenized in cold PBS with a

protease inhibitor (Protease Inhibitor Cocktail Tablets, Mini Complete, Boehinger

Mannheim, Germany) at a ratio of 0.1g of tissue/ml (133). The homogenates were

centrifuged at 4C at 2000 rpm for 10 minutes and the supematants were frozen at -80C

until use. The total IL-12 (p70 heterodimer protein, p40 monomers, and p40 dimers) in

the lung lavage and homogenate was determined using an ELISA kit (murine IL-12,

Biosource International, Camarillo, CA) according to the manufacturer's instructions.

Due to the ability of IL-12 p40 monomers and dimers to antagonize the effects of

biologically active IL-12 p70 and the potential roles of IL-12 p40 observed in mouse

Sendai viral infections, the IL-12 p40 protein was measured in the lung homogenates

(17,127). The level of IL-12 p40 protein was determined using a mouse ELISA kit

(Quantikine M Murine Mouse EL-12 p40 ELISA, R&D Systems, Minneapolis, MN).








These results were compared to the IL-12 total protein levels to determine the

contribution of the protein forms of IL-12.

Immunohistochemistry

Frozen lung sections from BN and F344 rats were dried at room temperature and

then fixed in cold acetone for 10 minutes at 4C. After washing in 1 X PBS, the

endogenous peroxidases were blocked using 1% hydrogen peroxide rinse for 10 minutes.

The slides were then blocked with either normal goat serum (IL-12 p40 assay) (Santa

Cruz Biotechnology ABC Staining Systems, Santa Cruz, CA) or normal mouse serum

(OX-6, detection of dendritic cell assay) (Santa Cruz Biotechnology ABC Staining

Systems, Santa Cruz, CA) for 1 hour in a humidified slide chamber at room temperature.

In the IL-12 p40 assay, the sections were incubated with either polyclonal, goat, anti-

mouse IL-12 p40 (indicator of IL-12 p70 cellular production) (Santa Cruz Biotechnology,

Santa Cruz, CA) at 3.5 [tg/pl or goat immunoglobulin G (IgG) (Sigma, St. Louis, MO) at

0.25 jig/tl in a humidified slide chamber, overnight at 4C. In the OX-6 (major

histocompatibility complex determinant on B lymphocytes, dendritic cells, some

macrophages, and certain epithelial cells) assay, the sections were incubated under the

same conditions with either monoclonal, mouse anti 1-A (OX-6) (Serotec, Raleigh, NC)

at 0.02 pig/itl or mouse IgG (Sigma, St. Louis, MO) at 0.2 pg/gl. In both assays antibody

binding was detected using an avidin-biotin, chromogen diaminobenzidine system

according to the manufacturer's instructions (Santa Cruz Biotechnology ABC Staining

Systems, Santa Cruz, CA). The density of cells expressing the IL-12 p40 protein was

determined by counting and classifying the number of labeled cells per millimeter (mm)

of bronchiolar basement membrane in an average of 10.3 mmn/section. Round cells with








central round/oval nuclei and abundant cytoplasm were classified as macrophages, and

cells with round/ asymmetrically placed nuclei with foamy, dendritic cytoplasmic

extensions were classified as dendritic cells. The density of OX-6 positive cells was

determined similarly with an average of 12.3 mm/section being counted.

Data Analysis

For all experiments, group means were compared by one-way analysis of variance

(ANOVA) using a computer-based statistical program (Sigmrna-Stat, Jandel Corp. San

Rafael, CA). Kruskal-Wallis analysis of variance was used on ranks if the normality test

or equal variance tests of group means failed. Multiple comparison procedures were used

to isolate the group or groups that differ from others. The Student-Newman-Kuel's test

was used if the sample sizes were equal; otherwise Dunn's test was used to compare

groups of unequal sample size.

Results

Virus-Resistant F344 Rats Have Increased Expression of Pulmonary IL-12 mRNA

Relative amounts of IL-12 p40 mRNA were significantly higher in the lung tissue

of the F344 rats compared to the BN rats beginning at the non-infected control level

(p<0.01) (n = 4) (Figure 3-1). The expression of IL-12 p40 mRNA in the F344 rats

remained significantly higher at 1,2, and 3 days after viral inoculation with the greatest

difference being 3.4-fold at 2 days after virus inoculation (p<0.02) (Figure 3-1).









25

MeantSEM
n=4
20
0
15 M BN
F344

oV
>10-

5-

0-'


0 1 2 3 5 7 10 14
Days After Inoculation

Figure 3-1. Real-time PCR analysis of IL-12 p 40 mRNA in whole lung samples of BN
and F344 rats after Sendai virus infection. (s = significant difference between strains,
p<0.04) (V = significant virus-induced difference compared to strain control, p<0.01)

There was a virus-induced increase of IL-12 p40 mRNA in both strains of rats,

however it reached statistical significance earlier in the F344 strain (2 days after

inoculation, p<0.01). Relative amounts, of the more constitutively expressed IL-12 p35

mRNA, were also significantly increased in the lungs of F344 rats at control, 2 and 3

days after inoculation (p<0.03) (n = 2) (Figure 3-2). However, there was not a virus-

induced increase in either strain above the non-infected controls. The expression of IL-

12 p35 mRNA significantly decreased as analyzed by real-time PCR (Figure 3-2).

The same trends were detectable in mRNA IL-12 p40 expression at the airway

level. IL-12 p40 mRNA expression was increased 3.7-fold in the F344 tracheas at 2 days

after inoculation as compared to the BN tracheas, and a virus-induced increase in IL-12

p40 mRNA expression was observed only in the F344 strain (p<0.03) (n = 4-7) (Figure

3-3). There were no IL-12 p35 mRNA expression differences detected at the








airway/tracheal level between rat strains or induced by virus (p>0.05) (n = 4-7) (Figure 3-

4).


0 1 2 3 5 7 10 14


Days After Inoculation


Figure 3-2. Real-time PCR analysis of L-12 p35 mRNA in whole lung samples of BN
and F344 rats after Sendai virus infection. (s = significant difference between strains,
p<0.03) (v = significant virus-induced difference compared to strain control, p<0.04)


0 1 2 3 5 7
Days After Inoculation
Figure 3-3. Real-time PCR analysis of IL-12 p40 mRNA in trachea samples of BN and
F344 rats after Sendai virus infection. (s = significant difference between strains,
p<0.03) (v = significant virus-induced difference compared to strain control, p<0.03)









0.12
) BN Mean SEM
U 010- F344 n=4-7
I p 10
) 0.08

0.06
0

0.04
0.02



0 1 2 3 5 7

Days After Inoculation

Figure 3-4. Real-time PCR analysis of IL-12 p35 mRNA in trachea samples of BN and
F344 rats after Sendai virus infection.

F344 Rats Have Increased Pulmonary IL-12 Protein After Sendai Virus Infection

As measured by ELISA, the F344 rats had 86 pg/ml 12.72 of pulmonary IL-12

total protein as compared to 54.8 pg/ml 5.82 in the BN strain (p<0.05) (n = 6-7) (Figure

3-5). The levels of IL-12 total protein increase with viral infection over the non-infected

controls in the F344 rats at all time points measured and in the BN rats on days 1, 3, and

5 after Sendai inoculation (Figure 3-5) (p<0.05).

In both BN and F344 rats the concentration of pulmonary IL-12 p40 protein

monomerss and homodimers) was approximately half that of the total IL-12 (Figure 3-6,

y-axis). Strain differences in pulmonary IL-12 p40 protein were only detected in the non-

infected controls (p<0.05) (n = 6-7) (Figure 3-6). Concentrations of IL-12 p40 protein

increased only in the BN strain with viral infection starting at 2 days after viral

inoculation but never reached statistical significance (p>0.05) (Figure 3-6).










140
BN n=6-7
E 120 M F344 MeanSEM
L100 S
V
80



S1 2 3 5



Days After Inoculation

Figure 3-5. ELISA analysis of IL-12 total protein in F344 and BN rats strains after
Sendai virus inoculation. (s = significant difference between strains, p<0.05) (V =
significant virus-induced difference compared to strain control, p<0.05)
0
.40

'~20

0
0 1 2 3 5 7
Days After Inoculation

Figure 3-5. ELISA analysis of IL-12 total protein in F344 and BN rats strains after
Sendai virus inoculation. (s = significant difference between strains, p<0.05) (V
significant virus-induced difference compared to strain control, p<0.05)


'25

20
0.
O 15

CM 10

! 5

0


0 1 2 3 5 7
Days After Inoculation


Figure 3-6. ELISA analysis ofIL-12 p40 protein in F344 and BN rats strains after Sendai
virus inoculation. (s = significant difference between strains, p<0.05)

F344 Rats Have Increased Numbers of IL-12 p40 Expressing Cells in the
Bronchioles After Sendal Virus Inoculation

Density of OX-6 positive dendritic cells was significantly increased in the

bronchioles of F344 rats at 2 and 3 days after virus inoculation as compared to BN rats

(p<0.03) (n = 4) (Figures 3-7 and 3-8). Dendritic cell numbers in the F344 rats increased









87.5% above the non-infected controls and almost 2-fold above the BN rats at 2 days

after inoculation. There were no significant virus-induced increases in the numbers of

bronchiolar dendritic cells measured in the BN rats at any of the time points after

inoculation.


S


Th


Figure 3-7. OX-6 immunohistochemistry in the bronchiole of a F344 rat three days after
inoculation (54X Magnification). There are several OX-6 positive dendritic cells,
indicated by dark brown staining, located within the bronchiole wall.


7
6
E

.il4
o 3

C

0


S 2 3
Days After Inoculation


Figure 3-8. Density of OX-6 positive dendritic cells in the bronchioles ofF344 and BN
rats. (a = significant difference between strains, p<0.03) (V = significant virus-induced
difference compared to strain control, p<0.01)









IL-12 p40 protein was expressed in dendritic cells and macrophages in the

bronchioles of both strains. The average number of IL-12 p40 positive cells/mm of

bronchiole wall was significantly higher in the F344 rat strain at 2 and 3 days after

inoculation as compared to the BN strain (p<0.03) (n = 4) (Figures 3-9 Figure 3-11).

E 3.0
-BN Mean SEM
2.5 F344 n

V
V
1.2


e0'1.0 a

0.5

0.0
0 2 3
Days After Inoculation

Figure 3-9. Density of IL-12 p40 positive dendritic cells (s = significant difference
between strains, p<0.03) (v = significant virus-induced difference compared to strain
control, p<0.01)


OP
-eA


V




two days after Sendai inoculation (08X Magnification). Several inflammatory cells are
ii














indicated by dark brown staining inflammatory cells (macrophages and dendritic cells)
within the airway wall.










4
: BN Mean SEM
E -F344 n-4
o V
81 3
U
=m S






0
0 S;S


0 2 3
Days After Inoculation
Figure 3.11. Density of IL-12 p40 positive macrophages in the bronchioles of BN and
F344 rats. (s = significant difference between strains, p<0.03) (v = significant virus-
induced difference compared to strain control, p<0.01)

F344 and BN Rats Do Not Have Detectable Differences in the Expression of IL-18
mRNA

Due to the co-stimulatory role of IL-18 with IL-12 in response to intracellular

infections, we examined the IL-18 mRNA expression in the lung tissues of both rat

strains (1). There were no differences detected by real-time PCR in the pulmonary IL-18

mRNA expression between BN and F344 rats at the time points measured (p>0.05) (n =

4-5) (Figure 3-12). Additionally, Sendai virus-induced increases in IL-18 were not

detected in either strain (Figure 3-12).









10
MeanSEM
M BN n=4-5
8 F344 P>0.05
0
C,
0


) 4


S2


0 1 2 3 7 10 14
Days After Inoculation

Figure 3-12. Real-time PCR analysis of IL-18 mRNA expression in lung samples of BN
and F344 rats after Sendai virus infection.

Discussion

The purpose of this study was to determine whether there is differential expression

of IL-12 between rat strains that are susceptible and resistant to the pulmonary sequelae

of Sendai virus infection. The critical role of cytokines expressed acutely following virus

infection in this model and the importance of IL-12 in the response to intracellular

infections made this an important component of this disease model to investigate. The

results indicate that there are significant differences in total pulmonary IL-12 p40 and

p35 mRNA and IL-12 total protein levels expressed between virus-resistant F344 and

virus-susceptible BN rats following Sendai virus infection. There are also significant

differences in the numbers of cells expressing IL-12 p40 protein in the bronchioles

between these two strains.

This data indicates that total IL-12 protein expression increases earlier than the

expression of IL-12 p40 mRNA. This discrepancy may be due to several factors in the








measurement of protein and mRNA. Although both were measured in lung homogenates,

the blood and serum proteins are more likely to have remained viable after processing for

the ELISA and could have contributed to the concentrations of total IL-12 protein.

Additionally, RT-PCR, using conventional primers, is unable to detect differences in the

isoforms of IL-12 p35 mRNA (Chapter 1). There are differences in the isoforms of IL-12

p35 mRNA expressed depending on whether the production is constitutive or pathogen-

induced (14). Primers specific for the 5'untranslated region of the p35 gene can only

distinguish the untranslatable form. Additionally, there can be several translatable

isoforms with slight variations in this region. So, there may be more exaggerated

differences in IL-12 p35 expression (both virus-induced and between strains) than we are

able to detect with these primers. Therefore, it is difficult to make comparisons between

IL-12 mRNA expression and the IL-12 total protein levels.

Two pathways have been established for the production of IFN-y in viral infection.

Some infections, such as lymphocytic choriomeningitis virus (LCMV), have an IL-12

independent induction of IFN-y through IFNa/p pathway with very low, non-inducible,

levels of IL-12 expression (92). In addition, differential expression of the co stimulatory

cytokine IL-18 has been shown to be critical to the level of IFN-y induced in many

models ofintracellular infection (55-57, 79, 92). In the model examined here, both rat

strains have virus induced increases in the level of IL-12 p40 mRNA and IL-12 total

protein with no changes in IL-18 expression, suggesting that IFN-y levels are being up

regulated by IL-12-dependent mechanisms in Sendai viral infection.

We also wanted to determine if the differences expressed at the total lung level

were consistent with the inflammatory response at the airway level. The IL-12 p40








immunohistochemistry (an indicator of IL-12 p70 cellular protein expression) results do

indicate that bronchiolar macrophage and dendritic cell protein expression is higher at

early time points in the F344 rats following Sendai virus inoculation. Therefore, the

differential expression of IL-12 does appear to be evident throughout the lung, even at the

local airway level. Additionally, the levels of pulmonary IL-12 p40 protein (homodimers

and monomers) measured in whole lung homogenates indicate that the possible role of

over-produced IL-12 p40 protein as an IL-12RP32 antagonist is likely not a factor in this

model. However, other possible roles of IL-12 p40 protein, such as a chemoattractant for

macrophages, have not been elucidated by these methods.

P.G. Holt and others have established that a network of resident airway epithelial

dendritic cells exists in throughout the respiratory tract of rodents and humans, where

these cells process antigen and initiate the generation of protective local immune

responses (121,134-135). The most prominent populations are present in the conducting

airways, such as the trachea (600-800/mm2), and decrease further down the respiratory

tract (75/ mm2) (134). This population also appears to be dynamic with changes

observed at steady state and increases seen in the trachea after Moraxella catarrhalis

bacteria and Sendai virus exposures (121,134). These numbers are much higher than the

dendritic cell numbers that we were able to detect at 2 and 3 days after inoculation in the

bronchioles. However, bronchiolar airways were not singled-out in previous experiments

(more focus on the interalveolar septal junctions) and the peak time point for tracheal

dendritic cell numbers was not observed until 5 days after inoculation (134).

Additionally, the experiments examining the lower airways were performed hours (not

days) after the exposure to pathogen before the dendritic cells would be migrating from








the local site of inflammation to the regional lymph nodes (134). Similar to the results

seen in previous studies, our results show an increase in dendritic cell numbers after

Sendai infection in the F344 rat strain.

An increasing amount of evidence suggests that dendritic cell population changes

and cytokine expression may be a factor in the susceptibility to allergic respiratory

disease (135). Examination of dendritic cells in the lungs of several rat strains suggests

that the Th-2 polarity of the resting mucosal immune system may also be a property of

the resident dendritic cell population (121, 136). Initiation of the Th-1 immune response

in these cells requires appropriate costimulation (such as CD40 ligation) from the

microenvironment (136). Based on our results, a change in IL-12 expression is occurring

at the whole lung and airway levels. F344 rats have virus-induced increases in dendritic

cell numbers and increased expression of IL-12 p40 in their bronchioles by both

macrophages and dendritic cells as compared to BN rats early after Sendai infection. The

levels of IL-12 p40 mRNA are also increased in the tracheas of the F344 strain at 2 and 3

days after Sendai inoculation. However, in order to assess whether the IL-12 mRNA

expression is from cells (macrophages and dendritic cells) already present or from a

cellular influx further experimentation will need to be done to compare the magnitude of

the response in the airways of both strains. A component of the F344 rat strain's

resistance to Sendai virus -induced airway damage may be that their dendritic cells and

macrophages receive an earlier signal from the microenvironment to become Th-1 type

cells secreting more EL-12 as compared to the BN rat strain. Further examinations of the

airway dendritic cells, their interactions with the local airway mucosal immune system,

and the magnitude of this response are needed to confirm this possibility.








The differential expression of IL-12 appears to be a factor in the resistance to

Sendai virus-induced airway disease. This differential expression appears to occur early

after infection and may be the source of the establishment of a predominately Th- 1

cytokine response in the F344 rat strain. Similar to this rat model, paramyxoviral

(respiratory syncytial virus and parainfluenza virus) infections during infancy are

potential risk factors for the development of asthma in children (104-108). Additionally,

cytokine imbalance during early life has been considered to be an important risk factor in

the development of asthma and atopy (117). The differences in IL-12 expression by

these rat strains in response to Sendai virus infection may give insight into the

mechanisms of asthma development and potential asthma therapies in children.













CHAPTER 4
EXOGENOUS INTERLEUKIN-12 (IL-12) ADMINISTRATION REDUCES THE
SEVERITY OF SENDAI VIRUS-INDUCED CHRONIC AIRWAY FIBROSIS AND
REMODELING IN BN RATS

Summary

Sendai virus infection in virus-susceptible BN rats causes persistent bronchiolar

inflammation and fibrosis that is associated with increased airway resistance and airway

hyperresponsiveness. In contrast, F344 rats have earlier viral clearance, increased IL-12

pulmonary expression, and are resistant to post viral airway function abnormalities. This

study determined whether the exogenous administration of interleukin-12 (IL- 12) could

confer resistance to Sendai virus-induced airway disease in virus-susceptible BN rats.

BN rats were treated with 3 Rg of recombinant IL-12 (rIL-12) or an equivalent volume of

saline intraperitoneally (IP) at the time of virus inoculation (day 0) or two days after virus

inoculation (day 2). Proliferating fibroblasts were labeled with bromodeoxyuridine

(BrdU) and detected by immunohistochemical staining. In comparison to infected rats

given saline, infected rats treated with rIL-12 at day 0 had 22.1% lower levels of chronic

airway inflammation and 23.8% lower levels of airway fibrosis as detected by

histological criteria. Rats treated on day 0 with r IL-12 had a 42% and 62.5% decrease in

BrdU-labeled fibroblasts in their bronchioles at 10 and 14 days after inoculation

respectively as compared to saline-treated virus-inoculated controls (p<0.05). There was

a 4-fold increase in pulmonary IFN-y mRNA and a 77% increase in pulmonary IFN-y

protein detected in the lungs of day 0 treated rats when compared to the virus-inoculated,

saline-treated control rats (P<0.05). In contrast, day 2 rIL-12 treatment induced a 20%








increase in bronchiolar airway wall thickness and a 12.5% increase in BrdU-labeled

fibroblasts at 14 days after inoculation (p<0.05). Day 2 treatment resulted in increased

pulmonary IL-4 mRNA levels compared to saline-treated virus-inoculated controls

(p<0.05). In conclusion, early IL-12 treatment reduces Sendai virus-induced bronchiolar

inflammation and fibrosis in virus-susceptible BN rats. This effect may be mediated, in

part, by the induction of IFN-y.

Introduction

Parainfluenza type I (Sendai) virus infection in rats is an animal model of virus-

induced airway abnormalities with similar characteristics to childhood asthma, such as

increased airway resistance and hyperresponsiveness (109-112). Previous studies have

demonstrated that virus-resistant Fischer (F344) rats have increased expression of

pulmonary interleukin-12 (IL-12), as well as increased numbers of IL-12 producing cells

dendriticc cells and macrophages) in their bronchioles as compared to virus-susceptible

Brown Norway (BN) rats after Sendai virus infection (Chapter 3). The biological

significance of this increased IL-12 expression in the F344 strain has not been

established.

The heterodimeric cytokine, IL-12 is produced by antigen presenting cells during

intracellular infections to up-regulate cell-mediated immune responses and the T helper-1

(Th-1) cytokines, principally interferon-y (IFN-y)(1-3). IL-12 works to enhance the

cytotoxic properties of natural killer (NK) cells and cytotoxic CD8 T lymphocytes

(CTLs) (2). Furthermore, IL-12 can down-regulate the T helper type-2 (Th-2) cytokine

response by decreasing the production of interleukin-4 (EL-4) and Th-2 type antibodies

(2). Increased expression of IL-12 in animal models of infectious as well as allergic








disease has conferred resistance to the particular pathogenic phenotype (79, 97-98). The

exogenous administration of IL-12 to susceptible animals at certain time points during

infection or sensitization has also been shown to provide resistance in many of these

models (79, 97-98).

In Sendai virus rat model, F344 rats have an early Th-1 type immune response to

infection with higher IFN-y production and their NK and CTL cell types have an

increased capacity compared with the BN strain to produce IFN-y in response to infection

(115, 117, 119). Due, at least in part, to the higher IFN-y expression F344 rats have a

greater CD8+ T cell response, earlier pulmonary viral clearance, and a reduced capacity to

develop airway fibrosis after viral infection (115, 119). However, BN in rats produce

Th-2 type cytokines, such as EL-4, interleukin-5 (EL-5), and the profibrotic cytokines

tumor necrosis factor-ax (TNF-a) and transforming growth factor-p-1 (TGF-P1i) after

virus infection (113, 115, 118). This response to Sendai infection involves the

persistence of airway inflammation (macrophages, lymphocytes, and eosinophils),

delayed viral clearance, and chronic bronchiolar fibrosis (112-113). Treatment of BN

rats with IFN-y reduced the amount of chronic bronchiolar inflammation and fibrosis,

thus protecting them from pulmonary function abnormalities (117).

Based on the previous 1L-12 studies (Chapter 3) and the IFN-y treatment results, we

investigated the possible protective role of IL-12 in this rat model. We hypothesized that

the lower EL-12 response during acute viral infection is an important factor in the

development of the Sendai-induced post viral sequelae in BN rats. The objective of this








study was to determine if the administration of exogenous IL-12 at early time points

during Sendai virus infection would prevent the development of post viral persistent

bronchiolar inflammation and fibrosis.

Materials and Methods

Animals

Weanling (22 days old), male, pathogen-free BN/RijHsd rats (94 total rats) were

purchased from Harlan Sprague Dawley, Inc. Madison, WI. The control and virally

infected animals were housed separately in adjacent, identical Micro-Isolator VCL-HDTM

individually HEPA filtered/ventilated cages (#10419ZTGA ZytemTM plastic Micro-

IsolatorTM system, Lab Products, Inc. Seaford, DE). The University of Florida Animal

Care and Use Committee approved all procedures.

Viral Procedures and Sample Collection

The rats were inoculated with aerosolized Sendai virus strain P3193 five days after

arrival. Briefly, rats in the virus-inoculation group were exposed to an aerosol (Tri-R

Aerosol Exposure Apparatus, Glas-Col, Terre Haute, IN) of virus at a concentration of 1-

3 plaque-forming units (PFU) per ml of gas for 15 minutes. At 0, 3, 7, 10, and 14 days

after inoculation, rats from each group were immobilized via deep anesthetization with

sodium pentobarbital (approximately 200Jtg/g of body weight) or urethane (1.5g/kg body

weight) and killed by exsanguination via cardiac puncture. Lung lavages were performed

through intratracheal cannulation with phosphate buffered saline (PBS). The right lungs

were frozen in liquid nitrogen and stored at -80C. The left lungs were tied off, perfused

with 4% paraformaldehyde PBS (pH 7.4) (30 cm H20 pressure for 2 hours) and

embedded in paraffin. Viral titers were measured in homogenates of frozen lung at seven








days after inoculation, by plaque assay using Madin-Darby bovine kidney cells as

described previously, and expressed as plaque forming units (pfu)/g lung tissue (128).

IL-12 Treatment Protocol

Groups of BN rats (n=8/group) were treated with mouse recombinant IL-12p70

(rIL-12) (Biosource International, Camarillo, CA). The dose of 3ptg was determined to

be the lowest dose administered intraperitoneally (IP) that provided a measurable

biological response (Appendix B). IP injections of 3 jtg of rIL-12 were given to control

and infection groups of rats on the day of viral inoculation (3 hours after inoculation)

(day 0) and to control and infection groups 2 days (day 2) after inoculation. A

comparable volume of sterile saline was given to a separate control and infection groups

IP as a negative control at the same time points. Groups of BN rats were injected IP at

200 jtg/g of rat with 5-bromo-2'-deoxyuridine (BrdU)(Sigma, St. Louis, MO) 10 and 14

days after inoculation twelve hours prior to necropsy to detect epithelial and stromal cell

fibrosis.

Cytokine mRNA

Frozen lungs were weighed and RNA was extracted by phenol/chloroform

extraction or using the RNeasy midi kit (Qiagen Inc, Valencia, Ca) (121,122). The

RNA samples were pre-treated with DNase I using the Deoxyribonuclease I,

amplification grade kit (Invitrogen, Carlsbad, CA) to remove genomic DNA. The

Reverse Transcriptase (RT) reactions were preformed using the AdvantageM RT-for-

PCR Kit (Clontech Laboratories, Inc, Palo Alto, CA). Polymerase chain reaction (PCR)

primers and probes for rat interleukin- 18 (IL- 18), and for the housekeeping gene rat

glyceraldehyde-3-phosphate dehydrogenase (GADPH) were TaqMan pre-developed








assay reagents for gene expression quantification (Applied Biosystems, Foster City, CA).

