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ADOPTIVE TRANSFER STUDIES TO ESTABLISH A
MODEL OF PHASE II EXOCRINE GLAND DYSFUNCTION IN THE NOD MODEL
OF SJOGREN'S SYNDROME
VINETTE B. BROWN
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
Vinette B. Brown
This document is dedicated to the graduate students of the University of Florida.
Working on this project has been a wonderful experience. As a master's student I
had the opportunity to learn from very knowledgeable professors and researchers. Many
people assisted me in the completion of my degree, for which I am very thankful.