The primers and TaqMan probe for BN rat IL-4 were designed using the Primer Express

software (Applied Biosystems, Foster City, CA). The sense and antisense primers were

made in the mRNA sequence to ensure discrimination between cDNA and genomic

DNA. The probe was labeled at the 5'-end with the reporter dye FAM (6-

carboxyfluorescein) and at the 3' end with a minor groove binder (TaqMan MGB) and a

non-fluorescent quencher prevent extension by AmpliTaq Gold DNA polymerase

(Forward Primer 5'-CAGGGTGCTTCGCAAATTTT-3'; Reverse Primer 5'-

CGAGAACCCCAGACTTGTTGTT-3'; and Probe 5'- TCCCACGTGATGTACCTCCGTGCTT-3').

Dilutional curves were evaluated to assure that the amplification efficiency of the IL-4

primers compared to the efficiency of the GADPH primers. Every time cDNA was

synthesized, parallel TaqMan assays were run for GADPH and the target cytokine in

separate wells. The PCR reactions contained 900 nM of each primer, 250 nM of the

TaqMan probe, PCR Mastermix (TaqMan Universal PCR Mastermix, Applied

Biosystems) containing 10 mM Tris-HCl, 50 mM KC1, 5 mM MgC12, 2.5 mM

deoxynucleotide triphosphates, 0.625 U AmpliTaq Gold DNA polymerase per reaction,

0.25 U AmpErase UNG per reaction, and 2 .1 of the cDNA sample in a final volume of

25 pil. The samples were amplified in an automated fluorometer (ABI Prism 7700

Sequence Detection System, Applied Biosystems). Amplification conditions were 2 min

at 50C, 10 min at 95C, 40 cycles of 15 s at 95C and 60 s at 60C (124).

IFN-y was detected in lung tissue by a competitive RT-PCR method as described

previously (126). Primers for IFN-y and for the housekeeping gene product,

hypoxanthine-guanine phosphoribosyltransferase (HPRT) were prepared as described by








the Interdisciplinary Center for Biotechnology and Research at the University of Florida
2+
(125). Each assay was optimized for temperature, Mg concentration, and primers. For

each cDNA sample reactions comparisons were made to 0, 0.5, 5, and 50 femptograms

(fg) of a competitive fragment (126). PCR reactions were performed at cycler programs

consisting of 1 minute at 94C, annealing temperature of 56C for 15 minutes, 72C for 2

minutes for 4 cycles. Then 36 cycles were run at the same temperatures and times except

for a 2-minute annealing time at 56C. PCR products were stained with ethidium

bromide and separated electrophoretically on 1.5-% agarose gels. The data are reported

as non-normalized mRNA abundance in competitive fragment units (117).

Enzyme-Linked Immunosorbent Assay (ELISA)

Lungs were harvested as previously described and homogenized in cold PBS with

a protease inhibitor (Protease Inhibitor Cocktail Tablets, Mini Complete, Boehinger

Mannheim, Germany) at a ratio of 0.lg of tissue/ml (133). The homogenates were

centrifuged at 4C at 2000 rpm for 10 minutes and the supematants were frozen at -80C

until use. IFN-y protein in the lung homogenates was determined using a rat IFN-y

ELISA kit (rat IFN-y, Biosource International, Camarillo, CA) according to the

manufacturer's instructions.

BrdU Immunohistochemistry

In previous studies, Sendai virus-induced fibroblast proliferation has been detected

by BrdU incorporation beginning at 9 days after inoculation (113). Paraffin sections of

lung at 10 and 14 days after Sendai inoculated rats were deparaffinized in xylene,

rehydrated through a graded series of ethanol washes, and washed in distilled water.

Immunohistochemical staining to detect BrdU labeling was performed using a method








previously described (137). Briefly, slides were placed in 3.0% H202 for 10 minutes to

quench endogenous peroxidase, washed twice in PBS, and pretreated in both 2N HC1 (30

minutes at 37C) and 0.1% w/v trypsin in PBS (20 minutes at 37C). Sections were

rinsed in PBS, covered with antibody diluent (1.0% BSA and 0.5% Tween 20 in PBS)

(Sigma, St. Louis, MO) for 30 minutes to block nonspecific binding, blotted and

incubated overnight at 4C covered in mouse anti-BrdU monoclonal antibody (Sigma, St.

Louis, MO) diluted 1:10 in antibody diluent. The next day the sections were washed in

PBS and covered with peroxidase-conjugated, goat, anti-mouse IgG (Fc specific) (Sigma,

St. Louis, MO) secondary antibody for one hour at room temperature. Bound peroxidase-

conjugated antibody was detected by development in the chromogen diaminobenzidine

(Sigma, St. Louis, MO) in 0.02 mg/ml in 0.25 mol/L Tris, pH 7.6, and 0.01% H202 for 7-

20 minutes as monitored with light microscopy. An average of 17 bronchioles per rat

(range 10-24) at 10 days after-inoculation and 14 bronchioles per rat (range 9-19) at 14

days after-inoculation were examined. Fibroblast proliferation was assessed by counting

the number of BrdU-labeled fibroblasts per mm of bronchiolar basement membrane.

Analysis of Bronchiolar Inflammation and Fibrosis

Previously studies have found that virus-induced bronchiolar fibrosis and collagen

deposition is present in BN rats by 14 days after-viral infection (113). Serial paraffin

sections of rat lungs 14 days after-inoculation (5 per rat) were stained with hematoxylin

and eosin (H&E). Each branch of bronchiole cut in transverse, longitudinal, or oblique

planes was counted and was evaluated for both the presence of inflammation and for the

presence of fibrosis/remodeling. An average of 42.7 (range 21-76) bronchioles per rat

were evaluated. Bronchioles were scored as positive for inflammation if the wall had








five or more inflammatory cell types eosinophilss, lymphocytes, or macrophages).

Bronchioles were scored as positive for fibrosis/remodeling if the walls were thickened

with increased fibroblasts and deposition of collagen. Collagen was identified using

Masson's Trichrome and Manuel's Reticulin stains. Numbers ofbronchioles with

inflammation or fibrosis/remodeling were divided by the number of total bronchioles

examined to calculate the percentage of bronchioles with each pathologic change.

Additionally, the area of the bronchiolar wall from the bronchiolar epithelial basement

membrane to basement membrane of the surrounding alveolar walls was measured.

Bronchiolar wall area was divided by the perimeter of bronchiolar basement membrane to

calculate the thickness of the wall (square micrometers of bronchiolar wall per

micrometer of bronchiolar basement membrane) (113).

Data Analysis

The final quantitation of cytokine mRNA levels detected by real-time PCR was

done using the comparative CT (cycle threshold) method and was reported as relative

transcription of the n-fold difference relative to a calibrator cDNA (BN rat high IL-4

responder lung cDNA for IL-4 mRNA and LPS-stimulated lung cDNA for IL-18 mRNA)

(Table 3-1) (124). For all experiments, group means were compared by one-way analysis

of variance (ANOVA) using a computer-based statistical program (Sigma-Stat, Jandel

Corp. San Rafael, CA). Kruskal-Wallis analysis of variance on ranks was used on ranks

if the normality test or equal variance tests of group means failed. Multiple comparison

procedures were used to isolate the group or groups that differ from others. The Student-

Newman-Kuel's test was used if the sample sizes were equal; otherwise Dunn's test was

used to compare groups of unequal sample size.









Results

IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Amount of Bronchiolar Inflammation and Fibrosis

Virus infection in BN rats induced bronchiolar inflammation and fibrosis (Figure 4-

1). Administration ofrIL-12 at the time of Sendai viral inoculation (Day 0) reduced the

number of inflamed bronchioles by 22.1% and the number of bronchioles with mural

fibrosis by 23.8% as compared with virus-inoculated, saline-treated controls (n = 8)

(Figure 4-1).

S120-
U Mean SE Inflammation
S100 n=8 Fibrosis


.- I L
U)
,80

60

0
0- 40o

.20
0
I- 0 l
Non- Virus Virus Virus
Infected Infected Infected Infected
IL-12 Saline IL-12 IL-12
DayO 0 DayO 0 Day 0 Day 2
S*p
Figure 4-1. The percent ofbronchioles containing evidence of inflammation and/or
fibrosis at 14 days after inoculation. Treatment of BN rats at the time of virus infection
resulted in the significant reduction of bronchiolar inflammation and fibrosis (p<0.01).

In contrast, IL-12 treatment 2 days after viral inoculation did not lead to a

significant decrease in the numbers of inflammatory cells or in the severity of fibrosis

observed. Previously it has been shown that non-infected BN rats normally have low-

density aggregates of lymphocytes, macrophages, mast cells, and eosinophils in

bronchiolar walls by 30 days of age (Figure 4-1) (112).









IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Bronchiolar Wall Thickness

Increases in bronchiolar wall thickness occur by 14 days after Sendai virus

inoculation due to edema, the accumulation of inflammatory cells, fibroblast

proliferation, and the intramural deposition of collagen and extracellular matrix (113).

The BN rats treated at time of virus-inoculation had a 15% decrease in the bronchiolar

wall thickness as compared with the virus-inoculated, saline-treated controls (p<0.02) (n

= 8) (Figure 4-2). A significant virus-induced increased in bronchiolar wall thickness

was observed above the non-infected BN rats in the virus-inoculated, saline treatment

group (p
30
MeanSEM *p S25 tp<0.0001oool
25 n=8 +p 00

# #.
I 15





=L 0

Non- Infected Infected Infected
Infected Saline IL-12 IL-12
IL-12 DayO 0 DayO 0 Day 2
Day 0

Figure 4-2. Airway morphometric analysis of bronchiolar wall thickness at 14 days after
virus inoculation. IL-12 administration at the time of virus inoculation reduced the
bronchiolar wall thickness to non-infected control levels (p<0.02).

In contrast rats treated on day 2 after inoculation had a 22.3% increase in the

bronchiolar wall thickness as compared to virus-inoculated, saline-treated controls

(p







IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Number of BrdU Labeled Fibroblasts in the Bronchiolar Walls

Bronchiolar fibrosis and other remodeling events were evaluated by counting

bronchiolar mural fibroblasts and epithelial cells labeled with BrdU at 10 and 14 days

after virus inoculation (Figure 4-3). Treatment of BN rats with rIL-12 at day 0

significantly reduced the magnitude of increase in mural fibroblast labeling by BrdU.

There was decreased mural fibroblast labeling by BrdU by 42% at 10 days after

inoculation (n= 8) (Figure 4-4) and 62.5% at 14 days following inoculations (n = 8)

(Figure 4-5).

Treatment of BN rats with rIL-12 at day 2 had no effect on virus-induced increases

in BrdU labeling of fibroblasts at 10 days after inoculation and increased labeling of

fibroblasts by 12.5% at 14 days after inoculation (Figure 4-4). Virus infection resulted in

increased labeling of bronchiolar epithelial cells with BrdU. However, rIL-12 treatment

had no statistical effect on this virus-induced labeling of epithelial cells.

IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Increases the
Pulmonary Expression of IFN-y

Virus infection increases the amount of IFN-y mRNA in whole lung tissues of BN

rats (n = 4-5) (Figure 4-6). There were significant increases in IFN-y mRNA in all

treatment groups infected with Sendai virus over non-infected controls (p<0.05). BN rats

treated with IL-12 at day 0 had 4-fold more IFN-y mRNA in lungs (p<0.002) when

compared to the saline-treated, virus-inoculated controls. IL-12 treatment at day 2 after

virus inoculation decreased the production of IFN-y mRNA by 4-fold as compared to

saline-treated, virus-inoculated controls (Figure 4-6).














4 ..--


4e NO


Figure 4-3. BrdU labeled bronchiole of a saline-treated, virus-inoculated BN rat. Labeled bronchiolar
epithelium is indicated by arrows/ several labeled fibroblasts are indicated with (*)










10 Days After Inoculation


Non- Virus Virus Virus
Infected Infected Infected Infected
Saline IL-12 IL-12
Day 0 Day 0 Day 2


Figure 4-4. Immunohistochemical analysis of fibroblast BrdU labeling at 10 days after
Sendai inoculation. BN rats treated at the time of virus inoculation reduced the number
of labeled fibroblasts significantly as compared to virus-inoculated, saline-treated
controls.

14 Days After Inoculation


SNon- Virus Virus Virus
Infected Infected Infected Infected
Saline IL-12 IL-12
Day 0 Day 0 Day 2

Figure 4-5. Immunohistochemical analysis of fibroblast BrdU labeling at 14 days after
Sendai inoculation. The treatment of BN rats with IL-12 at the time of virus infection
reduced the number of BrdU labeled fibroblasts significantly as compared to virus-
inoculated, saline-treated controls (p<0.0001).









500 -
MeanSEM tp (An=4-5 t *p<0.01
400
4

E 300
E
I)
U.
( 200
>.
0100
| 100 -. t- .***'"
E
0
0 *
0
Non-Infected Infected Infected Infected
IL-12 Saline IL-12 IL-12
DayO 0 DayO 0 DayO 0 Day 2

Figure 4-6. Competitive PCR analysis of IFN-y mRNA in BN rats 3 days after
inoculation treated at day 0 or day 2 with IL-12. IFN-y mRNA levels in rats treated with
IL-12 at the time of virus infection increased significantly above the saline-treated, virus-
inoculated controls (p<0.002). Day 2 treatment decreased the IFN-y mRNA response to
levels significantly below the saline-treated, virus-inoculated controls (p<0.01).

There was a significant increase (73.5%) in the level of IFN-y protein in the total

lung tissue of the BN rats (7 days after inoculation) treated at day 0 as compared to the

virus-inoculated, saline-treated rats (p<0.05) (n = 7-8) (Figure 4-7). Day 2 IL-12

treatment did not increase IFN-y over the saline-treated rats measured seven days after

virus inoculation. A virus-induced increase in IFN-y protein was detected in all treatment

groups at 7 days after inoculation as compared to the non-infected controls.

IL-12 Treatment of BN Rats on Day 2 After Sendai Virus Inoculation Alters the
Levels of IL-18 or IL-4 mRNA

The levels of mRNA of the co stimulatory cytokine IL-18 were not increased by

IL-12 treatment at any of the time points measured (n = 3-5) (Figure 4-8).










500
Mean SEM
Tn=7-8
400 *p<0.05
E ;:

C300


0
2200-
a.N

z 100 ,
LI.


Non- 7 7 Day 7 Day 10 10 Day 10 Day
Infected Day Infected Infected Day Infected Infected
Controls Infected IL-12 IL-12 Infected IL-12 IL-12
DayO Day2 Day0 Day2
Treatment Groups


Figure 4-7. ELISA analysis of whole lung homogenates from BN rats at 7 and 10 days
after virus inoculation. Day 0 IL-12 treatment induced a significant increase in the levels
of IFN-y as compared to the virus-inoculated, saline treated controls (p<0.05).

However, L-12 treatment at day 2 caused a decrease in the amount ofIL-18 mRNA

measured at three days after inoculation as compared to day 0 treated animals (p<0.05).

No significant differences were measured between either of these groups and the non-

infected controls.

The treatment of BN rats at the time of virus inoculation did not reduce the levels

ofIL-4 mRNA at any time points measured after inoculation. However, IL-4 mRNA

levels were significantly elevated (3.2-fold increase) in the virus inoculated rats treated at

2 days after inoculation as compared to virus-inoculated, saline-treated controls (n = 4-6,

p<0.02) (n = 4-6) (Figure 4-9). Virus infection increased the level of IL-4 mRNA in all

treatment groups at 3 days after Sendai virus inoculation (p<0.05).













20

0
U
15

10
Q io
0

- 5



0


Figure 4-8. Analysis of real-time PCR for the detection of IL-18 mRNA. IL-12
treatment at 2 days after inoculation significantly reduced the expression of IL-18 mRNA
as compared to the rats treated the day of virus inoculation.


Non- Infected
Infected 3 Days
Saline Saline
Day 0 Day 0


Infected
3 Days
IL-12
Day 0


Infected Infected Infected
3Days 7Days 7Days
IL-12 Saline IL-12
Day 2 Day 0 Day 0


Figure 4-9. Analysis of real-time PCR for the detection of L-4 mRNA. BN rats treated
with IL-12 at day 2 had a significant increase in the level of IL-4 mRNA at three days
after inoculation compared to virus-inoculated, saline-treated rats 3 days after inoculation
(p<0.02). IL-4 mnRNA expression significantly increased in all of the virus-infected
groups assessed at 3 days after Sendai inoculation (p<0.05).


1.8

1.6-

1.4-
0
I 12-
0
1.2

S1.0-
Q
S0.8-

S0.6-

0.4-

0.2-

0.0


n= 4-6
Mean SE

*a.'







i : r :
.. y .
!"; :

___________ ,_ ,:, ___________,____


Infected
7 Days
IL-12
Day 0









IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Does Not Alter
the Respiratory Clearance of Sendai Virus

F344 rats have been shown to clear Sendai virus from their lungs at 7 days after

inoculation as compared to virus-susceptible BN rats (115). Viral clearance was not

affected in BN rats treated with rIL-12 on day 0 as compared to saline-treated, virus-

inoculated controls (p>0.05). At seven days after inoculation, the mean value of virus

recovered from the saline-treated, virus inoculated controls was 6.19 X 104 PFU/g of

lung, and the BN rats treated on day 0 had a mean titer of 7.33 X 104 PFU/g of lung (n =

8) (Figure 4-10).

14 -
13 p>0.05
e n=8
0
,J 12-012

10

I1( 100
-I 9 o o



4W 0 0
oo
_j 8 8

I- -
> 6 8

0
5 -
Virus Virus -
Infected Infected
IL-12 Saline
Day 0 Day 0

Figure 4-10. Viral titer results from plaque assays at 7 days after inoculation. IL-12
treatment does not increase the rate of viral clearance from the lungs of BN rats after
Sendai infection (P>0.05).

Discussion

The results indicate early treatment of virus-susceptible BN rats with IL-12 inhibits

the development of virus-induced chronic inflammation and bronchiolar fibrosis. IL-12

treatment at the time of Sendai virus inoculation decreased the amount of airway wall









inflammation and fibrosis, decreased the thickness of the bronchiolar walls, and increased

the expression of the Th-1 cytokine IFN-y. This protection is not observed if IlL-12

treatment is delayed as little as two days after virus-inoculation. In fact several of the

viral sequelae such as airway wall thickening and fibrosis were exacerbated.

Mechanisms by which IL-12 treatment early in the Sendai virus infection may

reduce virus-induced persistent bronchiolar inflammation and bronchiolar fibrosis and

other remodeling may be direct through IL-12 or indirect through the induction of IFN-y.

IL-12 can act to increase the cytotoxicity and mitogenic activity of T and NK cells, and to

inhibit B cell functions while enhancing the conventional, B cell-dependent antibody

responses (1-4). IL- 12 also has direct stimulatory effects on hematopoietic progenitor

cells (2). The main biological effects of IL-12 are attributed to the induction of the

cytokines TNF-a, granulocyte-macrophage colony-stimulatory factor, IL-10, IL-2, and,

most importantly, the induction and maintenance of IFN-y (1-4). IL-12 also acts to

reduce the levels of IL-4 and the establishment of the Th2 phenotype (2-3). IL-12

treatment did increase the levels of IFN-y mRNA and protein significantly above the

levels seen with viral infection alone (saline-treatment). IL-12 treatment however, did

not reduce the levels of IL-4 mRNA at the time points measured in this model. Still,

there may be reductions in IL-4 at a later time point due to the prevention of the

proliferation and development of Th2 cells (3). The viral titer data does not suggest that

IL-12 is directly inhibiting viral replication. This is consistent with previous studies in

which IFN-y treatment reduced bronchiolar inflammation and fibrosis without altering

Sendai virus replication in BN rats (119).








The effects of IL-12 treatment in reducing the pulmonary fibrosis observed in BN

rats after Sendai virus infection may be mediated by increases in IFN-y expression. Early

IL-12 treatment was associated with increases in total lung IFN-y mRNA at three days

after inoculation and in total lung IFN-y protein at seven days after inoculation. IFN-y

can induce angiostatic chemokines such as IFN-inducible protein 10 (IP-10), macrophage

inflammatory protein-2 (MIP-2), and monokine induced by IFN-y (MIG). These

chemokines have been shown importance in the down-regulation of angiogenesis and

fibrosis in a mouse bleomycin-induced pulmonary fibrosis model (132). IL-12

administration to bleomycin-treated CBA/J mice decreased levels ofhydroxyproline and

increased lung IFN-y, IP-10, and MIG (138). Additionally, IFN-y treatment has been

shown to attenuate increases ofTGF-P1, procollagen mRNA, and total lung collagen in

after bleomycin challenge in mice (132).

The time frame for the protective effects of IL-12 appears to be narrow and only

very early after Sendai infection. Administration of IL-12 just 2 days after viral

inoculation failed to protect against increased virus-induced bronchiolar wall thickening,

bronchiolar airway wall fibroblast proliferation, increased EL-4 mRNA production, and

suppressed the expression of total lung IFN-y mRNA. These results are similar to the

responses of exogenous IL-12 treatment seen in other infection models. Exogenous IL-

12 administered at times when T-cell responses are known to be effecting clearance of

marine lymphocytic choriomeningitis virus caused reduced cytotoxic T cell lytic

capabilities, inhibition of virus-induced expansion of CD8+ T cells, and increased

production of TNF (136). IL-12 immunotherapy of murine leishmaniasis infection is

only effective during the first week of infection. Delayed treatment is ineffective and can








enhance IL-4 production and susceptibility (79). The molecular basis for this loss of IL-

12 sensitivity is hypothesized to be due to a disruption in Th-2 cells of the IL-12

dependent activation of the Janus kinases (JAK) and signal transducers and activators of

transcription (STAT) intermediates through the IL-12RP2 that are preserved in Th-1 or

Th-0 cells (2, 71). Early IL-12 treatment however, modifies the course of leishmaniasis

by inhibiting the development of Th-2 type responses and promoting the Th-1 cell

responses dependent on IFN-y (79). The dynamics observed in these experimental

models may be true in this model, in that once a Th-2 response is already established; it

can be difficult to reverse.

The protective effects of IL-12 administration in this model are consistent with

effects seen in other studies of infectious and allergic airway disease (Chapter 1).

Furthermore, this model also exhibits many of the complications observed in other

models regarding the dependence on the timing of IL-12 administration and protective

immunomodulatory effects (79, 97-99). These results are consistent with the conclusion

that airway dysfunction in childhood asthma may partially be the result of slight

differences in the immune cytokine response that control the inflammatory and repair

processes to viral disease. Based on these experiments, it may be possible to interrupt the

progression of viral injury to chronic airway damage with early immunomodulatory

cytokine administration.













CHAPTER 5
GENERAL SUMMARY AND FUTURE DIRECTIONS

The goal of this research was to determine the role ofinterleukin-12 (IL-12) in the

development of resistance to chronic airway disease induced by parainfluenza (Sendai)

virus during early life. The hypothesis tested was that F344 rats are more resistant to

virus-induced airway damage and fibrosis because they produce high levels of IL-12

early in response to virus that up-regulates Th-1 cytokine responses, antiviral immunity,

and reduces airway wall fibrosis. Fulfilling 4 specific objectives tested this hypothesis:

Objective 1

1) To compare the pulmonary IL-12 mRNA and protein responses of virus- resistant
F344 and virus-susceptible BN rats following Sendai virus infection.

Real-time PCR revealed that both rat strains have early virus-induced increases in

IL-12 p40 mRNA (2-5 days after inoculation). In addition, it was demonstrated that

virus-inoculated F344 rats have significantly increased pulmonary IL-12 p40 and IL-12

p35 mRNA at early time points after inoculation as compared to the BN rat strain (0-3

days after inoculation). This is just prior to the increased expression of IFN-y mRNA

observed in F344 during previous studies at 3, 5, and 7 days after Sendai inoculation

(117). ELISA of whole lung tissue, revealed that IL-12 protein levels are increased

significantly in the F344 at two days following inoculation, and that this increase was not

due to over-production of potentially antagonistic IL-12 p40 monomers and dimers

(2,13).








Objective 2

2) To determine ifF344 rats have greater numbers of pulmonary cells and differing
cell types that express IL-12 in response to Sendai virus infection than BN rats.

Protein immunohistochemistry demonstrated that F344 rats have higher numbers of

bronchiolar dendritic cells and macrophages expressing IL-12 p40 protein as compared to

the virus-inoculated BN rats. Although many attempts were made to establish the

location and differential levels of IL-12 p40 mRNA expression by in situ hybridization,

none of the experimental results were conclusive. Observationally, the IL-12 p40 mRNA

message was detected sporadically in the bronchiolar macrophages, dendritic cells, in the

lymphocytes of the BALT, and in the airway epithelial layer.

Objective 3

3) To determine if Sendai virus-induced airway damage in BN rats can be reduced by
IL-12 treatment early in the virus infection.

The treatment of BN rats with exogenous IL-12 at the time (within 3 hours) of viral

inoculation does reduce the chronic sequelae of Sendai virus infection. BN rats treated

with IL-12 on the day of inoculation-displayed decreases in bronchiolar inflammation

and fibrosis, decreases in airway wall fibroblast proliferation, and increases in IFN-y

expression at various time points after Sendai infection. Viral clearance in BN rats was

not affected by the treatment of IL-12 at the time of virus inoculation, and viral clearance

after treatment on day two after inoculation was not assessed. In contrast, IL-12

treatment two days after virus inoculation significantly increased airway wall thickness,

decreased IFN-y mRNA expression, and increased the expression of IL-4 mRNA.








Objective 4

4) To compare the airway IL-12 p35 and p40 mRNA responses of virus-resistant F344
and virus-susceptible BN rats following Sendai virus infection (This specific aim is
contingent on the results from the second specific aim. If there is differential
expression of IL-12 in the dendritic cell or other cells types in the large airways
based on the results of the in situ hybridization and immunohistochemistry, then
this specific aim will be explored).

When the experiments to determine the differential expression of IL-12 in this

model were begun, real-time PCR was in its very early stages. Therefore, I was

demonstrating only marginal differences in the amount of IL-12 p40 mRNA expression

by dilutional PCR. At the time, it seemed that this was possibly due to the dilution of

differences that may be at the airway level being masked by the inclusion of the total lung

tissue. Additionally, the in situ hybridization and the immunohistochemistry techniques

were very time consuming to resolve. Based on these difficulties, this specific aim was

added. Real-time PCR of the tracheal tissue revealed that IL-12 p40 mRNA does

increase in both rat strains early after virus inoculation, and is significantly elevated at

two days following inoculation in the F344 rat strain. There were no significant

alterations in the IL-12 p35 mRNA possibly due to the low levels of tissue used or due to

the limits of this procedure at detecting all of the IL-12 p35 isoforms (Chapters 1 and 3).

Conclusions

The results of these studies supports the hypothesis that F344 rats are more resistant

to virus-induced airway damage and fibrosis because they produce high levels of IL-12

early in response to virus that up-regulates Th-1 cytokine responses, antiviral immunity,

and reduces airway fibrosis. It is concluded that:

1) Virus-resistant F344 rats express higher pulmonary IL-12 gene expression early
after Sendai virus infection as compared to virus-susceptible BN rats.









2) Virus-resistant F344 rats have more bronchiolar dendritic cells and macrophages
expressing IL-12 than BN rats at early time points after inoculation.

3) Treatment of virus-susceptible BN rats with IL-12 early after Sendai inoculation
reduces the severity of airway wall fibrosis and remodeling.

4) IL-12 has a critical role in the immune response to Sendai virus infection in F344
rats.

Future Studies

In previous experiments by P.G. Holt and others, have identified dendritic cells as

the principal resident APC of the rat, mouse, and human lung and that these cells form a

network throughout the epithelium to alert the immune response to inhaled antigens

(134). Furthermore, the examination of these resident cells in BN rats suggests that the

resting Th-2 polarity of the resting mucosal immune system may also be a property of the

resident dendritic cell population (121,136). Initiation of the Th-1 immune response

seems to require additional signals such as TNF-a expression and/or CD40 ligation from

the microenvironment (136).

The results these studies using OX-6 immunohistochemistry indicate that there are

more dendritic cells located in the F344 bronchiolar airways at two and three days after

Sendai virus inoculation compared to the BN rats. Further studies to map the kinetics of

the dendritic cell numbers and turnover in the context of this model are indicated. It may

be that the dendritic cell numbers persist longer and in different locations within the

airways of F344 rats after Sendai infection than in BN rats. These results also indicate

that these cells are expressing differing levels of cytokines at the airway level depending

on the rat strain infected. Using techniques such as laser microdissection, real-time PCR,

and double-staining immunohistology the important differences may be elucidated at the








dendritic cell network level that influence the susceptibility to Sendai virus infection and

possibly asthma.

Another aspect in this model that may affect the susceptibility of the rats in this

model to Sendai virus is the expression of the IL-1 2Rp2 receptor protein. The expression

of the IL-12Rp32 protein is limited to Th-1 cells and may correlate with IL-12

responsiveness (29,30). In the development ofT cells, IL-4 can inhibit the expression of

this subunit, thus these cells lose the ability to respond to IL-12 after TCR binding (30).

Recently, differential expression of the IL- 12Rp2 subunit between predominately Th-2

responding BN rats and Th-1 responding Lewis rats (139). Within the model, F344 rats

may have increased expression of the IL-12Rp2 protein, therefore not only producing

more IL-12 but may be more responsive to its immunologic effects. Preliminary data

using IL-12Rp2 RT-PCR on pulmonary tissue, an increase in the expression of this

subunits mRNA message in the F344 rat tissue (Figure 5-1).






288 bp --I


t BF BF BF BF Negative
Controls
100 bp Ladder

Figure 5-1. RT-PCR of IL-12Rp2 in BN and F344 rats at non-infected control levels.
Observationally, there are brighter bands in the non-infected control F344 rats (F) rats as
compared to the BN (B) rat strain at control levels. The band size of lL-12Rp2 is 288
base pairs (bp) as indicated above.

The differential expression of the IL-12Rp2 may indicate differences in the ability

of the rats in this model to respond to IL-12. This aspect of IL-12 regulation may need to






85


be addressed in this rat model and in human asthma patients before the potential

effectiveness of IL-12 immunomodulation can be fully assessed.













APPENDIX A
PRELIMINARY DATA

Experiment 1: Pulmonary Expression of IL-12 in Sendai Virus-Infected BN and
F344 Rats

Dilutional RT-PCR

Preliminary pulmonary mRNA levels were measured in a small number of rats

using 10-fold dilutional RT-PCR (Chapter 2). IL-12 p40 mRNA levels were increased

over levels in BN rats at two, three, and seven days after inoculation with Sendai virus

(Figure A- 1).

10000
4 = BN
S8000 F344
E Mean SE
0 8 n=4-7
74
= o6000 *p
4000
g :4ooo

5 2000 1 tj

0 1 2 3 5 7
Days After Inoculation
Figure A-I. Dilutional RT-PCR analysis of mRNA from virus-infected BN and F344
rats. F344 rats have increased pulmonary IL-12 p40 mRNA over BN rats at 2, 3, and 7
days post-inoculation. This difference is statistically significant at 3 days after viral
inoculation (p<0.05).

Lung Lavage Fluid ELISA

Preliminary ELISAs using lung lavage fluid detected very low levels of IL-12 p70

and IL-12 p40 protein with minimal differences between the two strains (Figures A-2, A-






87


3). Based on the low levels detected, whole lung homogenates were used in further

studies to measure the expression of IL-12 protein.


0 3 5


Days After Inoculation

Figure A-2. ELISA analysis of IL-12 p70 protein in concentrated lavage samples from
small numbers virus-infected and control rats. The level ofIL-12 p70 increases with
viral infection, however there are no significant differences between the BN and F344 rat
strains.

$,
-BN
E F344
0) 4
2 Mean SE
n=2-5
3. *p<0.05
0



N


0-
0 3 5

Days After Inoculation

Figure A-3. ELISA analysis of IL-12 p40 protein (homodimers and monomers) in
concentrated lavage samples from small numbers virus-infected and control rats. The
level of L-12 p40 is higher at control levels in the F344 rat strain, however extremely
low amounts were detected in both strains at all three time points.









Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats

IFN-y protein was evaluated in concentrated lung lavage samples from saline-

treated, virus-infected BN rats, control BN rats, and IL-12-treated, virus-infected BN rats.

Preliminary analysis using concentrated lung lavage fluid samples detected an increase in

levels of IFN-y after viral infection, but no significant differences in the IL-12 treatment

groups (Figure A-4). This analysis was repeated using the supernatant from whole lung

homogenates.

50
Mean SE
40- n=5-8
CO *p<0.003
0.
30 "'
S ..

20

10-
X,20



"0: .; ,, -, .".

IL 10 0 >.0t
"4.,
Cn 'n t on P a
S; S1 % 0 0 C 0 -- i




differences between the treatment groups in the levels of protein detected (p>0.05).

However, IFN-y protein levels increased at seven days after infection above the previous
(three day) levels in the saline-treated and IL-12 (day 0) treated animals, but not in the
IL-12 (day 2) treated BN rats (P<0.003), ANOVA.




Full Text
THE ROLE OF INTERLEUKIN-12 IN THE PATHOGENESIS OF SENDAI VIRUS-
INDUCED AIRWAY DISEASE
BY
AMY ELIZABETH SEYMOUR STONE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2002

This work is dedicated to my mother, Dr. Sandra Fields Seymour, who instilled me with
the courage and desire to become an educated woman.

ACKNOWLEDGMENTS
I would like to start by thanking my family; my parents, Andy, and Sydne were all
so supportive through this whole thing. I really appreciate their never wavering support.
I thank all of my friends for never letting me take myself too seriously when things
were tough. I appreciate my coworkers/fellow graduate students Diane Hulse and Tracy
Jack for sticking with me through the highs (happy hour) and lows (molecular biology) of
the last few years.
I would not have been able to complete this work without the support of my life
partner, Andy. I cannot thank him enough for his love, patience, and ability to make me
laugh at the times when I needed it.
Special thanks go to William Castleman for giving me the opportunity to embark on
this dual degree program, for giving me guidance through the system, and for setting a
quality example as a scientist and a leader.
I thank the members of my committee, Mary Brown, Thomas Brown, Steeve
Giguére, Elizabeth Uhl, and former committee members Ammon Peck, Trenton Schoeb,
and Rosalia Simmen for their ideas and assistance.
Additionally, I appreciate all of the editorial and motivational help that Carol
Detrisac provided in the completion of this document. I would also like to acknowledge
the other graduate student in my lab, Xuezhong Cai, who provided expertise,
encouragement, and humor whenever needed.
iii

Many thanks go to Karen Dukes, Heather Sorenson, Jeff Mans, Kristen Ruest, Mark
Hickey, Timothy Holloway, and Christy Voakes, who provided help with laboratory
work as well as much friendship.
Special appreciation and admiration go to Tracy Jack, who was my listening ear,
my shoulder, my colleague, and my friend through this whole process.
IV

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
TABLE OF CONTENTS v
LIST OF TABLES viii
LIST OF FIGURES ix
ABSTRACT xi
CHAPTER
1 LITERATURE REVIEW 1
Interleukin-12 1
IL-12 in Bacterial Infections 12
IL-12 in Protozoal Infections 13
IL-12 in Fungal Infections 15
IL-12 in Viral Infections 16
The Role of IL-12 in Allergy and Asthma 18
Rodent Model for Virus-Induced Pulmonary Disease 21
2 RESEARCH PLAN AND PROTOCOL 24
Hypothesis and Specific Aims 24
Background/Significance 24
Gaps in Knowledge to Be Addressed by This Research 26
Research and Design Methods 26
Overview of Experiments and Schedule 26
Experiment 1: Pulmonary Expression of IL-12 in Sendai Virus-infected BN and F344
Rats 26
Objectives 26
Rationale 27
Design and Methods 28
Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats 31
Objectives 31
v

Rationale 32
Design and Methods 33
3 INCREASED EXPRESSION OF PULMONARY INTERLEUKIN-12 (IL-12) IN
SENDAI VIRUS-RESISTANT F344 RATS 38
Summary 38
Introduction 39
Materials and Methods 42
Animals 42
Viral Procedures and Sample Collection 42
Cytokine mRNA 43
Protein Analysis 44
Enzyme-linked immunosorbent assay (ELISA) 44
Immunohistochemistry 45
Data Analysis 46
Results 46
Virus-Resistant F344 Rats Have Increased Expression of Pulmonary IL-12
mRNA 46
F344 Rats Have Increased Pulmonary IL-12 Protein After Sendai Virus Infection
49
F344 Rats Have Increased Numbers of IL-12 p40 Expressing Cells in the
Bronchioles After Sendai Virus Inoculation 50
F344 and BN Rats Do Not Have Detectable Differences in the Expression of IL-
18 mRNA 53
Discussion 54
4 EXOGENOUS INTERLEUKIN-12 (IL-12) ADMINISTRATION REDUCES THE
SEVERITY OF SENDAI VIRUS-INDUCED CHRONIC AIRWAY FIBROSIS
AND REMODELING IN BN RATS 59
Summary 59
Introduction 60
Materials and Methods 62
Animals 62
Viral Procedures and Sample Collection 62
IL-12 Treatment Protocol 63
Cytokine mRNA 63
Enzyme-Linked Immunosorbent Assay (ELISA) 65
BrdU Immunohistochemistry 65
Analysis of Bronchiolar Inflammation and Fibrosis 66
Data Analysis 67
Results 68
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Amount of Bronchiolar Inflammation and Fibrosis 68
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Bronchiolar Wall Thickness 69
vi

IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Number of BrdU Labeled Fibroblasts in the Bronchiolar Walls 70
IL-12 Treatment of BN Rats at the Time of Sendai Vims Inoculation Increases the
Pulmonary Expression of IFN-y 70
IL-12 Treatment of BN Rats on Day 2 After Sendai Vims Inoculation Alters the
Levels of IL-18 or IL-4 mRNA 73
IL-12 Treatment of BN Rats at the Time of Sendai Vims Inoculation Does Not
Alter the Respiratory Clearance of Sendai Virus 76
Discussion 76
5 GENERAL SUMMARY AND FUTURE DIRECTIONS 80
Objective 1 80
Objective 2 81
Objective 3 81
Objective 4 82
Conclusions 82
Future Studies 83
APPENDIX
A PRELIMINARY DATA 86
Experiment 1: Pulmonary Expression of IL-12 in Sendai Virus-Infected BN and F344
Rats 86
Dilutional RT-PCR 86
Lung Lavage Fluid ELISA 86
Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats 88
B IL-12 DOSAGE FOR TREATMENT TRIAL 89
Experimental Design 89
Results and Conclusions 90
C IN SITU HYBRIDIZATION FOR INTERLEUKIN-12 93
Protocol For In Situ Hybridization 93
Problems Solving For Difficulties With In Situ Hybridization 94
Observations of In Situ Hybridization Experiments 95
LIST OF REFERENCES 98
BIOGRAPHICAL SKETCH 111
vii

LIST OF TABLES
Table page
2-1 Table of Experimental Design: Experiment 1 28
2-2 Table of Experimental Design: Experiment 2 34
3-1 Comparative Ct Method of cDNA Relative Quantitation 44
B-l Experimental Design for IL-12 Dosage Trial 89
B-2 Percentages of Inflammatory Cells in the IL-12 Treated BN Rats 91
vin

LIST OF FIGURES
Figure page
2-1 Experimental design diagram for experiment 2 34
3-1 Real-time PCR analysis of IL-12 p 40 mRNA in whole lung samples of BN and
F344 rats after Sendai vims infection 47
3-2 Real-time PCR analysis of IL-12 p35 mRNA in whole lung samples of BN and F344
rats after Sendai virus infection 48
3-3 Real-time PCR analysis of IL-12 p40 mRNA in trachea samples of BN and F344 rats
after Sendai vims infection 48
3-4 Real-time PCR analysis of IL-12 p35 mRNA in trachea samples of BN and F344 rats
after Sendai vims infection 49
3-5 ELISA analysis of IL-12 total protein in F344 and BN rats strains after Sendai vims
inoculation 50
3-6 ELISA analysis of IL-12 p40 protein in F344 and BN rats strains after Sendai vims
inoculation 50
3-7 OX-6 immunohistochemistry in the bronchiole of a F344 rat three days after
inoculation 51
3-8 Density of OX-6 positive dendritic cells in the bronchioles of F344 and BN rats 51
3-9 Density of IL-12 p40 positive dendritic 52
3-10 IL-12 p40 immunohistochemistry in the wall of bronchiole of a F344 rat at two
days after Sendai inoculation 52
3.11 Density of IL-12 p40 positive macrophages in the bronchioles of BN and F344 rats.
53
3-12 Real-time PCR analysis of IL-18 mRNA expression in lung samples of BN and
F344 rats after Sendai virus infection 54
4-1 The percent of bronchioles containing evidence of inflammation and/or fibrosis at 14
days after inoculation 68
IX

4-2 Airway morphometric analysis of bronchiolar wall thickness at 14 days after virus
inoculation 69
4-4 Immunohistochemical analysis of fibroblast BrdU labeling at 10 days after Sendai
inoculation 72
4-5 hnmunohistochemical analysis of fibroblast BrdU labeling at 14 days after Sendai
inoculation 72
4-6 Competitive PCR analysis of IFN-y mRNA in BN rats 3 days after inoculation
treated at day 0 or day 2 with 1L-12 73
4-7 ELISA analysis of whole lung homogenates from BN rats at 7 and 10 days after
virus inoculation 74
4-8 Analysis of real-time PCR for the detection of IL-18 mRNA 75
4-9 Analysis of real-time PCR for the detection of IL-4 mRNA 75
4-10 Viral titer results from plaque assays at 7 days after inoculation 76
5-1 RT-PCR of IL-12R|32 in BN and F344 rats at non-infected control levels 84
A-l Dilutional RT-PCR analysis of mRNA from virus-infected BN and F344 rats 86
A-2. ELISA analysis of IL-12 p70 protein in concentrated lavage samples from small
numbers vims-infected and control rats 87
A-3 ELISA analysis of IL-12 p40 protein (homodimers and monomers) in concentrated
lavage samples from small numbers virus-infected and control rats 87
A-4 ELISA analysis of IFN-y in concentrated lavage samples 88
B-l The percentage of cells identified as macrophages, neutrophils, lymphocytes, and
epithelial cells in all treatment groups 91
B-2 Pneumonia indices of BN rats in all treatment groups 92
C-l Anti-Sense IL-12 p40 mRNA In Situ Hybridization of bronchiole wall in a F344 rat
at 5 days after Sendai inoculation 96
C-2 Anti-Sense IL-12 p40 mRNA In Situ Hybridization of inflammatory aggregate in the
bronchiole wall of an F344 rat at 3 days after inoculation 96
C-3 Sense IL-12 p40 mRN A In Situ Hybridization in the airway of a F344 rat 5 days
after inoculation 97
x

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE ROLE OF INTERLEUKIN-12 IN THE PATHOGENESIS OF SENDAI VIRUS-
INDUCED AIRWAY DISEASE
By
Amy Elizabeth Seymour Stone
December 2002
Chair: William L. Castleman
Major Department: Veterinary Medicine
Brown Norway (BN) rats are susceptible to Sendai virus-induced chronic airway
inflammation that results in fibrosis and functional abnormalities resembling asthma.
Fischer (F344) rats are resistant to these virus-induced changes and have earlier viral
clearance, increased expression of Th-1 cytokines (e.g., interferon-y, IFN-y), and do not
develop pulmonary function abnormalities. In contrast, BN rats are Th-2 type cytokine
responders (e.g., interleukin-4, IL-4) and have delayed viral clearance. Due to the critical
role of interleukin-12 (EL-12) in regulating the IFN-y cytokine response in intracellular
infections, I hypothesized that virus-resistant F344 rats are higher IL-12 gene responders
than BN rats. Levels of IL-12 p40 messenger (mRNA) were measured by real-time
polymerase chain reaction (RT-PCR) and IL-12 protein was detected by lung
homogenate enzyme-linked immunosorbent assay (ELISA) at several time points after
xi

viral inoculation. Although virus infection resulted in increased IL-12 production in both
strains, F344 rats had significantly more IL-12 p40 mRNA than BN rats at 0-3 days
(early) after virus inoculation (p<0.05). Furthermore, IL-12 total protein levels were
elevated in F344 rats as early as 2 days following viral challenge, and the numbers of EL-
12 p40 protein expressing cells were also significantly increased in their bronchioles at 2
and 3 days following Sendai inoculation (p<0.05). To evaluate the potential protective
role of IL-12 in virus-induced airway injury, BN rats were given IL-12 intraperitoneally
at either the time of (day 0) or two days after viral inoculation (day 2). In contrast to
infected rats given saline, infected rats treated with IL-12 at day 0 had 22.1% lower levels
of chronic airway inflammation, 23.8% lower levels of airway fibrosis, and 42% and
62.5% decrease in bromodeoxyuridine (BrdU)-labeled fibroblasts at 10 and 14 days after
inoculation respectively (p<0.05). Day 0 treated BN rats had a 4-fold increase in the
pulmonary IFN-y mRNA and a 77% increase in IFN-y protein as compared to saline-
treated, virus-inoculated controls. In contrast, day 2 IL-12 treatment induced a 20%
increase in bronchiolar airway wall thickness, a 12.5% increase in BrdU-labeled
fibroblasts at 14 days after inoculation, and an increase in pulmonary IL-4 mRNA
(p<0.05). The results are consistent with the hypothesis that resistance to virus-induced
airway damage in F344 rats is due, at least in part, to their high virus-induced IL-12 gene
expression.
Xll

CHAPTER 1
LITERATURE REVIEW
Interleukin-12
Interleukin-12 (IL-12) is a heterodimeric cytokine, which plays a role in the
induction of cell-mediated and T helper type-1 (Th-1, CD4+ T cells) immune responses
(1). This cytokine is produced primarily by antigen presenting cells in response to
intracellular bacterial, viral, protozoal, and fungal infections (1). The main function of
IL-12 is to direct the “cross talk” between the phagocytic antigen presenting cells and
effector lymphocytes by inducing the production of cytokines, particularly interferon-y
(IFN-y), and by enhancing lymphocyte cytotoxic activity (2). Directly or indirectly, IL-
12 is involved in the activation of macrophages, the generation and survival of Th-1 cells,
the generation of cytotoxic T lymphocytes (CTL, CD8+ T cells), and the suppression of
IgGl and IgE production (2). IL-12, synergistically with interleukin-2 (IL-2), tumor
necrosis factor-a (TNF-a), and interleukin-18 (IL-18) induces the maturation of Th-1
cells from Th-0 precursors by inducing the production of IFN-y from resting and
activated natural killer (NK) and T cells (1). IL-12 maintains high levels of IFN- y once
the CD4+ and CD8+ T cell types are established to resolve the infection (2). Additionally
IL-12 acts to prevent the outgrowth and development of Th-2 cells and their production
of Th-2 cytokines (3).
The structure of IL-12 is a unique heterodimer composed of two disulfide-linked
subunits, p35 and p40 (representing the approximate molecular weights) (4). These two
1

2
subunits are encoded on two unrelated genes residing at independent loci in the mouse,
human, and rat genomes (5-7). No sequence homology exists between the two subunits,
but the p35 subunit shares homology with interleukin-6 (IL-6), granulocyte colony
stimulating factor (G-CSF), and chicken myelomonocytic growth factor (8). The p40
subunit is not homologous to other cytokines, but is a part of the hemopoietin receptor
family most closely resembling the IL-6 receptor a-subunit and the ciliary neutrotrophic
factor receptor (5,9). Coexpression and covalent linkage of both chains of this cytokine
in the same cell is required for the generation of the functionally active heterodimer (10).
Expression of the p35 chain is ubiquitously constitutive. The secretion of free p35
subunit has not been demonstrated (11-12). Both subunits are induced by intracellular
infections via the subsequent activation of phagocytic cells (13). The p40 gene is located
only in IL-12 producing cells, and is produced at much higher levels than the p35 chain
or p70 heterodimer (11-12). The rate of p70 production is limited by p35 expression
because the p40 subunit is produced by phagocytic cells at a few-fold to 1000-fold higher
levels than the active heterodimer (14-15). Secretion of IL-12 p40 monomers and
homodimers can have an inhibitory effect on the expression and production of the p70
heterodimer in murine T and NK cells (10). The p40 homodimer may be acting as a
physiologic antagonistic regulator in the mouse system or may just be competing for the
IL-12 cellular receptor (2). In humans, the p40 homodimer has modest ability compete
with the heterodimer for IL-12 biological activity (16). Recently, human airway
epithelial cells have been shown to produce high levels of p40 subunit, the possible role
being to attract macrophages to the site of airway inflammation during mucosal defense
(17).

3
The predominately transcriptionally regulated p40 subunit gene spans 13 Kbp
containing eight exons preceded by a classical promoter (18,1). The murine p40 promoter
contains three essential transcription factor-binding sites, a nuclear factor kappa B (NF-
kB) site, a CCAAT enhancer-binding protein site (C/EBP), and an adaptor protein¬
binding site (AP-1), that are involved in lipopolysaccharide (LPS) and EFN-y promoter
activation (19-20). The human p40 promoter also has three essential cz's-acting elements
in its promoter, including a NF-kB site, an Ets (erythroblastosis virus oncogene
homologue) core element (Ets-2, interferon regulatory factor-1 [IRF-1], interferon
consensus sequence binding protein, and c-Rel), and a C/EBP site (18,21-22). The
regulatory mechanisms of this promoter appear to involve functional synergy between the
Ets and NF-kB transcription factors in the human gene, and the C/EBP factor also
interacts functionally with the NF-kB factor in the promoters of both species (21-22).
The ira/zs-acting proteins interacting with the binding sites mediating the activation of the
IL-12 p40 gene have not been well elucidated. However, a novel repressor element, GA-
12 binding protein (GAP-12), has been shown to reduce inducible IL-12 p40 gene
transcription in response to interleukin-4 (IL-4) and prostaglandin^ (PGE2) in human
monocytes (23).
Due to the constitutive expression of the IL-12 p35 gene in many tissue types, many
studies have focused on the p40 gene expression. However, p35 subunit production is
more tightly regulated as it appears to be controlled translationally and transcriptionally
(24). The murine p35 gene has multiple transcription start sites at either of two 5’ exons
resulting in mRNA iso forms with different untranslated regions (5’UTR) (25). The
human p35 gene also has multiple forms initiating from two separate exons, suggesting

4
that the p35 genes in both species are similarly regulated (14). Under non-stimulated
conditions, p35 transcripts contain an additional upstream ATG from a region whose
presence inhibits translation (24). Stimulated cells produce transcripts that lack this
upstream ATG that can be translated into p35 protein; the proportion of each set of
transcripts in vivo depends on the stimulus (14,24-25). The multiple transcription start
sites suggest the existence of two promoters, and the switch between the two may depend
on the initiating transcription factor (14). There are multiple NF-kB sites, IRF-1 sites,
and an AP-2 site within both promoter regions (14). Only the NF-kB, particularly c-Rel,
and the proximal most IRF-1 sites have proven critical for efficient transcription (13,14).
There are several other putative elements that may contribute to regulation, including
gamma-associated elements (GAS), IFN-stimulated response elements (ISRE), and
interferon consensus sequence binding sites (ICSBP) (14). Confusion regarding the gene
expression of IL-12 p35 remains, due to the inability of conventional methods, such as
northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), to
accurately distinguish between the at least four different mRNA isoforms.
The IL-12 receptor is composed of two subunits (IL-12Rpl and IL-12RP2) that are
members of the cytokine receptor super family most closely related to glycoprotein 130
(gpl30) (26). Coexpression of these two receptors is required for the formation of a
high-affinity IL-12 binding site in human cells; however in mouse cells the IL-12Rpi
subunit confers high and low-affinity binding (26-27). The IL-12RP2 subunit contains
three tyrosine residues and is essential for signal transduction in both species (26-28).
There is a 68% homology between the human and mouse receptors, and both are thought
to be regulated and expressed in a similar manner (26). Expression of EL-12Rp2 protein

5
may be limited to Thl cells and this expression may correlate with IL-12 responsiveness
(29-30). The IL-12R(31 subunit is present on resting NK and T cells and is up regulated
during activation (30). The IL-12R02 subunit is not present on resting CD4+ T cells but
is up regulated upon antigen activation of the T cell receptor (TCR) (30). Upon T cell
activation, the cytokines interleukin-4 (IL-4) and IFN-y can modify this subunit
expression. During T cell maturation, IL-4 can inhibit IL-12RP2 subunit expression, thus
losing the ability to respond to IL-12 after TCR binding (30). When low levels of IL-4
are present, IFN-y is required for IL-12RP2 expression (30). However, if there is no
detectable IL-4, the presence of IFN-y is not necessary (30). The IL-12Rp2 chain
appears also to be more highly regulated than the IL-12 Rpi by cytokines such as IL-10
and TGF-p, (31).
Once IL-12 is bound to its receptor, the complex induces the rapid tyrosine
phosphorylation of both Janus (JAK) and Tyrosine kinases (Tyk) (32). Following the
activation of the JAK and Tyk, three members of the signal transducers and activators of
transcription (STAT) family are phosphorylated and activated [STAT-1, STAT-3, and
STAT-4] (33-34). STAT-4 is not activated by any other cytokine except IFN-a in human
cells, so it is unique to the IL-12 signaling pathway (35). STAT-4 is directly involved in
the transcription of IFN-y, primarily through two binding sites in the first intron (36-37).
STAT-4 has been shown to be present and activated with translocation to the nucleus in T
cells, NK cells, and dendritic cells (38). STAT-4 knock out mice have an almost
identical phenotype to IL-12 or IL-12R knock out mice and produce no IFN-y in response
to IL-12 stimulation confirming this pathway in the IL-12 biological response (36,39).
Additionally, there is evidence that IL-12 can induce transcription of an Ets transcription

6
factor called ERM in differentiated Th-1 cells through the binding of the TCR (40). The
induction of this factor, although it does not activate STAT-4, is dependent on the
presence of STAT-4 within the cell (40). This pathway may regulate another aspect of
Thl behavior or may require cooperation to regulate IFN-y production (40).
The Role of Interleukin-12 in Regulating the Immune Response
The production of IL-12 in the immune response is complex and can be initiated by
several different pathways. Phagocytic cells, including dendritic cells, produce IL-12 by
T cell-independent and T cell-dependent means. Infectious agents, including bacteria,
bacterial products, both metazoan and protozoan parasites, fungi and viruses, induce the
production of IL-12 by phagocytic cells initiating the inflammatory process independent
of T lymphocytes (15, 41). Additionally during inflammation, but independent of
infectious agents or T cells, IL-12 production is induced by the interaction of adhesion
molecules with substrates of the inflammatory cascade, such as the interaction of the
CD44 adhesion molecule with low molecular weight hyalumonan (42). This mechanism
may contribute to macrophage activation due to the proinflammatory roles of IL-12 and
IFN-y (2, 42). The T cell-dependent mechanism of IL-12 production by antigen
presenting cells depends on the CD40/CD40 ligand interaction with activated T cells
(43). The antigen presenting cells induce IL-12 and up regulate receptors, such as B7 on
monocytes and dendritic cells, thus activating antigen specific T cells (43). During this
interaction, the p70 heterodimer is produced more or as efficiently as the p40 chain,
indicating the effective up regulation of both p35 and p40 peptide chains (14,24,43). The
T cells then produce IFN-y and the cytokine granulocyte/monocyte-colony stimulatory
factor (GM-CSF), which enhance the ability of the antigen presenting cells to produce IL-

7
12 (45-46). Two signals are required for both the T cells (CD40L and IFN-y) to produce
IFN-y and for most of the antigen presenting cells (innate signal from infection and
CD40L) to produce IL-12 in response to CD40 ligation (47). This bi-directional, two-
signal interaction functions to maintain the T cell-independent mechanisms of EL-12
production that initiated the inflammatory process (48).
The ability of these various pathways to induce IL-12 production is regulated by
both positive and negative feedback mechanisms. IL-12 induces the production of IFN-y
by T and NK cells; IFN-y then enhances the expression of IL-12 by phagocytic cells and
neutrophils (14,21,49). There are several other cytokines produced in response to EL-12
expression, such as TNF-a, GM-CSF, Interleukin-8 (IL-8), and interleukin-1(3 (EL-ip)
(43,50). TNFa and GM-CSF are also involved in positive feedback loops with IL-12.
Specifically, TNF-a enhances the ability of IFN-y to prime phagocytic cells for IL-12
production, and GM-CSF has a priming effect on IL-12 production from phagocytic cells,
primarily at the level of the p40 gene (51,52). Other Th-1 cytokines, such as IL-18, IL-2,
and IL-15, are costimulatory with IL-12 in the production of IFN-y. IL-2 or IL-12 alone
can stimulate the production of IFN-y from NK and T cells, but when acting together the
half-life of the IFN-y mRNA is doubled and IL-2 increases IL-12RP2 expression on
activated NK cells, thus enhancing the production of IFN-y (53, 54). IL-15 shares the
same biological functions with IL-2 and seems to interact with IL-12 through similar
pathways (55). IL-18 also acts with IL-12 to induce IFN-y in T cells and NK cells, but
this combination also is capable in vitro of stimulating IFN-y enhancement in mouse
dendritic cells and macrophages, suggesting the existence of an autocrine feedback loop
in these professional antigen presenting cells (55-57).

8
The counter mechanisms that down regulate the positive amplification of EL-12
production are mediated by EFN-y and they prevent uncontrolled cytokine production.
Interleukin-10 (IL-10), potent inhibitor of IFN-y production directly inhibits the
production of IL-12 by antigen presenting cells (58). IL-10 is able to block the
proliferation of Th-1 cells through the inhibition of IL-12 transcripts, other soluble
cytokines, and costimulatory surface molecules by antigen presenting cells (58). In both
mice and humans, IL-10 also prevents the development of mature, differentiated dendritic
cells (59-60). However, IL-12 induces the production of EL-10 in T cells and primes T
cell clones for high IL-10 production, thus inducing a negative feedback loop to reduce
its own expression (61). Other inhibitors of IL-12 include transforming growth factor-P
(TGF-P), PGE2, and partial inhibitors IL-4, TNF-a and IL-13 (62). Like IL-10, TGF-Pi
suppresses IL-12 at the transcriptional level; however it also appears to reduce the
stability of the IL-12 p40 mRNA (63). IL-4 and interleukin-13 (IL-13) can suppress IL-
12 expression when added simultaneously with a stimulus to cell cultures. However,
monocytes primed with IL-4 or IL-13 prior to stimulation can significantly enhance EL-12
expression (62, 64). These overlapping cytokine regulation pathways are postulated to be
extra backup mechanisms to prevent uncontrolled production of proinflammatory
cytokines.
Although B cells, neutrophils, microglial nerve cells, and macrophages produce
some IL-12, dendritic cells have been identified as extremely efficient producers of the
IL-12 that can act in inducing Th-1 responses upon antigen presentation (65-67). Due to
the heterogeneity of both human and mouse dendritic cells, it is not clear which type or
maturational state of dendritic cell is the major producer of IL-12. Human monocyte-

9
derived dendritic cells or myeloid dendritic cells (DC1) obtained from cultures treated
with GM-CSF and IL-4 produce high levels of the bioactive IL-12 p70 in response to
various stimuli (65). Alternatively, the plasmacytoid dendritic cells (lymphoid origin)
have been reported to produce lower levels of IL-12 (68). In the mouse, the CD8a-
positive dendritic cells, which may be comparable to the human plasmacytoid dendritic
cells, are more efficient producers of IL-12 when compared to myeloid dendritic cells
after intracellular infection in vivo (69). Dendritic cells from both species are unique
antigen presenting cells because 1) they produce bioactive IL-12 upon specific interaction
with T cells without additional stimuli; 2) their production of IL-12 is critical for optimal
proliferation and IFN-y production by activated Th-1 cells; 3) they prime resting, naive T
cells that, once restimulated, produce Th-1 cytokines (66). The ability of dendritic cells
to propagate Th-1 differentiation is due to their levels of STAT-4 (70). IL-4 can reduce
the amount of STAT-4 in maturing dendritic cells, reducing the amount of IL-12
dependent IFN-y produced as well as the Th-1 signaling capacity of these cells (70).
Once dendritic cells are mature, IL-4 can no longer inhibit the production of STAT-4
(70). Dendritic cells, depending on type and maturational status, are therefore capable of
initiating the antigen-activated immune response in the innate branch of the immune
system and then directing the Th-1 response to steer the humoral immune response.
Many parameters direct the development of Th-1 or Th-2 cells from a naive
precursor. These include the antigen presenting cells used for priming, the dose of
antigen encountered, the costimulatory cell-surface molecules, the genetic background of
the cells, and the cytokine milieu present in the environment (71). Changing one of these
factors can alter the resulting T cell phenotype. Ultimately the cytokines IL-4 and IL-12

10
act directly on T cells through STAT-6 and STAT-4 respectively to deliver final
differentiation signals (38,71). Naive CD4+ T cells are activated by interaction with
antigen presenting cells, primarily dendritic cells, through the TCR (30,66). This initiates
expression of the IL-12R(32 subunit and up-regulates the IL-12Rpi subunit already
present on the surface of T cells (30). The presence of even minute quantities of IL-4
will inhibit IL-12RP2 expression; however, the presence of IFN-y enhances this subunit’s
expression and can even reverse IL-4 inhibition (30, 71). The source of EFN-y is likely to
be NK cells, T cells, and IL-12 stimulated dendritic cells and macrophages (1, 38,57).
Once the capacity of T cells to respond or not to respond to IL-12 is established, IL-12
acts through STAT-4 via the IL-12R to determine the Th-1 or IFN-y producing phenotype
(39). This process is comparable between the human and mouse species except that
human Th-2 cells maintain their IL-12 responsiveness through low levels of IL-12P2
receptor subunit. In addition, IFN-a is as effective, and in some instances more effective,
an inducer of IL-12RP2 chain in human T cells (72). Generally, the choice between a
Th-1 and Th-2 phenotypic T cell response is dependent on the balance between the levels
of IL-4 and IL-12 during the maturation of naive T cells.
As previously noted, one of the main actions of IL-12 in the inflammatory response
to pathogens is to direct the development of Th-1 cells; however EL-12 has several other
very important functions. IL-12 is a potent inducer of IFN-y from CD4+ and CD8+ T
cells, NK cells, and y/5 T cells (2). IL-12 also directly enhances the cytotoxicity of NK
cells and CTLs by inducing the expression of genes encoding cytotoxic granules (e.g.,
perforin) and by endowing the CTLs with the ability to mediate antibody-redirected lysis
of target cells (2,73). These effects can be additive when synergistic cytokines such as

11
IL-2 and IFN-y are present, but IL-12 alone is capable of inducing these effects (2).
Additionally, IL-12 is the major factor required for the differentiation of CD8+ T cells
and y/8 T cells, priming them to be polarized to produce Th-lcyokines, similar to its
effect on their CD4+ counterparts (2). IL-12 can also function as an inhibitor of Fas-
mediated, non- B cell lymphoma oncogene-2- (BCL-2) -dependent T-cell apoptosis (2).
IL-12 is critical in the activation of macrophages through the Th-1 differentiation,
enabling them to produce bactericidal and anti-viral cytokines (67). Recent evidence
using infection models suggests that IL-12 is not only required to initiate these responses
but also maintains antimicrobial functions, such as the ability of memory T cells to
produce IFN-y at later stages of infection (3).
Investigations of the humoral side of the inflammatory response demonstrated that
IL-12 could suppress the production of Th-2 type antibodies (IgGl and IgE) and increase
the production (10- to 1000- fold) of Th-1 type antibodies IgG2a, IgG2b, and IgG3 (74).
This effect is mediated predominantly by the increased expression of BFN-y; however
removal of IL-12 alone also affects the type of antibodies produced (74). This regulation
is similar to the interaction of IL-12 with IL-4, in that if cells are primed or boosted, IL-
12 can modestly enhance the production of IgGl and IgE (74). Recent studies have also
shown that IL-12 increases the production of complement fixing antibodies. In addition
certain aspects of the complement cascade may directly modulate the production of IL-12
in various infections and delayed-type hypersensitivity reactions (75). IL-12 is at the
interface of all aspects of the immune response to intracellular infection with interacting
links between the innate and immune responses.

12
The Role of IL-12 in Infectious Diseases
IL-12 in Bacterial Infections
Intracellular infection of mice with Listeria monocytogenes has been used
extensively as a model to study the role of Th-1-dependent, cell-mediated immunity,
including the role of IL-12 in intracellular infections (76). L. monocytogenes resistant
mice, such as C57BL/6, have macrophages and dendritic cells with higher EL-12 (52%
more total IL-12 p70) producing capacities that activate NK and y/8 T cells to secrete
IFN-y, and promote the development of Th-1 immunity early during acute infection (77,
78). Additionally, administration of recombinant IL-12 (rIL-12) increases resistance to
this bacterium, and its antibody neutralization leads to increased bacterial susceptibility
(79). The role for IL-12 in sustaining the response in murine listeriosis is unclear (3).
The evidence for the critical need for early IL-12 production in these models is proven
whereas the evidence for late production is still controversial.
IL-12 is induced at high levels in response to Salmonella, mycobacteria, and other
bacterial components. Mice infected with attenuated S. dublin bacteria had increased IL-
12 production in the lymph nodes and Peyer’s patches and were protected against
subsequent infection with the virulent agent (79, 80). The emergence of a protective Th-
1 immune response is dependent on IL-12 in murine Mycoplasma tuberculosis infections
(2,79). As in Listeriosis, the addition of rIL-12 to susceptible B ALB/c mice increased
survival and delayed lung pathology (81). Blocking IL-12 with monoclonal antibodies
increases susceptibility in normally M. tuberculosis resistant mice, similar to the
phenotype observed in IL-12 p40 deficient mice (2, 81). The addition of rIL-12 to human
cell cultures increased the cytolytic activities ofNK and CD4+T cells against monocytes

13
infected with M. tuberculosis (82). Recombinant IL-12 also increases the proliferative
responses of peripheral blood lymphocytes and stimulates the antibacterial properties of
macrophages in patients with M. avium infection (83). Even the addition of a synthetic
oligonucleotide containing a palindromic sequence from mycobacteria, a sequence of
DNA from Escherichia coli, or the B subunit of E. coli can induce the expression of IL-
12 mRNA p40 and p35 in mouse splenocytes in cell culture and intestinal lymphoid
tissue in vivo (79). IL-12 has significant roles in other bacterial infections such as
Brucella abortus and Klebsiella pneumoniae, but research has not been as extensive in
these infections (79).
IL-12 in Protozoal Infections
The production of IL-12 is important in the initiation and the maintenance of the
Th-1 response in cutaneous and visceral leishmaniasis. The susceptibility of BALB/c
mice to the cutaneous, intracellular protozoan, Leishmania major, is in part due to the
genetic background of the T lymphocytes (84). Though the expression of IL-12 mRNA
and protein levels is similar between resistant and susceptible mouse strains, susceptible
BALB/c mice T cells lose the ability to generate IL-12-induced Th-1 responses and
instead form an IL-4-induced Th-2 response that is ineffective in clearing the pathogen
(84). The important role of IL-12 in resistant mouse strains, such as C57BL/6 and C3H,
seems to be its ability to function as a growth factor for Th-1 cells by the intensification
of IFN-y production and the suppression of IL-4 and EL-10 (79). Treatment of the
susceptible BALB/c mouse strain with IL-12 during the first week of infection with L.
major induces resistance to the infection with a shift in the immune system from Th-2 to
a Th-1 response and the cured animals are resistant to subsequent rechallenge (2). In

14
addition, during L. major infections resistant C3H mice upregulate the mRNA expression
of IL-12Rpl and -(32 subunits on CD4+and CD8+ T cells (85). In contrast, susceptible
BALB/c mice show no increase in IL-12 receptor subunits upon infection with L. major
(85). The antibody neutralization of IL-12 in Leishmania-resistmt strains converts these
mice to susceptibility (79).
The treatment of BALB/c mice with rIL-12 two weeks after L. major infection
abrogates its protective effects and can even enhance the expression of IL-4 (79). Thus,
it is difficult to reverse an already established Th-2 immune response. Alternatively, IL-
12 may be needed to maintain a Th-1 response in animals where a Th-1 response has
been initiated. Despite the development of a Th-1 response in IL-12 p40 knockout (IL-12
KO) mice transiently treated with rIL-12, the animals were unable to sustain a Th-1
response in the absence of IL-12 past the acute phase of infection (79, 86). These IL-12
KO mice in the absence of IL-12 treatment developed evidence of a Th-2 response (86).
IL-12 may also play an important role in visceral leishmaniasis in mice and humans. In
L. donovani mouse infections, in which susceptibility is associated with a failing Th-1
immune response, treatment with rIL-12 has been effective in reversing the disease
process (79). The addition of rIL-12 to cultures of human peripheral blood mononuclear
cells from patients with visceral leishmaniasis restores the proliferative and IFN-y
producing capacities of these cells (79). Therefore, in mice and in humans, IL-12 appears
to initiate and maintain cell-mediated immunity, as well as suppress the Th-2 response to
Leishmania infection.
The role of IL-12 in other protozoal infections appears to be quite similar to that in
leishmaniasis, but the differential expression of IL-12 is much greater. C3H mice have

15
twice the total IL-12 protein in serum and splenic tissues as compared to BALB/c mice in
response to Trypanosoma cruzi infection (87). This difference has been associated with
increased infection resistance due, most likely, to observed increases in NK cell
cytotoxicity and the levels of IL-12 dependent IFN-y protein (87). Additionally, rIL-12
treatment of the susceptible BALB/c mice led to resistance during the acute phase of the
disease, but was ineffective during the chronic infection (87). Cells from Toxoplasma
gondii infected, IL-12 KO mice, transiently treated with rIL-12, were unable to produce
IFN-y upon antigenic stimulation without the addition of IL-12 (3). There is evidence
that the Th-1 response was initiated and that Th-1 cells were developed, but the T cell
memory was not functional without IL-12 (3). These mechanisms, along with IL-12’s
ability to upregulate TNF-a, cell surface molecules, and to increase the phagocytic ability
of antigen presenting cells, are also important in resistance to the mouse protozoal
diseases Plasmodium chabaudi and Cryptosporidium parvum (79).
IL-12 in Fungal Infections
In many fungal infections, the establishment of a Th-1 type reaction is critical to
development of phagocyte dependent protection and the production of inhibitory
cytokines such as IL-4, IL-10, and the IgE antibody is associated with disease
progression (79). DBA/2 mice are genetically resistant to Coccidioides immitis and are
induced to produce five times more IL-12 p40 mRNA in their lungs as compared to C.
iww/tó-susceptible C57BL/6 mice (79, 88). Neutralization of IL-12 in the DBA/2 strain
by monoclonal antibodies to IL-12 leads to severe disease, and conversely administration
of rIL-12 to the fungus-susceptible strain (BALB/c) decreases susceptibility to clinical
disease progression (79).

16
Recombinant IL-12 administered at the time of murine Cryptococcus neoformans
infection results in protection from disseminated infection including pneumonia and then
meningitis (89). This effect seems to be mediated by an increase in the numbers of
pulmonary inflammatory cells, a decrease in the number of neural yeast cells, and
detectable IFN-y mRNA in the lungs of treated mice (89). Later administration of rIL-12
fails to protect these mice against dissemination of the infection with C. neoformans, with
no detectable pulmonary IFN-y mRNA (89). The role of EL-12 in human fungal
infections continues to be investigated due to the importance of human
immunodeficiency virus (HIV) related susceptibilities to opportunistic fungal infections.
IL-12 in Viral Infections
IL-12 plays an important role in viral defense; however its role is more complex
than in other types of intracellular infections. In murine viral infections such as murine
cytomegalovirus (MCMV), respiratory syncytial virus (RSV), influenza, and herpes
simplex virus (HSV), IL-12 is critical in the early activation of NK cells and the
establishment of a Th-1 antiviral immune response (2, 79, 90). During MCMV and RSV
infection, IL-12 p70 levels increase in serum (50% and four-fold respectively) at early
time points after infection (90, 91). However lymphocytic choriomeningitis virus
(LCMV) infection does not induce detectable IL-12 levels, but instead activates the T-
cell IFN-y responses through the IL-12 inhibitory cytokines IFN-aP (92). During LCMV
in the absence of EFN-ap the IL-12 response is inducible and indicates an alternative
pathway to NK cell activation and IFN-y production (92).
Low dose IL-12 administration has some protective effects in LMCV infection;
however higher doses can lead to decreases in T cell activity and increases in T cell

17
necrosis (92). Low doses of IL-12 increased NK cell cytotoxicity, CD4+ and CD8+ T
lymphocyte numbers, fFN-y production, and antiviral status (92). In other murine viral
infections, such as mouse hepatitis virus, encephalomyocarditis virus, and mouse
adenovirus infection rEL-12 administration induced protection (79). In mice transgenic
for the hepatitis B antigen, IL-12 suppressed autoantibody production (Th2 to Thl shift),
inhibited virus replication in the liver and kidneys, and increased EFN-y production (79).
In many of these infections peak IL-12 levels are noted 1-3 days (i.e., early) after
infection and are usually transient (2,79).
IL-12 may also play opposing roles in the outcome and/or associated pathology in
the same infection (79). In corneal HSV infection, local over-production of IL-12 leads
to a virus-specific Th-1 reactivity and immunopathologic disease (93). However, in
thermally injured mice, IL-12 promotes resistance to HSV infection (79). In humans, the
measles virus actually down regulates the expression of IL-12 in vivo. In human
monocyte cell cultures measles infection selectively impairs the expression of IL-12
without affecting other cytokines (94). This decreased IL-12 production is dependent on
the activation of the measles virus cellular receptor CD46, a regulator of the complement
gene cluster (94). Thus, it appears that there is some plasticity in the immune response to
viral infections depending on the genetics of the host, the cytokine environment, and the
type of viral pathogen.
IL-12 has also been significant in the pathology and possible treatment of HIV
infection in humans. During in vitro studies with peripheral blood mononuclear cells
(PBMC) and T cells from HIV-infected individuals, it was demonstrated that these cells
produce less IL-12 than non-infected controls (79). Although macrophages from these

18
individuals express low levels of IL-12, these cells do not respond to normal stimulation
(2, 79). IL-12 treatment in vitro has been shown to enhance NK cytotoxicity in HIV
infected cells, and is able to increase the cytotoxic activity of lymphocytes in non-
infected donors against HIV-infected target cells (2). IL-12 treatment has also improved
the ability for immune cells to recall antigens and to prevent T-cell-receptor-induced
apoptosis (79). The protective ability of IL-12 against the HIV virus may be to help
maintain the CD4+ T cell population from apoptotic destruction by shifting the T cells to
a Th-1 profile that is less permissive to HIV than the Th-2 cell type (2,79).
The Role of IL-12 in Allergy and Asthma
Asthmatic disease involves intermittent airway obstruction, bronchial smooth
muscle cell hyperreactivity to bronchoconstrictors, and chronic bronchial inflammation
(95). This persistent airway inflammation ultimately leads to remodeling of the airway
epithelial cells and the deposition of collagen by proliferating fibroblasts (95). The
primary lesion of asthma consists of the accumulation of CD4+ Th-2 cells and in some
cases eosinophils in the airway mucosa (95). The Th-2 cells direct the persistent
inflammation through the cytokines IL-4, IL-13, IL-5, and IL-9 (96). Although EL-4 is
the main cytokine responsible for Th-2 differentiation and the high IgE levels observed in
many asthmatics, IL-13 and IL-5 are involved in bronchoconstriction and eosinophilia
respectively (96). Chemokines such as RANTES (regulated upon activation, normal T-
cell expressed and secreted) eotaxin, and macrophage inflammatory protein la (MLP-la)
also act on eosinophils and T cells to enhance their recruitment and activation (95-96).
These inflammatory mediators have become proposed sites for therapeutic modulation
and have been studied in both human and animal models of allergic disease.

19
IL-12 appears to have an immunomodulatory effect on the predominantly Th-2
driven pulmonary inflammation in rodent (rat and mouse) models of allergic airway
disease and human asthma. IL-12 KO mice sensitized and challenged with ovalbumin
(OVA) have pronounced eosinophilic airway inflammation with enhanced IL-4 and TNF-
a levels in the bronchoalveolar lavage fluid (4). Recombinant IL-12 given
intraperitoneally in a murine model of ovalbumin-induced, allergic, airway inflammation
suppresses antigen-induced airway eosinophilia, circulating IgE levels, and airway
hyperresponsiveness in a dose dependent manner (97). The administration of rIL-12 was
timed during either allergic sensitization (early dosage) or the hypersensitivity of
inflammation in the lung (late dosage) (97-98). Early dosages or early and late dosages
combined were effective in C57BL/6 suppressing all signs of the asthma-like phenotype,
however the late doses alone were not as effective, especially in reducing IgE levels (97-
98). In addition, rIL-12 administration to rats by intraperitoneal dosing also inhibits
allergen-induced inflammation and the sensitization to allergens (99). Brusselle et al.
examined the mechanism of the inhibitory effects of IL-12 on airway inflammation using
IFN-y receptor deficient (IFN-yR-KO) mice (100). Recombinant IL-12 given by aerosol
to IFN-yR-KO and wild-type mice during sensitization inhibited airway eosinophilia and
specific IgE production in the wild-type mice and increased these parameters in IFN-yR-
KO mice possibly due to a more established Th-2 response (100). Similar to the previous
studies, rIL-12 given only during the hypersensitivity phase (late) inhibited the airway
eosinophilia, but not the circulating IgE in the wild-type mice (100). The inhibition of
eosinophil influx into the airways by IL-12 appears IFN-y dependent during initial
sensitization and IFN-y independent during the secondary allergic response. These

20
results suggest that endogenous and exogenous IL-12 play important roles in curtailing
the allergic response in the airways, and the timing of the expression is critical to
suppress the Th-2 response.
In humans, bronchial biopsies from asthma patients compared to normal controls
show decreases in the numbers of IL-12 producing cells, reduced airway EL-12 mRNA,
and a reduction in the ability of the PBMCs in vitro to produce IL-12 during stimulation
as compared to normal non-atopic controls (4). The expression of IL-12R02 on T cells of
asthmatics is also reduced, partly due to diminished production of IL-12 and enhanced
secretion of IL-4 by their PBMCs (101). Furthermore, there are intrinsic defects of the
CD4+ T cells, which reduce their ability to respond to IL-12 with IL-12R(32 expression
(101). Problematically, systemic administration of rIL-12 to human asthmatics has
several toxic effects such as general malaise/flu-like symptoms and cardiac arrhythmias
(99). Mucosal administration by airway aerosolization of rIL-12 in mouse and non¬
human primate models abrogates airway eosinophilia and airway hyperresponsiveness, as
well augmenting the expression of pulmonary EFN-y (4, 102). In addition, lung mucosal
IL-12 gene delivery via viral vectors prevents the development of allergic disease, airway
hyperresponsiveness, and suppressed established allergic responses (4,103). This form of
gene therapy also reversed the suppression of local antiviral cell-mediated immunity
resulting in rapid resolution of viral infection in previously susceptible mice (103). These
therapies may enable exogenous administration of rIL-12 to asthmatic individuals
without the side effects associated with systemic treatment.

21
Rodent Model for Virus-Induced Pulmonary Disease
Viral bronchiolitis during infancy (less than 1 year of age) in humans has been
associated with chronic airway dysfunction and may be a factor in the development of the
asthmatic phenotype (104-105). These pulmonary function abnormalities include
increased airway resistance and airway hyperresponsiveness to airway smooth muscle
agonists such as methacholine (104,106). Viral infections may be inducing persistent
structural abnormalities through direct inflammatory injury and repair mechanisms that
may lead to permanent structural abnormalities (107). Another factor in the development
of asthma is the association between elevated IgE levels and a predominating Th-2 type
cytokine response in some infants that develop asthma (108). Diminished IFN-y
production can be demonstrated in the cord blood mononuclear cells from infants with
increased risk of developing atopic diseases, such as asthma, and in the PBMCs of infants
that develop virus-associated airway function abnormalities (108). The asthmatic
phenotype then appears to be a combination of inheritable factors (cytokine dysregulation
and/or atopy) and environmental components (viral infections, allergens, and other lower
airway antigens).
Parainfluenza virus type 1 (Sendai) infection in weanling rats produces pulmonary
structural and functional abnormalities, such as bronchiolar hypoplasia and alveolar
dysplasia leading to increased airway resistance and hyperresponsiveness (109-110).
These abnormalities have been developed as an animal model of virus induced airway
disease with many features that resemble human asthma including; episodic, reversible
airway obstruction, airway hyperresponsiveness to methacholine, chronic airway wall
inflammation, and airway wall remodeling (111-113). Young rats infected with Sendai

22
virus develop severe bronchiolitis followed by pulmonary growth abnormalities,
including bronchiolar hypoplasia, alveolar dysplasia, and increases in bronchiolar airway
wall thickness (113). Sendai virus-induced increases in bronchiolar wall thickness are
due to increases in inflammatory cells (macrophages, mast cells, eosinophils and
lymphocytes), airway wall edema, fibroblast proliferation, and collagen and extracellular
matrix deposition (113). Previously, it has been determined that Brown Norway (BN)
rats are susceptible to virus-induced chronic inflammation and remodeling leading to
airway function abnormalities, whereas Fischer 344 (F344) rats are highly resistant to
these virus-induced effects (111).
The development of the Sendai virus-induced abnormalities may be related to the
initial immune response early after viral infection. The virus-susceptible BN rat strain
differs from the F344 in response to viral infection as it has a greater pulmonary
expression of the Th-2 cytokines IL-4 and IL-5, less IFN-y production, and fewer CD8+ T
lymphocytes at early time points after viral inoculation (112, 114-116). In addition, BN
rats have higher serum IgE levels with enhanced recruitment of airway mast cells,
eosinophils, and prolonged viral replication within the airways when compared to the
F344 rat strain (115-116). The immune effector cells from BN rats are also less
responsive to IL-12 stimulation. Splenocytes and NK cells from uninfected BN rats
secrete significantly less IFN-y upon stimulation with IL-12 or Sendai virus than
splenocytes and NK cells from uninfected F344 rats (117). In the viral repair process,
BN rats have increased and prolonged expression of the fibrosis-inducing cytokines TGF-
Pi and TNF-a in the macrophages surrounding the airways at 10 and 14 days after viral
infection (113, 118). Modulation of the immune response by the exogenous

23
administration of recombinant EFN-y, given 4 days before and during the first week of
viral infection, prevents the development of persistent airway inflammation, fibrosis, and
the associated chronic airway dysfunction in the BN rats (119). Thus, differences in the
genetic immune response (i.e., the cytokine response) to parainfluenza virus are critical in
determining whether chronic airway dysfunction and asthma-like disease will develop.
The above findings indicate that the cytokine response is an important component
of the Sendai virus-induced pulmonary damage associated with chronic airway
dysfunction. These results combined with the properties of IL-12 outlined in the previous
sections, indicate that F344 rats may be more resistant to Sendai virus-induced
bronchiolar damage and fibrosis because they produce higher levels of IL-12 early in
response to viral infection. The studies outlined here are designed 1) to investigate the
pulmonary expression of IL-12 in the F344 and BN rat strains in response to viral
infection and 2) determine if systemic treatment with IL-12 could abrogate or lessen the
severity of Sendai virus-induced bronchiolar inflammation and fibrosis that is associated
with airway dysfunction in BN rats.

CHAPTER 2
RESEARCH PLAN AND PROTOCOL
Hypothesis and Specific Aims
The goal of this research was to determine the role of interleukin-12 (IL-12) in the
development of resistance to chronic airway disease induced by parainfluenza (Sendai)
virus during early life. The hypothesis to be tested was that F344 rats are more resistant
to virus-induced airway damage and fibrosis because they produce high levels of IL-12
early in response to virus that up-regulates Th-1 cytokine responses, antiviral immunity,
and reduces airway fibrosis. There were four specific aims:
1) To compare the pulmonary IL-12 mRNA and protein responses of virus- resistant
F344 and virus-susceptible BN rats following Sendai virus infection.
2) To determine if F344 rats have greater numbers of pulmonary cells and differing
cell types that express IL-12 in response to Sendai virus infection than BN rats.
3) To determine if Sendai virus-induced airway damage in BN rats can be reduced by
IL-12 treatment early in the virus infection.
4) To compare the airway IL-12 p35 and p40 mRNA responses of virus-resistant F344
and virus-susceptible BN rats following Sendai virus infection (This specific aim is
contingent on the results from the second specific aim. If there is differential
expression of IL-12 in the dendritic cell or other cells types in the large airways
based on the results of the in situ hybridization and immunohistochemistry, then
this specific aim will be explored).
Background/Significance
Inbred rats differ in susceptibility to Sendai virus-induced chronic airway disease
that resembles human asthma. The BN rat strain is highly susceptible to this virus-
induced damage and produces a predominantly Th-2-type acute cytokine response to
virus with increased levels of IL-4 and IL-5 seven days after inoculation (115). In
24

25
contrast, the F344 rat strain is resistant and produces an earlier Th-l-type cytokine
response with high IFN-y mRNA levels three days after virus inoculation and higher IFN-
y protein levels in the bronchoalveolar lavage fluid seven days after inoculation (115).
BN rats have high serum IgE levels and persistent inflammation characterized by the
enhanced recruitment of mast cells and eosinophils in response to Sendai viral infection
(114,116,120). F344 rats have a higher CD8+ T cell response to Sendai infection and
have viral titers comparable to non-infected controls seven days after virus inoculation
(115-116). BN rats continue to have persistent airway inflammation with increased
numbers of macrophages expressing the pro-fibrotic cytokines TNF-a and TGF-Pi in
their airways at 10,14, and 30 days after inoculation (113,118). Additionally, BN rats
have virus-induced proliferation of fibroblasts in the bronchiolar walls chronically after
virus inoculation (118). These changes remodeling the airways are associated with virus-
induced increases in pulmonary resistance and hyperresponsiveness that persists for 28 to
65 days after inoculation.
IL-12 is a heterodimeric cytokine in the Th-1 cytokine group that is produced in
response to many intracellular infections including viral, protozoal, fungal, and bacterial
(1-3). This cytokine, produced mostly by antigen presenting cells (dendritic cells and
macrophages), promotes antiviral immune responses, in part by inducing IFN-y
production by immune effector cells (2). Differential IL-12 expression has been shown
to be important in susceptibility to intracellular pathogens in several rodent models (79).
Administration of recombinant IL-12 confers resistance in rodent and primate infectious
and allergic disease models (79,97-103). F344 rats have higher virus-induced pulmonary
IFN-y expression and increased NK and T lymphocyte responsiveness to IL-12 or Sendai

26
virus (117). Therefore, expression of IL-12 may play a major role in controlling the
difference in susceptibility to virus-induced airway inflammation, fibrosis, and
hyperresponsiveness in these two rat strains.
Gaps in Knowledge to Be Addressed by This Research
1) Is early IL-12 gene expression increased in the virus-resistant F344 rats as
compared to virus-susceptible BN rats in this model?
2) Can exogenous administration of IL-12 reduce the effects of virus-induced damage
and fibrosis in the pulmonary tissues?
3) Which cell types are responsible for producing IL-12, post-viral infection, in the
lungs?
4) Is the dendritic cell IL-12 response greater in the F344 rats early after Sendai virus
inoculation?
Research and Design Methods
Overview of Experiments and Schedule
Experiment 1: Comparison of IL-12 response in virus-susceptible BN rats and virus-
resistant F344 rats. (Specific Aims 1, 2, and 4) (Year 1 and 3)
Experiment 2: Can Sendai virus-induced airway damage be reduced in virus-susceptible
BN rats by exogenous administration of IL-12 early in viral infection? (Specific Aim 3)
(Year 2)
Experiment 1: Pulmonary Expression of IL-12 in Sendai Virus-infected BN and
F344 Rats
Objectives
1) To determine if virus-resistant F344 rats have higher levels of IL-12 pulmonary
expression in response to Sendai viral inoculation as compared to virus-susceptible
BN rats.
2) To determine the location and amounts of p70 protein in the pulmonary tissues in
the two rat strains.
3) To determine the level of the IL-12 synergistic cytokine, IL-18, mRNA in the
pulmonary tissues of both rat strains.

27
4) To compare the accumulation of dendritic cells, a primary IL-12 producing cell,
into the airways of both rat strains.
Rationale
Levels of IL-12 have not been compared in this rat model consisting of BN and
F344 inbred rat strains. It has been determined that F344 rats have higher EFN-y
expression than BN rats in response to viral infection and that IL-12 is a potent inducer of
IFN-y in many intracellular infections (119, 1-4). It is of interest to determine if the
levels in pulmonary tissue post Sendai viral infection differ between the two strains.
Previous studies have determined the protein expression of the IL-12 p70 heterodimer
locally in areas of intracellular infection (2, 79). Given that F344 rats have a higher
expression of IFN-y post viral infection, the IL-12 signaling pathway may be an
important factor in the antiviral response. This study is designed to determine the levels
and location (i.e., cell types) that are expressing IL-12.
IL-18 can act synergistically with IL-12 to induce and/or augment the production of
EFN-y. It has been shown to act specifically at the IFN-y promoter to augment production
from Thl cells (57). The combination of IL-12 and IL-18 together has been shown to
induce extremely high amounts of EFN-y protein (643-fold increase) and IFN-y gene
expression in NK cells above resting or non-activated human NK cells (55). It may be
possible that there is not differential expression of IL-12 in this model, but that an
augmenting cytokine like EL-18 is expressed at greater levels in the virus-resistant F344
rats.
Dendritic cells have been shown to be one of the earliest cells responders in the rat
airways after Sendai virus infection (121). Sendai virus initially replicates in airway
epithelial cells; the earliest IL-12 responses should be from intramural and epithelial

28
dendritic cells (121). Measurement of total lung mRNA may mute any differences at the
airway level by averaging all of the lung tissues together. Focused examination of
airway-specific expression may be required to elucidate these potentially important
differences in EL-12 (122). Previously most of the studies examining regulation of IL-12
have focused on the p40 subunit and its regulation at the transcriptional level in IL-12
producing cells. Recent studies have shown that in CD8+ dendritic cells the production
of IL-12 p70 heterodimer requires the induction of the p35 subunit (13). Since IL-12 p40
homodimers can bind to the IL-12RP2 subunit and antagonize IL-12 function, the
biological activity may be, at least in part, determined by the ratio of p70 to p40
homodimer. This indicates that p35 levels may be an important limiting factor in IL-12
production by dendritic cells (13). In this model it may important to examine the p40
subunit at the lung and airway level and the p35 subunit production to elucidate any
critical differences that may exist in IL-12 regulation between these two rat strains.
Design and Methods
Twenty-two day old weanling male BN and F344 rats were aerosol-inoculated with
Sendai virus or remained unexposed to Sendai virus (lung changes same as
chorioallantoic fluid exposed) in separate but equal housing, and studied at 0, 1, 2, 3, 5, 7,
10, and 14 days post inoculation.
Necropsy and tissue processing. The rats were anesthetized with sodium
pentobarbital (approximately 200pg/g body weight) or urethane (1.5g/kg body weight)
and killed by exsanguination via cardiac puncture. Lung lavages for ELISA were
performed through intratracheal cannulation with phosphate buffered saline (PBS). For
RT-PCR and ELISA, the lungs and tracheas were removed, immediately frozen in liquid

29
nitrogen, and then stored at -80°C until processed. Lungs and tracheas were removed and
fixed for 2 hours by tracheal perfusion with 4% paraformaldehyde-PBS (pH = 7.4) and
embedded in paraffin, for in situ hybridization. For immunohistochemistry lungs were
removed, inflated with O.C.T. embedding medium compound (Tissue-Tek®, Torrance,
CA), sectioned, and placed into molds. These lungs were then frozen immediately in
liquid nitrogen and then stored at -80°C until processing.
Table 2-1. Table of Experimental Design: Experiment 1
Assay
Sample
Reverse-Transcriptase
Polymerase Chain
Reaction (RT-PCR)
Immunohistochemistry
In situ Hybridization
RNA from BN and F344 rat
lungs and tracheas (frozen)/virus
exposed and non-infected
controls
Frozen lung sections from BN
and F344 rats/virus exposed and
non-infected controls
Paraffin lung and trachea sections
from BN and F344 rats/virus
exposed and controls
Enzyme-Linked
Immunosorbent Assay
(ELISA)
Lung homogenates and lung
lavage fluid from BN and F344
rats/virus exposed and controls
Purpose
Detection of differences in p35
and p40 IL-12 mRNA levels post
viral inoculation
Detection and location of cells
expressing IL-12 p40 protein and
the OX-6 dendritic cell marker
Location of the IL-12 p40
mRNA message within the
pulmonary tissues
Determination of the level of IL-
12 p70 and p40 in the total lung
tissue and secreted into the
airways
Dilutional and real-time RT-PCR. Frozen lungs and tracheas were weighed and
total RNA was extracted by phenol/chloroform extraction or using the RNeasy® midi kit
(Qiagen, Inc, Valencia, CA) (123,124). Preliminary pulmonary mRNA levels were
measured in a small number of rats using by 10-fold dilutional RT-PCR method with
RNA dilutions ranging from neat to 1:10,000 (125). The primers for IL-12 p40 and the
housekeeping gene product, hypoxanthine-guanine phosphoribosyltransferase (HPRT)
were designed from previously published sequences and all reactions were optimized for
temperature, Mg2+concentration, and primers (125,126) (Appendix A, Figure A-l).

30
Reverse transcriptase reactions and real-time PCR for IL-12 p35, IL-12 p40, IL-18
mRNA, and the housekeeping gene rat glyceraldehyde-3-phosphate dehydrogenase
(GADPH) were performed on total RNA extracted from the lungs and tracheas of
individual rats. The primers for each of these cytokines were TaqMan® pre-developed
assay reagents for gene expression quantification (Applied Biosystems, Foster City, CA).
Every time cDNA was synthesized, parallel TaqMan® assays were run for GADPH and
the target cytokine in separate wells (124).
Immunohistochemistry. Antibodies against IL-12 p40 and OX-6 major
histocompatibility complex (MHC), dendritic cell marker were used to detect bronchiolar
cell cytokine expression or to identify the cell type, respectively. The labeled cells were
quantitated and the numbers of positive cells were compared between treatment groups.
In situ hybridization. A portion of the dilutional IL-12 p40 PCR product was
subcloned and used as a template for the production of RNA probes (125, Appendix C).
The digoxigenin- IL-12 p40 labeled antisense riboprobe was constructed and used to
qualitatively determine which cell types were expressing p40 mRNA in lung and trachea
sections. Control sections were incubated with a labeled sense riboprobe. Northern
analysis was done to verify the success of the IL-12 p40 probe binding to rat RNA
samples with high levels of IL-12 p40 mRNA expression (Appendix C).
Enzyme-linked immunosorbent assay. The IL-12 p 70 protein levels were
determined preliminarily in concentrated lung lavage fluid samples (Millipore
Ultraffee®-4 centrifugal filter and tube Millipore Corporation, Bedford, MA) from
individual rats using an ELISA kit according to manufacturers instructions (murine IL-12
p70, Biosource International, Camarillo, CA) (Appendix A, Figure A-2). Due to the

31
ability of IL-12 p40 monomers and dimers to antagonize the effects of biologically active
IL-12 p70, the IL-12 p40 protein was measured in the lavage fluid using an ELISA kit
according to the manufacturers instructions (Quantikine® M Murine Mouse IL-12 p40
ELISA, R&D Systems, Minneapolis, MN) (17,125) (Appendix A, Figure A-3). Based on
the low levels of IL-12 p70 and p40 detected in concentrated lung lavage fluid ELISA
results, the same analyses were done using the supernatants from whole lung
homogenates from control and virus-infected rats of both strains.
Data analysis. The final quantitation of cytokine mRNA levels detected by real¬
time PCR was done using the comparative CT (cycle threshold) method and was reported
as relative transcription of the n-fold difference relative to a calibrator cDNA (LPS-
stimulated rat lung) (124). The density of protein labeling was assessed by counting and
identifying based on morphology the number of labeled cells per mm of bronchiolar
basement membrane. Group means for all assays were compared by one-way analysis of
variance (ANOVA) using a computer-based statistical program (Sigma-Stat, Jandel Corp.
San Rafael, CA). Kruskal-Wallis analysis of variance was used on ranks if normality test
of group means failed. In addition, t-tests, Wilcoxin-Mann-Whitney test, and Student-
Newman-Keuls method of pair wise multiple comparison procedures were used
depending on numbers of groups being compared as well as the variance within each of
the individual groups (113).
Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats
Objectives
1) To determine if administration of exogenous IL-12 early in viral infection increases
resistance to virus-induced chronic airway disease.

32
2) To determine the mechanism(s), if IL-12 administration does increase resistance,
by which IL-12 may be functioning to promote protection from virus-induced disease.
Rationale
The exogenous administration of recombinant IL-12 in numerous murine and
primate infectious disease models increases resistance to infection and/or to the
pathogen-associated, disease phenotype. Intraperitoneal (IP) administration of rLL-12 has
been the method used in many of these studies, additionally local and mucosal rIL-12
treatment has also been effective at the sites of pathogenesis (79,102-104). In most
disease models the administration of IL-12 directly, by the production of DFN-y, drives
the development of the Th-1 type immune response with simultaneous suppression of the
Th-2 immune response (2, 79).
IL-12 is elevated most often early in the disease process, particularly in viral
infections where peak IL-12 levels are noted 1-3 days after infection (2,79). The timing
of the cytokine treatment has been shown to be important in both the infectious models
and the allergic disease models (79, 97-98). If the Th-2 response or allergic sensitization
has already occurred, then IL-12 may actually serve to accentuate or reverse these
responses (79, 97-98). Once the T lymphocytes have lost the ability to respond to IL-12,
possibly due to the absence of the IL-12RP2 subunit, altering the immune response
appears to be very difficult (29, 30).
In the murine model of Leishmaniasis IL-12 the administration of neutralizing
antibody converts normally Leishmania-resisXanX C57BL/6 and CH3 mice to
susceptibility, and rIL-12 treatment confers resistance to the Leishmania-resistmt
BALB/c strain (79,127). The important role for IL-12 in resistant strains seems to be its
ability to function as a growth factor for Thl cells by the intensification of IFN-y

33
production and to suppress the production of IL-4 to undetectable levels (79,127). IL-12
treatment within our model could act in a similar manner increasing IFN-y levels and
reducing IL-4 levels after Sendai virus infection.
Preliminary results have shown that IL-12 p40 mRNA levels are elevated in the
early days post viral infection. This is consistent with previous studies in this model, in
which IFN-y mRNA and protein levels also peak at early time points (119). IFN-y has
been shown to be a potent mediator of the Th-1 response and treatment of BN rats before
and early after Sendai infection prevents post viral chronic bronchiolitis (119). This
experiment is designed to determine if early exogenous administrations of IL-12 to BN
rats will up regulate Th-1 type and reduce chronic airway fibrosis and remodeling.
Design and Methods
Twenty-two day old weanling male BN were aerosol-inoculated with Sendai virus
or remained unexposed to Sendai virus (lung changes same as chorioallantoic fluid
exposed) in separate but equal housing, and studied at 0, 3, 7, 10, and 14 days post
inoculation (DPI). IL-12 treatment groups were injected IP with 3pg of mouse
recombinant EL-12 or a comparable volume of saline at the time of virus inoculation (0
DPI) or two days after inoculation (2 DPI) (Biosource International, Camarillo, CA) (See
Figure 2-1). If rats were in the 10 or 14 days post inoculation, they were injected IP at
200 mg/g of rat with 5-bromo-2-deoxyuridine (BrdU)(Sigma, St. Louis, MO) twelve
hours prior to necropsy.

34
Table 2-2. Table of Experimental Design: Experiment 2
Assay Sample
Reverse-Transcriptase
Polymerase Chain
Reaction (RT-PCR)
Immunohistochemistry
Differential Cell Counts
Histology and
Bronchiole Wall
Morphometry
Enzyme-Linked
Immunosorbent Assay
(ELISA)
Viral Plaque Assays
RNA from BN rat lungs
(frozen)/virus-exposed and non-
infected controls with and
without rIL-12 treatment
Paraffin lung sections from BN
rats /virus exposed and non-
infected controls with and
without IL-12 treatment
Lung lavage fluid from BN rats
/virus-exposed and non-infected
controls with and without rIL-12
treatment
Paraffin lung sections from BN
/virus exposed and non-infected
controls with and without IL-12
treatment
Lung homogenates and lung
lavage fluid from BN rats/virus
exposed and controls with and
without rIL-12 treatment
Lung BN rats (frozen)/ virus
exposed with and without rIL-12
treatment
Purpose
Detection of differences in IFN-
y, IL-18, and IL-4 mRNA levels
after IL-12 treatment
Detection and evaluation of
fibroblast proliferation in the
bronchiolar walls
Determination of total numbers
of cells and types in BAL
Evaluate the extent of
inflammation and fibrosis in the
bronchioles
Determination of the level of IL-
12 p70 and p40 in the total lung
tissue and secreted into the
airways
Determination of the levels of
virus remaining in the treatment
groups at various time points
IL-12 or Saline Treatment of
BrdU Histology to
Identify
Fibrotic Cells in
the Airways
10 and 14 Days DPI
BN Rats (3ng IP)
0 or 2
Days After Virus inoculation
or Non-infected Controls
Injected
on Same Day
Airway Morphometry
to Compare the
thickness of Airways
10 and 14 Days DPI
V
Histology to
Characterize Airway
Inflammation
And Fibrosis
14 Days Pl
RT-PCR to Measure
IFN-y mRNA
0, 3, 7 Days DPI
Real-Time PCR to
Measure
IL-4 or IL-18 mRNA
0, 3, 7,10,14 Days DPI
Viral Titers to
Determine Viral
Clearance
7 Days DPI
Figure 2-1. Experimental design diagram for experiment 2

35
Necropsy and tissue processing. The rats were anesthetized with sodium
pentobarbital (approximately 200pg/g body weight) or urethane (1.5g/kg body weight)
and killed by exsanguination via cardiac puncture. Lung lavages for ELISA and cells
counts (differential and total) were performed through intratracheal cannulation with
phosphate buffered saline (PBS). For RT-PCR and ELISA, the right lungs were
removed, immediately frozen in liquid nitrogen, and then stored at -80°C until processed.
The left lungs were tied off, removed and fixed for 2 hours by tracheal perfusion with 4%
paraformaldehyde-PBS (pH = 7.4) and embedded in paraffin for immunohistochemistry
and histology.
Real-time and competitive RT-PCR. Reverse transcriptase reactions and real¬
time PCR for IL-18 mRNA and GADPH were performed on total RNA extracted from
the lungs of individual rats. The primers these cytokines were TaqMan® pre-developed
assay reagents for gene expression quantification (Applied Biosystems, Foster City, CA).
The primers and TaqMan probe for BN rat IL-4 were designed using the Primer Express
software (Applied Biosystems, Foster City, CA). The sense and antisense primers were
made in the mRNA sequence to ensure discrimination between cDNA and genomic
DNA. Dilutional curves were evaluated to assure that the efficiency of the IL-4 primers
compare to the GADPH primers. Every time cDNA was synthesized, parallel TaqMan®
assays were run for GADPH and the target cytokine in separate wells (124).
IFN-y was detected in lung tissue by a competitive RT-PCR method (126). Primers
for IFN-y and HPRT were constructed from published sequences and each assay was
optimized for temperature, Mg2+concentration, and primers (126). For each cDNA
sample reactions comparisons were made to 0, 0.5, 5, and 50 femptograms (fg) of a

36
competitive fragment (126). PCR products were stained with ethidium bromide and
separated electrophoretically on 1.5-% agarose gels.
BrdU immunohistochemistry. Transverse sections were taken from the fixed left
lungs of rats treated with BrdU: at the hilus, between the hilus and the cranial lobe
margin, and at the same distance from the hilus to the caudal lobe margin.
Immunohistochemical staining using the antibody to BrdU was preformed and the
numbers of BrdU-labeled fibroblasts per mm of bronchiolar basement membrane were
counted.
Histology and bronchiolar wall morphometry. Serial hematoxylin and eosin
(H&E) stained, paraffin sections were scored for airway wall inflammation and fibrosis.
Bronchioles were scored as positive for inflammation if the wall had five or more
inflammatory cell types (eosinophils, lymphocytes, or macrophages). Bronchioles were
scored as positive for fibrosis/remodeling if the walls were thickened with increased
fibroblasts and deposition of collagen. Collagen was identified using Masson’s
Trichrome and Manuel’s Reticulin stains. Additionally, the area of the bronchiolar wall
from the bronchiolar epithelial basement membrane to basement membrane of the
surrounding alveolar walls was measured. Bronchiolar wall area was divided by the
perimeter of bronchiolar basement membrane to calculate the thickness of the wall
(square micrometers of bronchiolar wall per micrometer of bronchiolar basement
membrane) (113).
Enzyme-linked immunosorbent assay. The IFN-y protein levels were determined
in concentrated lung lavage fluid (Millipore Ultrafree®-4 centrifugal filter and tube
Millipore Corporation, Bedford, MA) samples from individual rats using an ELISA kit

37
according to manufacturers instructions (rat IFN-y, Biosource International, Camarillo,
CA). Preliminary analysis using concentrated lung lavage fluid samples detected an
increase in levels of IFN-y after viral infected, but no significant differences in the IL-12
treatment groups (Appendix A) (Figure A-4). Based on the minimal effects detected in
concentrated lung lavage fluid, the same analyses were done using the supernatants from
whole lung homogenates from individual animals in the same treatment groups.
Virology. Viral titers, to determine viral clearance, were measured in homogenates
of frozen lung, using a standard plaque assay, and expressed as plague forming units/g
lung tissue (128).
Data analysis. The final quantitation of cytokine mRNA levels detected by real¬
time PCR was done using the comparative CT (cycle threshold) method and was reported
as relative transcription of the n-fold difference relative to a calibrator cDNA (LPS-
stimulated rat lung) (124). The competitive RT-PCR data are reported as non-normalized
mRNA abundance in competitive fragment units (119). Fibroblast proliferation was
assessed by counting the number of BrdU-labeled fibroblasts (identified due to spindled
shape) per mm of bronchiolar basement membrane. Group means for all assays were
compared by one-way analysis of variance (ANOVA) using a computer-based statistical
program (Sigma-Stat, Jandel Corp. San Rafael, CA). Kruskal-Wallis analysis of variance
was used on ranks if normality test of group means failed. In addition, several pair wise
multiple comparison procedures were used depending on numbers of groups being
compared as well as the variance within each of the individual groups (113).

CHAPTER 3
INCREASED EXPRESSION OF PULMONARY INTERLEUKIN-12 (IL-12) IN
SENDAI VIRUS-RESISTANT F344 RATS
Summary
Brown Norway (BN) and Fischer (F344) rats differ in their susceptibility in early
life to Sendai virus-induced persistent airway inflammation, chronic airway remodeling,
and airway hyperresponsiveness. These characteristics, as well as other phenotypic
characteristics serve as an experimental model of virus-induced asthma. Virus-
susceptible BN rats mount a predominantly Th-2 cytokine response (IL-4-dominated) to
Sendai virus, whereas virus-resistant F344 rats respond to infection with a Th-1 cytokine
pattern (IFN-y-dominated). F344 rats are more efficient in clearing the virus and in
resisting the induction of chronic airway lesions. We hypothesized that an earlier and
more robust IL-12 response was responsible for the differing IFN-y expression and viral
resistance of F344 rats. IL-12 mRNA and protein expression were evaluated by real-time
PCR, ELISA, and quantitative immunohistochemistry for IL-12 positive dendritic cells in
the lungs and tracheas of BN and F344 rats. F344 non-infected control rats had higher
pulmonary EL-12 p40 mRNA levels than the non-infected control BN rats. Virus-induced
increases in IL-12 p40 mRNA were detected as early as 2 days after inoculation, while
virus-induced increases in IL-12 p40 mRNA were not detected in BN rats until 3 days
after inoculation. F344 rats had higher concentrations of IL-12 total in the lung than BN
rats at 2 days after inoculation. Virus-induced increases in bronchiolar OX-6 positive and
IL-12 p40 dendritic cells were observed as early as 2 days following inoculation in F344
38

39
rats. No dendritic cell response was detected in BN rats. These results indicate that
resistance to the sequelae of Sendai virus infection by F344 rats is associated with their
earlier and higher production of IL-12.
Introduction
Viral bronchiolitis in infant children can be associated with chronic bronchiolar
dysfunction and is implicated as an important risk factor for the development of asthma
and other airway abnormalities (129,130). A rat model of virus-induced bronchiolar
damage has been developed in weanling and neonatal rats that has characteristics similar
to human asthma. These characteristics include episodic and reversible bronchiolar
obstruction, bronchiolar hyperresponsiveness to methacholine, chronic bronchiolar
inflammation (lymphocytes, macrophages, eosinophils, and mast cells) and bronchiolar
wall remodeling (111-113). This model can be used to examine the possible direct roles
that viral infections have in pulmonary function abnormalities and what genetic factors
may be contributing to the susceptibility to viral sequelae during early life (119).
Interleukin-12 (IL-12) has been associated with resistance to intracellular infections
(2). IL-12 is produced by antigen presenting cells, particularly dendritic cells, during
infection and induces IFN-y production by natural killer (NK) and cytotoxic T cells
(CD8+) (2,57). Through this direct up-regulation of IFN-y, EL-12 drives the development
of T helper-1 (Th-1) type immune response with simultaneous suppression of the T
helper-2 (Th-2) immune response without the requirement for IFN-y (1-3). IL-12 directly
increases the cytotoxic killing capacities of NK and cytotoxic T cells (2). The IL-12 p35
and p40 subunits covalently link to form the biologically active p70 heterodimer (2).
Many cells constitutively express the p35 chain, however the p40 gene is expressed only

40
in IL-12 producing cells (10). The expression of both chains is induced upon
intracellular infection and the subsequent CD40 ligand binding of antigen presenting cells
(14, 47).
Differential expression of IL-12 has been shown to be important in intracellular
infections in several rodent models. DBA/2 mice are genetically resistant to C. immitis
and produce five times more IL-12p40 mRNA in their lungs as compared to C. immitis-
susceptible C57BL/6 mice (79,88). Neutralization of IL-12 in the DBA/2 strain leads to
severe disease. Administration of rIL-12 to another fungus-susceptible mouse strain
(BALB/c) decreases susceptibility to clinical disease (79). Similarly, C3H mice that are
resistant to the protozoan T. cruzi have twice the IL-12 total protein in serum and splenic
tissue as compared to susceptible BALB/c mice. This differential IL-12 response has
been associated with increased resistance and an increased IFN-y protein response (87).
Additionally, resistant C57BL/6 mice have 52% more IL-12 total protein in splenic
dendritic cells early in murine listeriosis than susceptible BALB/c mice (78). Finally,
C57BL/6 mice have a four-fold greater increase in pulmonary IL-12 expression in
response to respiratory syncytial virus and mild disease as compared to DBA/2 and
BALB/c mice which have lower IL-12 levels and develop increased bronchiolar
hyperactivity and mucus production leading to a more severe disease process (91).
In viral infections such as mouse choriomeningitis (MCMV), comeal herpes
simplex vims and murine influenza, IL-12 is critical in the early activation and
maintenance of the Th-1 immune response (2,90,93). In many of these responses, IL-12
acts through up-regulation of IFN-y, which has been shown to control viral infections and
tissue alterations associated with exaggerated repair mechanisms, such as those

41
associated with bleomycin and immune complex-induced lung injury (90,131-132).
Recently it has been determined that the p40 chain of IL-12 may also play a role in
mouse Sendai viral infection systemically as a macrophage chemoattractant, as well as at
a local level where it is produced in by bronchiolar epithelial cells (17). IL-12 also has
been shown to inhibit bronchiolar hyperresponsiveness and bronchiolar eosinophil
recruitment in several rodent and primate models of allergic sensitization (79,97-98).
As previously noted, it has been determined that Brown Norway (BN) rats are
susceptible to Sendai virus-induced inflammation and remodeling, whereas Fischer 344
(F344) are highly resistant to the viral effects (111). The development of the Sendai
virus-induced abnormalities may be related to the early immune response after viral
infection. BN rats have greater expression of interleukin-4 (IL-4) and decreased levels of
IFN-y production, high mast cell and eosinophil response, fewer CD8+ T lymphocytes,
and prolonged levels of viral replication in comparison to F344 rats (119). BN rats also
have increased mRNA expression of profibrogenic cytokines TGF-Pi and TNF-a at 10
and 14 days after Sendai virus infection (113,118). Treatment of Sendai virus-inoculated
BN rats with EFN-y results in a reduction of virus-induced bronchiolar inflammation,
bronchiolar fibrosis, and ultimately results in less severe pulmonary dysfunction (119).
Given the critical role that IL-12 plays in inducing IFN-y in the regulation of the Th-1
immune response and its differential expression in several models of intracellular
infection, we hypothesized that F344 rats are more resistant to Parainfluenza type-1
(Sendai) virus-induced bronchiolar damage and fibrosis because they produce higher
levels of IL-12 early in the response to viral infection. The objectives of this study were

42
to determine the quantity of pulmonary IL-12 mRNA and protein expression and to
determine the structural and cellular location of this expression in the airways of both rat
strains after Sendai virus infection.
Materials and Methods
Animals
Weanling (22 days old), male, pathogen-free BN/RijHsd (24 rats) and F344/NHsd
(24 rats) rats were purchased from Harlan Sprague Dawley, Inc. Madison, WI and
Indianapolis, IN respectively. The control and virally infected animals were housed
separately in adjacent, identical Micro-Isolator VCL-HDâ„¢ individually HEP A
filtered/ventilated cages (#10419ZTGA Zytemâ„¢ plastic Micro-Isolatorâ„¢ system, Lab
Products, Inc. Seaford, DE). The University of Florida Animal Care and Use Committee
approved all procedures.
Viral Procedures and Sample Collection
The rats were inoculated with aerosolized parainfluenza (Sendai) virus type 1 strain
P3193 (117). The numbers of rats used for each experimental technique are indicated in
the figures located within the results section. Briefly, rats in the virus-inoculation group
were exposed to an aerosol of virus at a concentration of 1-3 plaque-forming units (PFU)
per ml of gas via a Glas-Col Aerosol Exposure Apparatus for 15 minutes (Tri-R) (Glas-
Col, Terre Haute, IN). At 0, 1, 2, 3, 5, 7, 10, and 14 days after inoculation, rats from each
group were immobilized via deep anesthetization with sodium pentobarbital
(approximately 200pg/g body weight) or urethane (1,5g/kg body weight) and killed by
exsanguination via cardiac puncture. Lung lavages were performed through intratracheal
cannulation with phosphate buffered saline (PBS). The lungs taken for homogenization

43
and RT-PCR were frozen in liquid nitrogen and stored at -80°C. The lungs and tracheas
used for in situ hybridization were tied off, perfused with 4% paraformaldehyde - PBS
(pH 7.4) (30 cm H20 pressure for 2 hours) and embedded in paraffin. Finally, the lungs
used for immunohistochemistry tied off, inflated with O.C.T. embedding medium
compound (Tissue-Tek®, Torrance, CA), sectioned, and placed into molds. These
tissues were stored at -80°C until processing.
Cytokine mRNA
Frozen lungs were weighed and RNA was extracted by a phenol/chloroform
method or by using the RNeasy® midi kit (Qiagen Inc, Valencia, Ca) (121,122). The
RNA samples were pre-treated with DNase I using the Deoxyribonuclease I,
amplification grade kit (Invitrogen, Carlsbad, CA) to remove genomic DNA. The
Reverse Transcriptase (RT) reactions were preformed using the Advantageâ„¢ RT-for-
PCR Kit (Clontech Laboratories, Inc, Palo Alto, CA). Polymerase chain reaction (PCR)
primers and probes for rat IL-12 p40, rat IL-12 p35, rat interleukin (IL-18), and for the
housekeeping gene rat glyceraldehyde-3-phosphate dehydrogenase (GADPH) were
TaqMan® pre-developed assay reagents for gene expression quantification (Applied
Biosystems, Foster City, CA). Every time cDNA was synthesized, parallel TaqMan®
assays were run for GADPH and the target cytokine in separate wells. The PCR
reactions contained 900 nM of each primer, 250 nM of the TaqMan probe, PCR
Mastermix (TaqMan Universal PCR Mastermix, Applied Biosystems) containing 10 mM
Tris-HCl, 50 mM KC1, 5 mM MgCh, 2.5 mM deoxynucleotide triphosphates, 0.625 U
AmpliTaq Gold DNA polymerase per reaction, 0.25 U AmpErase UNG per reaction, and
2 pi of the cDNA sample in a final volume of 25 pi. The samples were amplified in an

44
automated fluorometer (ABI Prism 7700 Sequence Detection System, Applied
Biosystems). Amplification conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of
15 s at 95°C and 60 s at 60°C. Final quantitation was done using the comparative Cx
(cycle threshold) method and was reported as relative transcription of the n-fold
difference relative to a calibrator cDNA (LPS-stimulated rat lung) (Table 3-1) (124).
Table 3-1 Comparative Cx Methoc
of cDNA Relative Quantitation
Average Cx
Average
GADPH CT
ACt
AACx
O-AAC
Z x
Target
cytokine Cx
average from
3 consecutive
wells
GADPH CT
average from 3
consecutive wells
(Same sample as
target cytokine)
Target CT -
GADPH CT
ACx - ACt Calibrator
Calibrator = LPS
Stimulated rat lung
cDNA
Relative
difference to
the calibrator
Protein Analysis
Enzyme-linked immunosorbent assay (ELISA)
Lungs were harvested as previously described and homogenized in cold PBS with a
protease inhibitor (Protease Inhibitor Cocktail Tablets, Mini Complete, Boehinger
Mannheim, Germany) at a ratio of 0.1 g of tissue/ml (133). The homogenates were
centrifuged at 4°C at 2000 rpm for 10 minutes and the supernatants were frozen at -80°C
until use. The total IL-12 (p70 heterodimer protein, p40 monomers, and p40 dimers) in
the lung lavage and homogenate was determined using an ELISA kit (murine IL-12,
Biosource International, Camarillo, CA) according to the manufacturer’s instructions.
Due to the ability of IL-12 p40 monomers and dimers to antagonize the effects of
biologically active IL-12 p70 and the potential roles of IL-12 p40 observed in mouse
Sendai viral infections, the IL-12 p40 protein was measured in the lung homogenates
(17,127). The level of IL-12 p40 protein was determined using a mouse ELISA kit
(Quantikine® M Murine Mouse IL-12 p40 ELISA, R&D Systems, Minneapolis, MN).

45
These results were compared to the IL-12 total protein levels to determine the
contribution of the protein forms of IL-12.
Immunohistochemistry
Frozen lung sections from BN and F344 rats were dried at room temperature and
then fixed in cold acetone for 10 minutes at 4°C. After washing in 1 X PBS, the
endogenous peroxidases were blocked using 1% hydrogen peroxide rinse for 10 minutes.
The slides were then blocked with either normal goat serum (IL-12 p40 assay) (Santa
Cruz Biotechnology ABC Staining Systems, Santa Cruz, CA) or normal mouse serum
(OX-6, detection of dendritic cell assay) (Santa Cruz Biotechnology ABC Staining
Systems, Santa Cruz, CA) for 1 hour in a humidified slide chamber at room temperature.
In the IL-12 p40 assay, the sections were incubated with either polyclonal, goat, anti¬
mouse IL-12 p40 (indicator of IL-12 p70 cellular production) (Santa Cruz Biotechnology,
Santa Cruz, CA) at 3.5 pg/pl or goat immunoglobulin G (IgG) (Sigma, St. Louis, MO) at
0.25 pg/pl in a humidified slide chamber, overnight at 4°C. In the OX-6 (major
histocompatibility complex determinant on B lymphocytes, dendritic cells, some
macrophages, and certain epithelial cells) assay, the sections were incubated under the
same conditions with either monoclonal, mouse anti 1-A (OX-6) (Serotec, Raleigh, NC)
at 0.02 pg/pl or mouse IgG (Sigma, St. Louis, MO) at 0.2 pg/pl. In both assays antibody
binding was detected using an avidin-biotin, chromogen diaminobenzidine system
according to the manufacturer’s instructions (Santa Cruz Biotechnology ABC Staining
Systems, Santa Cruz, CA). The density of cells expressing the EL-12 p40 protein was
determined by counting and classifying the number of labeled cells per millimeter (mm)
of bronchiolar basement membrane in an average of 10.3 mm/section. Round cells with

46
central round/oval nuclei and abundant cytoplasm were classified as macrophages, and
cells with round/ asymmetrically placed nuclei with foamy, dendritic cytoplasmic
extensions were classified as dendritic cells. The density of OX-6 positive cells was
determined similarly with an average of 12.3 mm/section being counted.
Data Analysis
For all experiments, group means were compared by one-way analysis of variance
(ANOVA) using a computer-based statistical program (Sigma-Stat, Jandel Corp. San
Rafael, CA). Kruskal-Wallis analysis of variance was used on ranks if the normality test
or equal variance tests of group means failed. Multiple comparison procedures were used
to isolate the group or groups that differ from others. The Student-Newman-Kuel’s test
was used if the sample sizes were equal; otherwise Dunn’s test was used to compare
groups of unequal sample size.
Results
Virus-Resistant F344 Rats Have Increased Expression of Pulmonary IL-12 mRNA
Relative amounts of IL-12 p40 mRNA were significantly higher in the lung tissue
of the F344 rats compared to the BN rats beginning at the non-infected control level
(p<0.01) (n = 4) (Figure 3-1). The expression of IL-12 p40 mRNA in the F344 rats
remained significantly higher at 1,2, and 3 days after viral inoculation with the greatest
difference being 3.4-fold at 2 days after virus inoculation (p<0.02) (Figure 3-1).

47
Figure 3-1. Real-time PCR analysis of IL-12 p 40 mRNA in whole lung samples of BN
and F344 rats after Sendai virus infection. (S = significant difference between strains,
p<0.04) (v = significant virus-induced difference compared to strain control, p<0.01)
There was a virus-induced increase of IL-12 p40 mRNA in both strains of rats,
however it reached statistical significance earlier in the F344 strain (2 days after
inoculation, p<0.01). Relative amounts, of the more constitutively expressed IL-12 p35
mRNA, were also significantly increased in the lungs of F344 rats at control, 2 and 3
days after inoculation (p<0.03) (n = 2) (Figure 3-2). However, there was not a virus-
induced increase in either strain above the non-infected controls. The expression of IL-
12 p35 mRNA significantly decreased as analyzed by real-time PCR (Figure 3-2).
The same trends were detectable in mRNA IL-12 p40 expression at the airway
level. IL-12 p40 mRNA expression was increased 3.7-fold in the F344 tracheas at 2 days
after inoculation as compared to the BN tracheas, and a virus-induced increase in IL-12
p40 mRNA expression was observed only in the F344 strain (p<0.03) (n = 4-7) (Figure
3-3). There were no IL-12 p35 mRNA expression differences detected at the

48
airway/tracheal level between rat strains or induced by virus (p>0.05) (n = 4-7) (Figure 3-
4).
Figure 3-2. Real-time PCR analysis of IL-12 p35 mRNA in whole lung samples of BN
and F344 rats after Sendai virus infection, (s = significant difference between strains,
p<0.03) (v = significant virus-induced difference compared to strain control, p<0.04)
Days After Inoculation
Figure 3-3. Real-time PCR analysis of IL-12 p40 mRNA in trachea samples of BN and
F344 rats after Sendai virus infection, (s = significant difference between strains,
p<0.03) (v = significant virus-induced difference compared to strain control, p<0.03)

49
Days After Inoculation
Figure 3-4. Real-time PCR analysis of IL-12 p35 mRNA in trachea samples of BN and
F344 rats after Sendai virus infection.
F344 Rats Have Increased Pulmonary IL-12 Protein After Sendai Virus Infection
As measured by ELISA, the F344 rats had 86 pg/ml ± 12.72 of pulmonary IL-12
total protein as compared to 54.8 pg/ml ± 5.82 in the BN strain (p<0.05) (n = 6-7) (Figure
3-5). The levels of IL-12 total protein increase with viral infection over the non-infected
controls in the F344 rats at all time points measured and in the BN rats on days 1, 3, and
5 after Sendai inoculation (Figure 3-5) (p<0.05).
In both BN and F344 rats the concentration of pulmonary IL-12 p40 protein
(monomers and homodimers) was approximately half that of the total IL-12 (Figure 3-6,
y-axis). Strain differences in pulmonary IL-12 p40 protein were only detected in the non-
infected controls (p<0.05) (n = 6-7) (Figure 3-6). Concentrations of IL-12 p40 protein
increased only in the BN strain with viral infection starting at 2 days after viral
inoculation but never reached statistical significance (p>0.05) (Figure 3-6).

50
140
0 1 2 3 5 7
Days After Inoculation
Figure 3-5. ELISA analysis of IL-12 total protein in F344 and BN rats strains after
Sendai virus inoculation, (s = significant difference between strains, p<0.05) (v =
significant virus-induced difference compared to strain control, p<0.05)
_ 30
E
g 25
c
jB 20
2
o'151
n
CM
10 -
- 5 ^
0
m BN
a F344
Mean ± SEM
n=6-7
1 2 3 5 7
Days After Inoculation
Figure 3-6. ELISA analysis of IL-12 p40 protein in F344 and BN rats strains after Sendai
virus inoculation. (S = significant difference between strains, p<0.05)
F344 Rats Have Increased Numbers of IL-12 p40 Expressing Cells in the
Bronchioles After Sendai Virus Inoculation
Density of OX-6 positive dendritic cells was significantly increased in the
bronchioles of F344 rats at 2 and 3 days after virus inoculation as compared to BN rats
(p<0.03) (n = 4) (Figures 3-7 and 3-8). Dendritic cell numbers in the F344 rats increased

51
87.5% above the non-infected controls and almost 2-fold above the BN rats at 2 days
after inoculation. There were no significant virus-induced increases in the numbers of
bronchiolar dendritic cells measured in the BN rats at any of the time points after
inoculation.
Figure 3-7. OX-6 immunohistochemistry in the bronchiole of a F344 rat three days after
inoculation (54X Magnification). There are several OX-6 positive dendritic cells,
indicated by dark brown staining, located within the bronchiole wall.
E
E
«5
O
o
'¡i
XI
c
®
o
Days After Inoculation
Figure 3-8. Density of OX-6 positive dendritic cells in the bronchioles of F344 and BN
rats. (S = significant difference between strains, p<0.03) (v = significant virus-induced
difference compared to strain control, p<0.01)

52
IL-12 p40 protein was expressed in dendritic cells and macrophages in the
bronchioles of both strains. The average number of IL-12 p40 positive cells/mm of
bronchiole wall was significantly higher in the F344 rat strain at 2 and 3 days after
inoculation as compared to the BN strain (p<0.03) (n = 4) (Figures 3-9 - Figure 3-11).
Days After Inoculation
Figure 3-9. Density of IL-12 p40 positive dendritic cells (S = significant difference
between strains, p<0.03) (v = significant virus-induced difference compared to strain
control, p<0.01)
jamm i y .
«3
Figure 3-10. IL-12 p40 immunohistochemistry in the wall of bronchiole of a F344 rat at
two days after Sendai inoculation (108X Magnification). Several inflammatory cells are
indicated by dark brown staining inflammatory cells (macrophages and dendritic cells)
within the airway wall.

53
- o
Mean ± SEM
n=4
J-1S
V
ii
0 2 3
Days After Inoculation
Figure 3.11. Density of IL-12 p40 positive macrophages in the bronchioles of BN and
F344 rats, (s = significant difference between strains, p<0.03) (v = significant virus-
induced difference compared to strain control, p<0.01)
F344 and BN Rats Do Not Have Detectable Differences in the Expression of IL-18
mRNA
Due to the co-stimulatory role of IL-18 with IL-12 in response to intracellular
infections, we examined the IL-18 mRNA expression in the lung tissues of both rat
strains (1). There were no differences detected by real-time PCR in the pulmonary IL-18
mRNA expression between BN and F344 rats at the time points measured (p>0.05) (n =
4-5) (Figure 3-12). Additionally, Sendai virus-induced increases in IL-18 were not
detected in either strain (Figure 3-12).

54
10
8 -
wm BN
â–¡ F344
MeaniSEM
n=4-5
P>0.05
Q)
O
c
(1)
I
0)
>
ra
o
O'
0 1 2 3 7 10
Days After Inoculation
Figure 3-12. Real-time PCR analysis of IL-18 mRNA expression in lung samples of BN
and F344 rats after Sendai virus infection.
Discussion
The purpose of this study was to determine whether there is differential expression
of IL-12 between rat strains that are susceptible and resistant to the pulmonary sequelae
of Sendai virus infection. The critical role of cytokines expressed acutely following virus
infection in this model and the importance of IL-12 in the response to intracellular
infections made this an important component of this disease model to investigate. The
results indicate that there are significant differences in total pulmonary IL-12 p40 and
p35 mRNA and IL-12 total protein levels expressed between virus-resistant F344 and
virus-susceptible BN rats following Sendai virus infection. There are also significant
differences in the numbers of cells expressing IL-12 p40 protein in the bronchioles
between these two strains.
This data indicates that total EL-12 protein expression increases earlier than the
expression of IL-12 p40 mRNA. This discrepancy may be due to several factors in the

55
measurement of protein and mRNA. Although both were measured in lung homogenates,
the blood and serum proteins are more likely to have remained viable after processing for
the ELISA and could have contributed to the concentrations of total IL-12 protein.
Additionally, RT-PCR, using conventional primers, is unable to detect differences in the
isoforms of IL-12 p35 mRNA (Chapter 1). There are differences in the isoforms of IL-12
p35 mRNA expressed depending on whether the production is constitutive or pathogen-
induced (14). Primers specific for the 5’untranslated region of the p35 gene can only
distinguish the untranslatable form. Additionally, there can be several translatable
isoforms with slight variations in this region. So, there may be more exaggerated
differences in IL-12 p35 expression (both virus-induced and between strains) than we are
able to detect with these primers. Therefore, it is difficult to make comparisons between
IL-12 mRNA expression and the IL-12 total protein levels.
Two pathways have been established for the production of IFN-y in viral infection.
Some infections, such as lymphocytic choriomeningitis virus (LCMV), have an IL-12
independent induction of IFN-y through IFNa/p pathway with very low, non-inducible,
levels of IL-12 expression (92). In addition, differential expression of the co stimulatory
cytokine IL-18 has been shown to be critical to the level of IFN-y induced in many
models of intracellular infection (55-57, 79, 92). In the model examined here, both rat
strains have virus induced increases in the level of IL-12 p40 mRNA and IL-12 total
protein with no changes in IL-18 expression, suggesting that IFN-y levels are being up
regulated by IL-12-dependent mechanisms in Sendai viral infection.
We also wanted to determine if the differences expressed at the total lung level
were consistent with the inflammatory response at the airway level. The IL-12 p40

56
immunohistochemistry (an indicator of IL-12 p70 cellular protein expression) results do
indicate that bronchiolar macrophage and dendritic cell protein expression is higher at
early time points in the F344 rats following Sendai virus inoculation. Therefore, the
differential expression of IL-12 does appear to be evident throughout the lung, even at the
local airway level. Additionally, the levels of pulmonary IL-12 p40 protein (homodimers
and monomers) measured in whole lung homogenates indicate that the possible role of
over-produced IL-12 p40 protein as an IL-12R(32 antagonist is likely not a factor in this
model. However, other possible roles of IL-12 p40 protein, such as a chemoattractant for
macrophages, have not been elucidated by these methods.
P.G. Holt and others have established that a network of resident airway epithelial
dendritic cells exists in throughout the respiratory tract of rodents and humans, where
these cells process antigen and initiate the generation of protective local immune
responses (121,134-135). The most prominent populations are present in the conducting
airways, such as the trachea (600-800/mm2), and decrease further down the respiratory
tract (75/ mm2) (134). This population also appears to be dynamic with changes
observed at steady state and increases seen in the trachea after Moraxella catarrhalis
bacteria and Sendai virus exposures (121, 134). These numbers are much higher than the
dendritic cell numbers that we were able to detect at 2 and 3 days after inoculation in the
bronchioles. However, bronchiolar airways were not singled-out in previous experiments
(more focus on the interalveolar septal junctions) and the peak time point for tracheal
dendritic cell numbers was not observed until 5 days after inoculation (134).
Additionally, the experiments examining the lower airways were performed hours (not
days) after the exposure to pathogen before the dendritic cells would be migrating from

57
the local site of inflammation to the regional lymph nodes (134). Similar to the results
seen in previous studies, our results show an increase in dendritic cell numbers after
Sendai infection in the F344 rat strain.
An increasing amount of evidence suggests that dendritic cell population changes
and cytokine expression may be a factor in the susceptibility to allergic respiratory
disease (135). Examination of dendritic cells in the lungs of several rat strains suggests
that the Th-2 polarity of the resting mucosal immune system may also be a property of
the resident dendritic cell population (121, 136). Initiation of the Th-1 immune response
in these cells requires appropriate costimulation (such as CD40 ligation) from the
microenvironment (136). Based on our results, a change in IL-12 expression is occurring
at the whole lung and airway levels. F344 rats have virus-induced increases in dendritic
cell numbers and increased expression of IL-12 p40 in their bronchioles by both
macrophages and dendritic cells as compared to BN rats early after Sendai infection. The
levels of IL-12 p40 mRNA are also increased in the tracheas of the F344 strain at 2 and 3
days after Sendai inoculation. However, in order to assess whether the IL-12 mRNA
expression is from cells (macrophages and dendritic cells) already present or from a
cellular influx further experimentation will need to be done to compare the magnitude of
the response in the airways of both strains. A component of the F344 rat strain’s
resistance to Sendai virus -induced airway damage may be that their dendritic cells and
macrophages receive an earlier signal from the microenvironment to become Th-1 type
cells secreting more IL-12 as compared to the BN rat strain. Further examinations of the
airway dendritic cells, their interactions with the local airway mucosal immune system,
and the magnitude of this response are needed to confirm this possibility.

58
The differential expression of IL-12 appears to be a factor in the resistance to
Sendai virus-induced airway disease. This differential expression appears to occur early
after infection and may be the source of the establishment of a predominately Th-1
cytokine response in the F344 rat strain. Similar to this rat model, paramyxoviral
(respiratory syncytial virus and parainfluenza virus) infections during infancy are
potential risk factors for the development of asthma in children (104-108). Additionally,
cytokine imbalance during early life has been considered to be an important risk factor in
the development of asthma and atopy (117). The differences in IL-12 expression by
these rat strains in response to Sendai virus infection may give insight into the
mechanisms of asthma development and potential asthma therapies in children.

CHAPTER 4
EXOGENOUS INTERLEUKIN-12 (IL-12) ADMINISTRATION REDUCES THE
SEVERITY OF SENDAI VIRUS-INDUCED CHRONIC AIRWAY FIBROSIS AND
REMODELING IN BN RATS
Summary
Sendai virus infection in virus-susceptible BN rats causes persistent bronchiolar
inflammation and fibrosis that is associated with increased airway resistance and airway
hyperresponsiveness. In contrast, F344 rats have earlier viral clearance, increased IL-12
pulmonary expression, and are resistant to post viral airway function abnormalities. This
study determined whether the exogenous administration of interleukin-12 (IL-12) could
confer resistance to Sendai virus-induced airway disease in virus-susceptible BN rats.
BN rats were treated with 3pg of recombinant IL-12 (rIL-12) or an equivalent volume of
saline intraperitoneally (IP) at the time of virus inoculation (day 0) or two days after virus
inoculation (day 2). Proliferating fibroblasts were labeled with bromodeoxyuridine
(BrdU) and detected by immunohistochemical staining. In comparison to infected rats
given saline, infected rats treated with rIL-12 at day 0 had 22.1% lower levels of chronic
airway inflammation and 23.8% lower levels of airway fibrosis as detected by
histological criteria. Rats treated on day 0 with r IL-12 had a 42% and 62.5% decrease in
BrdU-labeled fibroblasts in their bronchioles at 10 and 14 days after inoculation
respectively as compared to saline-treated virus-inoculated controls (p<0.05). There was
a 4-fold increase in pulmonary IFN-y mRNA and a 77% increase in pulmonary IFN-y
protein detected in the lungs of day 0 treated rats when compared to the virus-inoculated,
saline-treated control rats (P<0.05). In contrast, day 2 rIL-12 treatment induced a 20%
59

60
increase in bronchiolar airway wall thickness and a 12.5% increase in BrdU-labeled
fibroblasts at 14 days after inoculation (p<0.05). Day 2 treatment resulted in increased
pulmonary IL-4 mRNA levels compared to saline-treated virus-inoculated controls
(p<0.05). In conclusion, early IL-12 treatment reduces Sendai virus-induced bronchiolar
inflammation and fibrosis in virus-susceptible BN rats. This effect may be mediated, in
part, by the induction of IFN-y.
Introduction
Parainfluenza type I (Sendai) virus infection in rats is an animal model of virus-
induced airway abnormalities with similar characteristics to childhood asthma, such as
increased airway resistance and hyperresponsiveness (109-112). Previous studies have
demonstrated that virus-resistant Fischer (F344) rats have increased expression of
pulmonary interleukin-12 (IL-12), as well as increased numbers of IL-12 producing cells
(dendritic cells and macrophages) in their bronchioles as compared to virus-susceptible
Brown Norway (BN) rats after Sendai virus infection (Chapter 3). The biological
significance of this increased IL-12 expression in the F344 strain has not been
established.
The heterodimeric cytokine, IL-12 is produced by antigen presenting cells during
intracellular infections to up-regulate cell-mediated immune responses and the T helper-1
(Th-1) cytokines, principally interferon-y (IFN-y)(l-3). IL-12 works to enhance the
cytotoxic properties of natural killer (NK) cells and cytotoxic CD8+ T lymphocytes
(CTLs) (2). Furthermore, IL-12 can down-regulate the T helper type-2 (Th-2) cytokine
response by decreasing the production of interleukin-4 (IL-4) and Th-2 type antibodies
(2). Increased expression of IL-12 in animal models of infectious as well as allergic

61
disease has conferred resistance to the particular pathogenic phenotype (79, 97-98). The
exogenous administration of IL-12 to susceptible animals at certain time points during
infection or sensitization has also been shown to provide resistance in many of these
models (79, 97-98).
In Sendai virus rat model, F344 rats have an early Th-1 type immune response to
infection with higher EFN-y production and their NK and CTL cell types have an
increased capacity compared with the BN strain to produce IFN-y in response to infection
(115, 117, 119). Due, at least in part, to the higher IFN-y expression F344 rats have a
greater CD8+ T cell response, earlier pulmonary viral clearance, and a reduced capacity to
develop airway fibrosis after viral infection (115, 119). However, BN in rats produce
Th-2 type cytokines, such as IL-4, interleukin-5 (IL-5), and the profibrotic cytokines
tumor necrosis factor-a (TNF-a) and transforming growth factor-P-1 (TGF-Pi) after
virus infection (113, 115, 118). This response to Sendai infection involves the
persistence of airway inflammation (macrophages, lymphocytes, and eosinophils),
delayed viral clearance, and chronic bronchiolar fibrosis (112-113). Treatment of BN
rats with IFN-y reduced the amount of chronic bronchiolar inflammation and fibrosis,
thus protecting them from pulmonary function abnormalities (117).
Based on the previous IL-12 studies (Chapter 3) and the IFN-y treatment results, we
investigated the possible protective role of IL-12 in this rat model. We hypothesized that
the lower IL-12 response during acute viral infection is an important factor in the
development of the Sendai-induced post viral sequelae in BN rats. The objective of this

62
study was to determine if the administration of exogenous IL-12 at early time points
during Sendai virus infection would prevent the development of post viral persistent
bronchiolar inflammation and fibrosis.
Materials and Methods
Animals
Weanling (22 days old), male, pathogen-free BN/RijHsd rats (94 total rats) were
purchased from Harlan Sprague Dawley, Inc. Madison, WI. The control and virally
infected animals were housed separately in adjacent, identical Micro-Isolator VCL-HDâ„¢
individually HEPA filtered/ventilated cages (#10419ZTGA Zytemâ„¢ plastic Micro-
Isolatorâ„¢ system, Lab Products, Inc. Seaford, DE). The University of Florida Animal
Care and Use Committee approved all procedures.
Viral Procedures and Sample Collection
The rats were inoculated with aerosolized Sendai virus strain P3193 five days after
arrival. Briefly, rats in the virus-inoculation group were exposed to an aerosol (Tri-R
Aerosol Exposure Apparatus, Glas-Col, Terre Haute, IN) of virus at a concentration of 1-
3 plaque-forming units (PFU) per ml of gas for 15 minutes. At 0, 3, 7, 10, and 14 days
after inoculation, rats from each group were immobilized via deep anesthetization with
sodium pentobarbital (approximately 200pg/g of body weight) or urethane (1.5g/kg body
weight) and killed by exsanguination via cardiac puncture. Lung lavages were performed
through intratracheal cannulation with phosphate buffered saline (PBS). The right lungs
were frozen in liquid nitrogen and stored at -80°C. The left lungs were tied off, perfused
with 4% paraformaldehyde - PBS (pH 7.4) (30 cm H2O pressure for 2 hours) and
embedded in paraffin. Viral titers were measured in homogenates of frozen lung at seven

63
days after inoculation, by plaque assay using Madin-Darby bovine kidney cells as
described previously, and expressed as plaque forming units (pfu)/g lung tissue (128).
IL-12 Treatment Protocol
Groups of BN rats (n=8/group) were treated with mouse recombinant EL-12p70
(rIL-12) (Biosource International, Camarillo, CA). The dose of 3pg was determined to
be the lowest dose administered intraperitoneally (IP) that provided a measurable
biological response (Appendix B). IP injections of 3 pg of rIL-12 were given to control
and infection groups of rats on the day of viral inoculation (3 hours after inoculation)
(day 0) and to control and infection groups 2 days (day 2) after inoculation. A
comparable volume of sterile saline was given to a separate control and infection groups
IP as a negative control at the same time points. Groups of BN rats were injected IP at
200 pg/g of rat with 5-bromo-2'-deoxyuridine (BrdU)(Sigma, St. Louis, MO) 10 and 14
days after inoculation twelve hours prior to necropsy to detect epithelial and stromal cell
fibrosis.
Cytokine mRNA
Frozen lungs were weighed and RNA was extracted by phenol/chloroform
extraction or using the RNeasy® midi kit (Qiagen Inc, Valencia, Ca) (121,122). The
RNA samples were pre-treated with DNase I using the Deoxyribonuclease I,
amplification grade kit (Invitrogen, Carlsbad, CA) to remove genomic DNA. The
Reverse Transcriptase (RT) reactions were preformed using the Advantageâ„¢ RT-for-
PCR Kit (Clontech Laboratories, Inc, Palo Alto, CA). Polymerase chain reaction (PCR)
primers and probes for rat interleukin-18 (IL-18), and for the housekeeping gene rat
glyceraldehyde-3-phosphate dehydrogenase (GADPH) were TaqMan® pre-developed

64
assay reagents for gene expression quantification (Applied Biosystems, Foster City, CA).
The primers and TaqMan probe for BN rat IL-4 were designed using the Primer Express
software (Applied Biosystems, Foster City, CA). The sense and antisense primers were
made in the mRNA sequence to ensure discrimination between cDNA and genomic
DNA. The probe was labeled at the 5'-end with the reporter dye FAM (6-
carboxyfluorescein) and at the 3' end with a minor groove binder (TaqMan® MGB) and a
non-fluorescent quencher prevent extension by AmpliTaq Gold DNA polymerase
(Forward Primer 5’-CAGGGTGCTTCGCAAATTTT-3’; Reverse Primer 5’-
CGAGAACCCCAGACTTGTTGTT-3 ’; and Probe 5’- TCCCACGTGATGTACCTCCGTGCTT-3 ’).
Dilutional curves were evaluated to assure that the amplification efficiency of the IL-4
primers compared to the efficiency of the GADPH primers. Every time cDNA was
synthesized, parallel TaqMan® assays were run for GADPH and the target cytokine in
separate wells. The PCR reactions contained 900 nM of each primer, 250 nM of the
TaqMan probe, PCR Mastermix (TaqMan Universal PCR Mastermix, Applied
Biosystems) containing 10 mM Tris-HCl, 50 mM KC1, 5 mM MgCh, 2.5 mM
deoxynucleotide triphosphates, 0.625 U AmpliTaq Gold DNA polymerase per reaction,
0.25 U AmpErase UNG per reaction, and 2 pi of the cDNA sample in a final volume of
25 pi. The samples were amplified in an automated fluorometer (ABI Prism 7700
Sequence Detection System, Applied Biosystems). Amplification conditions were 2 min
at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 60 s at 60°C (124).
IFN-y was detected in lung tissue by a competitive RT-PCR method as described
previously (126). Primers for IFN-y and for the housekeeping gene product,
hypoxanthine-guanine phosphoribosyltransferase (HPRT) were prepared as described by

65
the Interdisciplinary Center for Biotechnology and Research at the University of Florida
(125). Each assay was optimized for temperature, Mg2+concentration, and primers. For
each cDNA sample reactions comparisons were made to 0, 0.5, 5, and 50 femptograms
(fg) of a competitive fragment (126). PCR reactions were performed at cycler programs
consisting of 1 minute at 94°C, annealing temperature of 56°C for 15 minutes, 72°C for 2
minutes for 4 cycles. Then 36 cycles were run at the same temperatures and times except
for a 2-minute annealing time at 56°C. PCR products were stained with ethidium
bromide and separated electrophoretically on 1.5-% agarose gels. The data are reported
as non-normalized mRNA abundance in competitive fragment units (117).
Enzyme-Linked Immunosorbent Assay (ELISA)
Lungs were harvested as previously described and homogenized in cold PBS with
a protease inhibitor (Protease Inhibitor Cocktail Tablets, Mini Complete, Boehinger
Mannheim, Germany) at a ratio of O.lg of tissue/ml (133). The homogenates were
centrifuged at 4°C at 2000 rpm for 10 minutes and the supernatants were frozen at -80°C
until use. IFN-y protein in the lung homogenates was determined using a rat IFN-y
ELISA kit (rat IFN-y, Biosource International, Camarillo, CA) according to the
manufacturer’s instructions.
BrdU Immunohistochemistry
In previous studies, Sendai virus-induced fibroblast proliferation has been detected
by BrdU incorporation beginning at 9 days after inoculation (113). Paraffin sections of
lung at 10 and 14 days after Sendai inoculated rats were deparaffinized in xylene,
rehydrated through a graded series of ethanol washes, and washed in distilled water.
Immunohistochemical staining to detect BrdU labeling was performed using a method

66
previously described (137). Briefly, slides were placed in 3.0% H2O2 for 10 minutes to
quench endogenous peroxidase, washed twice in PBS, and pretreated in both 2N HC1 (30
minutes at 37°C) and 0.1% w/v trypsin in PBS (20 minutes at 37°C). Sections were
rinsed in PBS, covered with antibody diluent (1.0% BSA and 0.5% Tween 20 in PBS)
(Sigma, St. Louis, MO) for 30 minutes to block nonspecific binding, blotted and
incubated overnight at 4°C covered in mouse anti-BrdU monoclonal antibody (Sigma, St.
Louis, MO) diluted 1:10 in antibody diluent. The next day the sections were washed in
PBS and covered with peroxidase-conjugated, goat, anti-mouse IgG (Fc specific) (Sigma,
St. Louis, MO) secondary antibody for one hour at room temperature. Bound peroxidase-
conjugated antibody was detected by development in the chromogen diaminobenzidine
(Sigma, St. Louis, MO) in 0.02 mg/ml in 0.25 mol/L Tris, pH 7.6, and 0.01% H2O2 for 7-
20 minutes as monitored with light microscopy. An average of 17 bronchioles per rat
(range 10-24) at 10 days after-inoculation and 14 bronchioles per rat (range 9-19) at 14
days after-inoculation were examined. Fibroblast proliferation was assessed by counting
the number of BrdU-labeled fibroblasts per mm of bronchiolar basement membrane.
Analysis of Bronchiolar Inflammation and Fibrosis
Previously studies have found that virus-induced bronchiolar fibrosis and collagen
deposition is present in BN rats by 14 days after-viral infection (113). Serial paraffin
sections of rat lungs 14 days after-inoculation (5 per rat) were stained with hematoxylin
and eosin (H&E). Each branch of bronchiole cut in transverse, longitudinal, or oblique
planes was counted and was evaluated for both the presence of inflammation and for the
presence of fibrosis/remodeling. An average of 42.7 (range 21-76) bronchioles per rat
were evaluated. Bronchioles were scored as positive for inflammation if the wall had

67
five or more inflammatory cell types (eosinophils, lymphocytes, or macrophages).
Bronchioles were scored as positive for fibrosis/remodeling if the walls were thickened
with increased fibroblasts and deposition of collagen. Collagen was identified using
Masson’s Trichrome and Manuel’s Reticulin stains. Numbers of bronchioles with
inflammation or fibrosis/remodeling were divided by the number of total bronchioles
examined to calculate the percentage of bronchioles with each pathologic change.
Additionally, the area of the bronchiolar wall from the bronchiolar epithelial basement
membrane to basement membrane of the surrounding alveolar walls was measured.
Bronchiolar wall area was divided by the perimeter of bronchiolar basement membrane to
calculate the thickness of the wall (square micrometers of bronchiolar wall per
micrometer of bronchiolar basement membrane) (113).
Data Analysis
The final quantitation of cytokine mRNA levels detected by real-time PCR was
done using the comparative Ct (cycle threshold) method and was reported as relative
transcription of the n-fold difference relative to a calibrator cDNA (BN rat high IL-4
responder lung cDNA for IL-4 mRNA and LPS-stimulated lung cDNA for EL-18 mRNA)
(Table 3-1) (124). For all experiments, group means were compared by one-way analysis
of variance (ANOVA) using a computer-based statistical program (Sigma-Stat, Jandel
Corp. San Rafael, CA). Kruskal-Wallis analysis of variance on ranks was used on ranks
if the normality test or equal variance tests of group means failed. Multiple comparison
procedures were used to isolate the group or groups that differ from others. The Student-
Newman-Kuel’s test was used if the sample sizes were equal; otherwise Dunn’s test was
used to compare groups of unequal sample size.

68
Results
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Amount of Bronchiolar Inflammation and Fibrosis
Virus infection in BN rats induced bronchiolar inflammation and fibrosis (Figure 4-
1). Administration of rIL-12 at the time of Sendai viral inoculation (Day 0) reduced the
number of inflamed bronchioles by 22.1% and the number of bronchioles with mural
fibrosis by 23.8% as compared with virus-inoculated, saline-treated controls (n = 8)
(Figure 4-1).
IL-12 Saline IL-12 IL-12
Day 0 Day 0 Day 0 Day 2
1 *p<0.01 1
Figure 4-1. The percent of bronchioles containing evidence of inflammation and/or
fibrosis at 14 days after inoculation. Treatment of BN rats at the time of virus infection
resulted in the significant reduction of bronchiolar inflammation and fibrosis (p<0.01).
In contrast, IL-12 treatment 2 days after viral inoculation did not lead to a
significant decrease in the numbers of inflammatory cells or in the severity of fibrosis
observed. Previously it has been shown that non-infected BN rats normally have low-
density aggregates of lymphocytes, macrophages, mast cells, and eosinophils in
bronchiolar walls by 30 days of age (Figure 4-1) (112).

69
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Bronchiolar Wall Thickness
Increases in bronchiolar wall thickness occur by 14 days after Sendai virus
inoculation due to edema, the accumulation of inflammatory cells, fibroblast
proliferation, and the intramural deposition of collagen and extracellular matrix (113).
The BN rats treated at time of virus-inoculation had a 15% decrease in the bronchiolar
wall thickness as compared with the virus-inoculated, saline-treated controls (p<0.02) (n
= 8) (Figure 4-2). A significant virus-induced increased in bronchiolar wall thickness
was observed above the non-infected BN rats in the virus-inoculated, saline treatment
group (p<0.001) (Figure 4-2).
IL-12 Day 0 Day 0 Day 2
Day 0
Figure 4-2. Airway morphometric analysis of bronchiolar wall thickness at 14 days after
virus inoculation. IL-12 administration at the time of virus inoculation reduced the
bronchiolar wall thickness to non-infected control levels (p<0.02).
In contrast rats treated on day 2 after inoculation had a 22.3% increase in the
bronchiolar wall thickness as compared to virus-inoculated, saline-treated controls
(p<0.01) (Figure 4-2).

70
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Reduces the
Number of BrdU Labeled Fibroblasts in the Bronchiolar Walls
Bronchiolar fibrosis and other remodeling events were evaluated by counting
bronchiolar mural fibroblasts and epithelial cells labeled with BrdU at 10 and 14 days
after virus inoculation (Figure 4-3). Treatment of BN rats with rEL-12 at day 0
significantly reduced the magnitude of increase in mural fibroblast labeling by BrdU.
There was decreased mural fibroblast labeling by BrdU by 42% at 10 days after
inoculation (n= 8) (Figure 4-4) and 62.5% at 14 days following inoculations (n = 8)
(Figure 4-5).
Treatment of BN rats with rIL-12 at day 2 had no effect on virus-induced increases
in BrdU labeling of fibroblasts at 10 days after inoculation and increased labeling of
fibroblasts by 12.5% at 14 days after inoculation (Figure 4-4). Virus infection resulted in
increased labeling of bronchiolar epithelial cells with BrdU. However, rIL-12 treatment
had no statistical effect on this virus-induced labeling of epithelial cells.
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Increases the
Pulmonary Expression of IFN-y
Virus infection increases the amount of IFN-y mRNA in whole lung tissues of BN
rats (n = 4-5) (Figure 4-6). There were significant increases in IFN-y mRNA in all
treatment groups infected with Sendai virus over non-infected controls (p<0.05). BN rats
treated with IL-12 at day 0 had 4-fold more IFN-y mRNA in lungs (p<0.002) when
compared to the saline-treated, virus-inoculated controls. IL-12 treatment at day 2 after
virus inoculation decreased the production of IFN-y mRNA by 4-fold as compared to
saline-treated, virus-inoculated controls (Figure 4-6).

Figure 4-3. BrdU labeled bronchiole of a saline-treated, virus-inoculated BN rat. Labeled bronchiolar
epithelium is indicated by arrows/ several labeled fibroblasts are indicated with (*)

72
10 Days After Inoculation
Saline IL-12 IL-12
Day 0 Day 0 Day 2
Figure 4-4. Immunohistochemical analysis of fibroblast BrdU labeling at 10 days after
Sendai inoculation. BN rats treated at the time of virus inoculation reduced the number
of labeled fibroblasts significantly as compared to virus-inoculated, saline-treated
controls.
14 Days After Inoculation
Saline IL-12 IL-12
Day 0 Day 0 Day 2
Figure 4-5. Immunohistochemical analysis of fibroblast BrdU labeling at 14 days after
Sendai inoculation. The treatment of BN rats with IL-12 at the time of virus infection
reduced the number of BrdU labeled fibroblasts significantly as compared to virus-
inoculated, saline-treated controls (p<0.0001).

73
Day 0 Day 0 Day 0 Day 2
Figure 4-6. Competitive PCR analysis of IFN-y mRNA in BN rats 3 days after
inoculation treated at day 0 or day 2 with IL-12. IFN-y mRNA levels in rats treated with
IL-12 at the time of virus infection increased significantly above the saline-treated, virus-
inoculated controls (p<0.002). Day 2 treatment decreased the IFN-y mRNA response to
levels significantly below the saline-treated, virus-inoculated controls (p<0.01).
There was a significant increase (73.5%) in the level of IFN-y protein in the total
lung tissue of the BN rats (7 days after inoculation) treated at day 0 as compared to the
virus-inoculated, saline-treated rats (p<0.05) (n = 7-8) (Figure 4-7). Day 2 IL-12
treatment did not increase IFN-y over the saline-treated rats measured seven days after
virus inoculation. A virus-induced increase in IFN-y protein was detected in all treatment
groups at 7 days after inoculation as compared to the non-infected controls.
IL-12 Treatment of BN Rats on Day 2 After Sendai Virus Inoculation Alters the
Levels of IL-18 or IL-4 mRNA
The levels of mRNA of the co stimulatory cytokine EL-18 were not increased by
IL-12 treatment at any of the time points measured (n = 3-5) (Figure 4-8).

74
Day 0 Day 2 Day 0 Day 2
Treatment Groups
Figure 4-7. ELISA analysis of whole lung homogenates from BN rats at 7 and 10 days
after virus inoculation. Day 0 IL-12 treatment induced a significant increase in the levels
of IFN-y as compared to the virus-inoculated, saline treated controls (p<0.05).
However, IL-12 treatment at day 2 caused a decrease in the amount of EL-18 mRNA
measured at three days after inoculation as compared to day 0 treated animals (p<0.05).
No significant differences were measured between either of these groups and the non-
infected controls.
The treatment of BN rats at the time of virus inoculation did not reduce the levels
of IL-4 mRNA at any time points measured after inoculation. However, IL-4 mRNA
levels were significantly elevated (3.2-fold increase) in the virus inoculated rats treated at
2 days after inoculation as compared to virus-inoculated, saline-treated controls (n = 4-6,
p<0.02) (n = 4-6) (Figure 4-9). Virus infection increased the level of IL-4 mRNA in all
treatment groups at 3 days after Sendai virus inoculation (p<0.05).

75
Figure 4-8. Analysis of real-time PCR for the detection of IL-18 mRNA. IL-12
treatment at 2 days after inoculation significantly reduced the expression of IL-18 mRNA
as compared to the rats treated the day of virus inoculation.
Saline Saline IL-12 IL-12 Saline IL-12 IL-12
Day 0 Day 0 Day 0 Day 2 Day 0 Day 0 Day 0
Figure 4-9. Analysis of real-time PCR for the detection of IL-4 mRNA. BN rats treated
with IL-12 at day 2 had a significant increase in the level of IL-4 mRNA at three days
after inoculation compared to virus-inoculated, saline-treated rats 3 days after inoculation
(p<0.02). IL-4 mRNA expression significantly increased in all of the virus-infected
groups assessed at 3 days after Sendai inoculation (p<0.05).

76
IL-12 Treatment of BN Rats at the Time of Sendai Virus Inoculation Does Not Alter
the Respiratory Clearance of Sendai Virus
F344 rats have been shown to clear Sendai virus from their lungs at 7 days after
inoculation as compared to virus-susceptible BN rats (115). Viral clearance was not
affected in BN rats treated with rIL-12 on day 0 as compared to saline-treated, virus-
inoculated controls (p>0.05). At seven days after inoculation, the mean value of virus
recovered from the saline-treated, virus inoculated controls was 6.19 X 104 PFU/g of
lung, and the BN rats treated on day 0 had a mean titer of 7.33 X 104 PFU/g of lung (n =
8) (Figure 4-10).
IL-12 Saline
Day 0 Day 0
Figure 4-10. Viral titer results from plaque assays at 7 days after inoculation. IL-12
treatment does not increase the rate of viral clearance from the lungs of BN rats after
Sendai infection (P>0.05).
Discussion
The results indicate early treatment of virus-susceptible BN rats with IL-12 inhibits
the development of virus-induced chronic inflammation and bronchiolar fibrosis. IL-12
treatment at the time of Sendai virus inoculation decreased the amount of airway wall

77
inflammation and fibrosis, decreased the thickness of the bronchiolar walls, and increased
the expression of the Th-1 cytokine IFN-y. This protection is not observed if EL-12
treatment is delayed as little as two days after virus-inoculation. In fact several of the
viral sequelae such as airway wall thickening and fibrosis were exacerbated.
Mechanisms by which IL-12 treatment early in the Sendai virus infection may
reduce virus-induced persistent bronchiolar inflammation and bronchiolar fibrosis and
other remodeling may be direct through IL-12 or indirect through the induction of IFN-y.
IL-12 can act to increase the cytotoxicity and mitogenic activity of T and NK cells, and to
inhibit B cell functions while enhancing the conventional, B cell-dependent antibody
responses (1-4). IL-12 also has direct stimulatory effects on hematopoietic progenitor
cells (2). The main biological effects of IL-12 are attributed to the induction of the
cytokines TNF-a, granulocyte-macrophage colony-stimulatory factor, IL-10, IL-2, and,
most importantly, the induction and maintenance of IFN-y (1-4). IL-12 also acts to
reduce the levels of IL-4 and the establishment of the Th2 phenotype (2-3). IL-12
treatment did increase the levels of IFN-y mRNA and protein significantly above the
levels seen with viral infection alone (saline-treatment). IL-12 treatment however, did
not reduce the levels of IL-4 mRNA at the time points measured in this model. Still,
there may be reductions in IL-4 at a later time point due to the prevention of the
proliferation and development of Th2 cells (3). The viral titer data does not suggest that
IL-12 is directly inhibiting viral replication. This is consistent with previous studies in
which IFN-y treatment reduced bronchiolar inflammation and fibrosis without altering
Sendai virus replication in BN rats (119).

78
The effects of EL-12 treatment in reducing the pulmonary fibrosis observed in BN
rats after Sendai virus infection may be mediated by increases in EFN-y expression. Early
IL-12 treatment was associated with increases in total lung IFN-y mRNA at three days
after inoculation and in total lung IFN-y protein at seven days after inoculation. EFN-y
can induce angiostatic chemokines such as EFN-inducible protein 10 (IP-10), macrophage
inflammatory protein-2 (MEP-2), and monokine induced by IFN-y (MIG). These
chemokines have been shown importance in the down-regulation of angiogenesis and
fibrosis in a mouse bleomycin-induced pulmonary fibrosis model (132). EL-12
administration to bleomycin-treated CBA/J mice decreased levels of hydroxyproline and
increased lung EFN-y, IP-10, and MIG (138). Additionally, IFN-y treatment has been
shown to attenuate increases of TGF-pi, procollagen mRNA, and total lung collagen in
after bleomycin challenge in mice (132).
The time frame for the protective effects of IL-12 appears to be narrow and only
very early after Sendai infection. Administration of IL-12 just 2 days after viral
inoculation failed to protect against increased virus-induced bronchiolar wall thickening,
bronchiolar airway wall fibroblast proliferation, increased EL-4 mRNA production, and
suppressed the expression of total lung EFN-y mRNA. These results are similar to the
responses of exogenous IL-12 treatment seen in other infection models. Exogenous IL-
12 administered at times when T-cell responses are known to be effecting clearance of
murine lymphocytic choriomeningitis virus caused reduced cytotoxic T cell lytic
capabilities, inhibition of virus-induced expansion of CD8+ T cells, and increased
production of TNF (136). IL-12 immunotherapy of murine leishmaniasis infection is
only effective during the first week of infection. Delayed treatment is ineffective and can

79
enhance IL-4 production and susceptibility (79). The molecular basis for this loss of IL-
12 sensitivity is hypothesized to be due to a disruption in Th-2 cells of the IL-12
dependent activation of the Janus kinases (JAK) and signal transducers and activators of
transcription (STAT) intermediates through the IL-12RP2 that are preserved in Th-1 or
Th-0 cells (2, 71). Early IL-12 treatment however, modifies the course of leishmaniasis
by inhibiting the development of Th-2 type responses and promoting the Th-1 cell
responses dependent on IFN-y (79). The dynamics observed in these experimental
models may be true in this model, in that once a Th-2 response is already established; it
can be difficult to reverse.
The protective effects of IL-12 administration in this model are consistent with
effects seen in other studies of infectious and allergic airway disease (Chapter 1).
Furthermore, this model also exhibits many of the complications observed in other
models regarding the dependence on the timing of IL-12 administration and protective
immunomodulatory effects (79, 97-99). These results are consistent with the conclusion
that airway dysfunction in childhood asthma may partially be the result of slight
differences in the immune cytokine response that control the inflammatory and repair
processes to viral disease. Based on these experiments, it may be possible to interrupt the
progression of viral injury to chronic airway damage with early immunomodulatory
cytokine administration.

CHAPTER 5
GENERAL SUMMARY AND FUTURE DIRECTIONS
The goal of this research was to determine the role of interleukin-12 (IL-12) in the
development of resistance to chronic airway disease induced by parainfluenza (Sendai)
virus during early life. The hypothesis tested was that F344 rats are more resistant to
virus-induced airway damage and fibrosis because they produce high levels of IL-12
early in response to virus that up-regulates Th-1 cytokine responses, antiviral immunity,
and reduces airway wall fibrosis. Fulfilling 4 specific objectives tested this hypothesis:
Objective 1
1) To compare the pulmonary IL-12 mRNA and protein responses of virus- resistant
F344 and virus-susceptible BN rats following Sendai virus infection.
Real-time PCR revealed that both rat strains have early virus-induced increases in
IL-12 p40 mRNA (2-5 days after inoculation). In addition, it was demonstrated that
virus-inoculated F344 rats have significantly increased pulmonary IL-12 p40 and IL-12
p35 mRNA at early time points after inoculation as compared to the BN rat strain (0-3
days after inoculation). This is just prior to the increased expression of IFN-y mRNA
observed in F344 during previous studies at 3, 5, and 7 days after Sendai inoculation
(117). ELISA of whole lung tissue, revealed that IL-12 protein levels are increased
significantly in the F344 at two days following inoculation, and that this increase was not
due to over-production of potentially antagonistic IL-12 p40 monomers and dimers
(2,13).
80

81
Objective 2
2) To determine if F344 rats have greater numbers of pulmonary cells and differing
cell types that express IL-12 in response to Sendai virus infection than BN rats.
Protein immunohistochemistry demonstrated that F344 rats have higher numbers of
bronchiolar dendritic cells and macrophages expressing IL-12 p40 protein as compared to
the virus-inoculated BN rats. Although many attempts were made to establish the
location and differential levels of IL-12 p40 mRNA expression by in situ hybridization,
none of the experimental results were conclusive. Observationally, the IL-12 p40 mRNA
message was detected sporadically in the bronchiolar macrophages, dendritic cells, in the
lymphocytes of the BALT, and in the airway epithelial layer.
Objective 3
3) To determine if Sendai virus-induced airway damage in BN rats can be reduced by
IL-12 treatment early in the virus infection.
The treatment of BN rats with exogenous IL-12 at the time (within 3 hours) of viral
inoculation does reduce the chronic sequelae of Sendai virus infection. BN rats treated
with IL-12 on the day of inoculation-displayed decreases in bronchiolar inflammation
and fibrosis, decreases in airway wall fibroblast proliferation, and increases in IFN-y
expression at various time points after Sendai infection. Viral clearance in BN rats was
not affected by the treatment of IL-12 at the time of virus inoculation, and viral clearance
after treatment on day two after inoculation was not assessed. In contrast, IL-12
treatment two days after virus inoculation significantly increased airway wall thickness,
decreased IFN-y mRNA expression, and increased the expression of IL-4 mRNA.

82
Objective 4
4) To compare the airway IL-12 p35 and p40 mRNA responses of virus-resistant F344
and virus-susceptible BN rats following Sendai virus infection (This specific aim is
contingent on the results from the second specific aim. If there is differential
expression of IL-12 in the dendritic cell or other cells types in the large airways
based on the results of the in situ hybridization and immunohistochemistry, then
this specific aim will be explored).
When the experiments to determine the differential expression of IL-12 in this
model were begun, real-time PCR was in its very early stages. Therefore, I was
demonstrating only marginal differences in the amount of IL-12 p40 mRNA expression
by dilutional PCR. At the time, it seemed that this was possibly due to the dilution of
differences that may be at the airway level being masked by the inclusion of the total lung
tissue. Additionally, the in situ hybridization and the immunohistochemistry techniques
were very time consuming to resolve. Based on these difficulties, this specific aim was
added. Real-time PCR of the tracheal tissue revealed that IL-12 p40 mRNA does
increase in both rat strains early after virus inoculation, and is significantly elevated at
two days following inoculation in the F344 rat strain. There were no significant
alterations in the IL-12 p35 mRNA possibly due to the low levels of tissue used or due to
the limits of this procedure at detecting all of the IL-12 p35 isoforms (Chapters 1 and 3).
Conclusions
The results of these studies supports the hypothesis that F344 rats are more resistant
to virus-induced airway damage and fibrosis because they produce high levels of IL-12
early in response to virus that up-regulates Th-1 cytokine responses, antiviral immunity,
and reduces airway fibrosis. It is concluded that:
1) Virus-resistant F344 rats express higher pulmonary IL-12 gene expression early
after Sendai virus infection as compared to virus-susceptible BN rats.

83
2) Virus-resistant F344 rats have more bronchiolar dendritic cells and macrophages
expressing IL-12 than BN rats at early time points after inoculation.
3) Treatment of virus-susceptible BN rats with IL-12 early after Sendai inoculation
reduces the severity of airway wall fibrosis and remodeling.
4) IL-12 has a critical role in the immune response to Sendai virus infection in F344
rats.
Future Studies
In previous experiments by P.G. Holt and others, have identified dendritic cells as
the principal resident APC of the rat, mouse, and human lung and that these cells form a
network throughout the epithelium to alert the immune response to inhaled antigens
(134). Furthermore, the examination of these resident cells in BN rats suggests that the
resting Th-2 polarity of the resting mucosal immune system may also be a property of the
resident dendritic cell population (121, 136). Initiation of the Th-1 immune response
seems to require additional signals such as TNF-a expression and/or CD40 ligation from
the microenvironment (136).
The results these studies using OX-6 immunohistochemistry indicate that there are
more dendritic cells located in the F344 bronchiolar airways at two and three days after
Sendai virus inoculation compared to the BN rats. Further studies to map the kinetics of
the dendritic cell numbers and turnover in the context of this model are indicated. It may
be that the dendritic cell numbers persist longer and in different locations within the
airways of F344 rats after Sendai infection than in BN rats. These results also indicate
that these cells are expressing differing levels of cytokines at the airway level depending
on the rat strain infected. Using techniques such as laser microdissection, real-time PCR,
and double-staining immunohistology the important differences may be elucidated at the

84
dendritic cell network level that influence the susceptibility to Sendai virus infection and
possibly asthma.
Another aspect in this model that may affect the susceptibility of the rats in this
model to Sendai virus is the expression of the IL-12Rp2 receptor protein. The expression
of the IL-12RP2 protein is limited to Th-1 cells and may correlate with IL-12
responsiveness (29,30). In the development of T cells, IL-4 can inhibit the expression of
this subunit, thus these cells lose the ability to respond to IL-12 after TCR binding (30).
Recently, differential expression of the IL-12RP2 subunit between predominately Th-2
responding BN rats and Th-1 responding Lewis rats (139). Within the model, F344 rats
may have increased expression of the IL-12RP2 protein, therefore not only producing
more IL-12 but may be more responsive to its immunologic effects. Preliminary data
using IL-12RP2 RT-PCR on pulmonary tissue, an increase in the expression of this
subunits mRNA message in the F344 rat tissue (Figure 5-1).
288 bp-
t B F BF BF BF Negative
Controls
100 bp Ladder
Figure 5-1. RT-PCR of IL-12RP2 in BN and F344 rats at non-infected control levels.
Observationally, there are brighter bands in the non-infected control F344 rats (F) rats as
compared to the BN (B) rat strain at control levels. The band size of IL-12RP2 is 288
base pairs (bp) as indicated above.
The differential expression of the IL-12Rp2 may indicate differences in the ability
of the rats in this model to respond to IL-12. This aspect of IL-12 regulation may need to

85
be addressed in this rat model and in human asthma patients before the potential
effectiveness of IL-12 immunomodulation can be fully assessed.

APPENDIX A
PRELIMINARY DATA
Experiment 1: Pulmonary Expression of IL-12 in Sendai Virus-Infected BN and
F344 Rats
Dilutional RT-PCR
Preliminary pulmonary mRNA levels were measured in a small number of rats
using 10-fold dilutional RT-PCR (Chapter 2). IL-12 p40 mRNA levels were increased
over levels in BN rats at two, three, and seven days after inoculation with Sendai virus
(Figure A-l).
Figure A-1. Dilutional RT-PCR analysis of mRNA from virus-infected BN and F344
rats. F344 rats have increased pulmonary IL-12 p40 mRNA over BN rats at 2, 3, and 7
days post-inoculation. This difference is statistically significant at 3 days after viral
inoculation (p<0.05).
Lung Lavage Fluid ELISA
Preliminary ELIS As using lung lavage fluid detected very low levels of IL-12 p70
and IL-12 p40 protein with minimal differences between the two strains (Figures A-2, A-
86

87
3). Based on the low levels detected, whole lung homogenates were used in further
studies to measure the expression of IL-12 protein.
0 3 5
Days After Inoculation
Figure A-2. ELISA analysis of IL-12 p70 protein in concentrated lavage samples from
small numbers virus-infected and control rats. The level of IL-12 p70 increases with
viral infection, however there are no significant differences between the BN and F344 rat
strains.
0 3 5
Days After Inoculation
Figure A-3. ELISA analysis of IL-12 p40 protein (homodimers and monomers) in
concentrated lavage samples from small numbers virus-infected and control rats. The
level of IL-12 p40 is higher at control levels in the F344 rat strain, however extremely
low amounts were detected in both strains at all three time points.

88
Experiment 2:The Effects of Exogenous Interleukin-12 Administration on the
Development of Sendai Virus-Induced Airway Disease in BN Rats
IFN-y protein was evaluated in concentrated lung lavage samples from saline-
treated, virus-infected BN rats, control BN rats, and IL-12-treated, virus-infected BN rats.
Preliminary analysis using concentrated lung lavage fluid samples detected an increase in
levels of IFN-y after viral infection, but no significant differences in the IL-12 treatment
groups (Figure A-4). This analysis was repeated using the supernatants from whole lung
homogenates.
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i ^
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Q to .E
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T CM
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Figure A-4. ELISA analysis of IFN-y in concentrated lavage samples. No significant
differences between the treatment groups in the levels of protein detected (p>0.05).
However, IFN-y protein levels increased at seven days after infection above the previous
(three day) levels in the saline-treated and IL-12 (day 0) treated animals, but not in the
IL-12 (day 2) treated BN rats (P<0.003), ANOVA.

APPENDIX B
IL-12 DOSAGE FOR TREATMENT TRIAL
Experimental Design
The purpose of this trial was to determine the appropriate dose of recombinant IL-
12 (rIL-12) (mouse rIL-12, Biosource International, Camarillo, CA) to administer to BN
rats to achieve a biological effect. A starting point for dosage and the route of
administration were based on those previously used by others in IL-12 treatment
experiments (140-142). We chose an early time point after Sendai inoculation to give IL-
12 due to its probable role in the acute immune response (Chapter 1). The following
table describes the details of the experimental design used in this dosage trial (Table B-
1).
Table B-l. Experimental Design for IL-12 Dosage Trial
IL-12 Dose
Days of
Treatment
Day of Necropsy
± Virus
Number of BN
Rats
0 pg (Saline)
Day 0
Infected Day 7
4
3 ftg
Day 0
Infected Day 7
1
6pg
Day 0
Infected Day 7
1
12 pg
Day 0
Infected Day 7
1
3 ftg
Day 0 and Day 3
Infected Day 7
1
3 Pg
Day 0
Non-infected Day 7
1
Oftg
Day 0
Non-infected Day 7
3
The rats were injected intraperitoneally (BP) with IL-12 or an equal volume of saline
with a 20 gauge, 1-inch needle at the time point after aerosolization that was specified in
Table B-1. Non-infected rats were housed separately from the infected rats regardless of
treatment group. Lung lavages for airway inflammatory cell assessment were performed
through intratracheal cannulation with phosphate buffered saline (PBS). Lungs were
89

90
removed and fixed for 2 hours by tracheal perfusion with 4% paraformaldehyde-PBS (pH
= 7.4) and embedded in paraffin for histologic evaluation of the pneumonia.
The percentage of macrophages, neutrophils, lymphocytes, and epithelial cells were
determined from the total number of cells recovered in each lavage sample. These
percentages were compared to determine if there was a reduction in certain cell
populations during with IL-12 administration. Areas of pneumonia were measured by
projecting images of the sections with a photographic enlarger on a digitizing tablet
interfaced with a microcomputer and a morphometric program (Bioquant II, R and M
Biometrics, Nashville, TN). Areas of pneumonia were detected and outlined (areas
chosen based on 5 or more inflammatory cells/alveolus and thickening of interalveolar
septa). The indices of pneumonia were calculated by dividing the sum of the areas of
exudative pneumonia by the area of the section.
Results and Conclusions
In the examination of the lung lavage cell counts it was noted that there was no
statistical significance in the percentage of cells and cell types counted in all of the IL-12-
treated, virus-infected animals (Table 3-2). Therefore, these animals were grouped
together as the IL-12 treatment group. The percent of cells and distribution of the cell
types in the IL-12 treatment groups as compared to the other treatment groups is shown
in Figure B-l.
The trends noted in the IL-12 treatment groups were a reduction in the percent of
macrophages and increases in the numbers of neutrophils and lymphocytes. There were
no statistical differences detected in comparing the groups by percentages or raw data
analysis. The power of the test due to the low numbers of rats was too low to make a
statistical assessment.

91
Table B-2.
ercentages of Inflammatory Cells in the IL-12 Treated BN Rats
IL-12
Treatment
Dose
CHAPTER 1°/
of
Macrophages
%of
Neutrophils
%of
Lymphocytes
%of
Epithelial
Cells
Total
Number
Cells
3 Pg
60.5
25
14.5
0
10 X 105
6 pg
64.5
19
16
0.5
28 X 105
12 pg
78
13
8
1
15 X 105
3 pg at 0
and 3 days
82.5
9.5
7.5
0.5
10 X 105
Figure B-l. The percentage of cells identified as macrophages, neutrophils, lymphocytes,
and epithelial cells in all treatment groups.
In the IL-12 treatment group, pneumonia index was decreased as compared to the
saline-treated, virus-infected rats (p<0.05 using student’s t-test) (Figure B-2). Again, due
to the low numbers of rats used, the power of the test was too low to make a statistical
assessment using ANOVA.

92
Infected Saline- IL-12-
Controls Treated Treated
Figure B-2. Pneumonia indices of BN rats in all treatment groups. The difference
between the saline-treated and IL-12-treated groups was statistically significant using the
student’s t-test (p<0.05).
Based on the trends seen with IL-12 treatment, the lowest dose that achieved a
biological effect (reduction in inflammatory cells and reduction in pneumonia index) was
chosen for the IL-12 treatment experiments in BN rats. The dose of 3 pg (rIL-12 or an
equivalent volume of saline) was to be given IP three hours after inoculation with Sendai
virus or two days after inoculation with Sendai virus.

APPENDIX C
IN SITU HYBRIDIZATION FOR INTERLEUKIN-12
Protocol For In Situ Hybridization
For in situ hybridization, the rat IL-12 p40 PCR product from published primer
sequences was subcloned into pGEM -T Easy (Promega, Madison, WI) as a template for
the production of digoxigenin-labeled RNA probes (125,142). Sense and antisense
probes were made using the Genius System (Genius 4 Kit, Roche Diagnostics,
Indianapolis, IN) according to the manufacturer’s instructions. The sense probe was
made by linearizing with Sal I/Pst I and incubating with T7 polymerase. Similarly, the
antisense probe was made by linearizing with Sph I and incubating with SP6 polymerase.
Lung and trachea sections were deparaffinized in xylene, rehydrated through a
graded series of ethanols, re-fixed in 4% paraformaldehyde, and denatured in 0.2 N HC1.
Prehybridization treatment was as follows: incubation in 10 pg/ml proteinase K (30
minutes, 37°C), acetylation in acetic anhydride, and dehydration in ascending grades of
ethanol with a chloroform rinse. The probes were added to the hybridization solution
(62.5% formamide, 12.5% dextran sulfate, 0.3 M NaCl, 0.025X Denhart’s solution
[Sigma, St. Louis, MO], 12.5mM Tris-HCl pH 8.0, 1.25 mM EDTA pH 8.0) and
denatured for 10 minutes at 65°C. Approximately lOOng of probe was added to each
slide, coverslips were applied, and the slides were placed in a 65°C waterbath for 10
minutes. The slides were hybridized overnight at 37°C in a humidified chamber. The
slides were RNase treated to remove any unbound probe and placed in a series of
93

94
decreasing stringency SSC washes at 37°C. The sections were blocked in 2% normal
sheep serum (1 hour, room temperature) and incubated with an alkaline phosphatase-
conjugated antibody to digoxigenin (Roche Diagnostics, Indianapolis, IN). Signal was
detected with the colorimetric method using the 5-Bromo-4-chloro-3-
indolylphosphate/Nitro-blue tetrazolium (BCEP/NBT) reaction (2-6 hours, room
temperature, dark) (Roche Diagnostics, Indianapolis, IN). Specificity of the antisense
probe was determined by comparison with the sections hybridized with the sense probe.
Problems Solving For Difficulties With In Situ Hybridization
After the probes were constructed several attempts were made using sections from
both rat strains at two days after Sendai virus inoculation. No specific probe binding was
observed with hybridization of the sense or antisense probes. Several more attempts were
made using sections with more observable inflammation, but the results were similar.
Northern blot was attempted to verify the specificity of the antisense IL-12 p40 probe.
No binding was seen in the chemiluminescent blot in the first Northern blot
experiment, so the plasmid with the PCR IL-12 p40 insert was sequenced to determine if
the insert was in the correct orientation. The insert was in the correct orientation. Next,
an agarose gel was run to compare the size of both of the probes with the 100 base pair
ladder. If the polymerases (T7 and SP6) were accurate, the probes should measure to the
specific size of the insert. However, polymerases are not always accurate and the
enzymes may have formed a hairpin loop, thus making the probes bind to themselves and
unable to bind to the sections. The antisense probe was much larger than the size of the
insert, so the plasmid was redigested with Neo I (antisense) and Sal I (sense) restriction
enzymes.

95
The sizes of the inserts were correct using the new restriction sites and these new
probes were labeled. Several attempts were made at running the in situ hybridization
protocol, however no specific labeling was observed. Northern analysis was performed
to verify the binding of the antisense probe. A faint band was observed using the IL-12
p40 antisense probe that was comparable to the P-actin band used as a positive control for
the northern analysis.
At this juncture, the probes were reconstructed using the second set of restriction
enzymes and relabeled with digoxigenin. Seven more attempts were made to get the
probes to specifically bind varying hybridization temperatures, fixing conditions, the
stringency of the washes, and probe concentrations. In the final attempt, sporadic binding
of the antisense probe was observed, but there is no confidence in the accuracy of this
assay with this set of probes.
Observations of In Situ Hybridization Experiments
The detection of the IL-12 p40 mRNA by in situ hybridization was sporadic and
difficult to interpret. In the slides observed, the antisense probe intermittently bound to
inflammatory cells (macrophages, dendritic cells, and lymphocytes) in the bronchiolar
and tracheal walls including in the bronchiole-associated-lymphoid-tissue (BALT), but
there was also a very high level of nonspecific background observed (Figure C-l and C-
2). Staining was also observed occasionally in the bronchiolar epithelium.
The sense probe bound nonspecifically to all structures on each section observed
(Figure C-3). Based on these observations, no conclusions can be drawn from this assay
about the location or quantity of the IL-12 p40 mRNA message in the F344 or BN rats
after Sendai virus infection.

96
Figure C-l. Anti-Sense IL-12 p40 mRNA In Situ Hybridization of bronchiole wall in a
F344 rat at 5 days after Sendai inoculation (54X Magnification). Sporadic binding of
inflammatory cells is seen with in the wall. The level of nonspecific binding is very high.
Figure C-2. Anti-Sense IL-12 p40 mRNA In Situ Hybridization of inflammatory
aggregate in the bronchiole wall of an F344 rat at 3 days after inoculation (108X
Magnification). Sporadic binding of inflammatory cell nuclei is shown in this section
with obvious nonspecific background.

97
Figure C-3. Sense IL-12 p40 mRNA In Situ Hybridization in the airway of a F344 rat 5
days after inoculation (54X Magnification). Extremely high levels of nonspecific
binding are seen in this section

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14.

BIOGRAPHICAL SKETCH
I was bom on April 27th, 1973, to Larry and Sandra Seymour, and named Amy
Elizabeth Seymour. Both of my parents had inspirations to be veterinarians and passed
their love for animals to me. I was bom in Richmond, VA, but spent most of my life in
north/central (mostly Gainesville) Florida. I received my B.S. from Florida State
University and completed an undergraduate research project examining the genetics of
weevil and plant behavior. From there I came back to Gainesville to attend veterinary
and graduate school at the University of Florida. I am one of the first students at U.F. to
complete the combined Ph.D./D.V.M. program.
Ill

University of Florida Graduate School
Electronic Thesis and Dissertation (ETD) Submission
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Student Name: Amy E. S. Stone
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Document Type: Master’s Thesis X Doctoral Dissertation
Document Title: THE ROLE OF INTERLEUKIN-12 IN THE PATHOGENEIS OF
SENDAI VIRUS-INDUCED AIRWAY DISEASE.
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I hereby grant to the University of Florida and its employees the nonexclusive license to archive and make
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Signed: CL^ r.
(student) (date)
Committee:
Type Professor’s Name & Title
William L. Castleman, Chair
Professor of Veterinary Medicine
Mary B. Brown
Professor of Veterinary Medicine
Thomas A. Brown
Professor of Dentistry
Steeve Giguére
Assistant Professor of Veterinary Medicine
Elizabeth W. Uhl
Assistant Professor of Veterinary Medicine
Month and Year of Graduation:
Professor’s signature
YY\aAM —
V
(-Á .
AÉ
U4
^
LUJh ki IÁJL
December 2002
Graduate Dean:

3 1262 08667 710 0




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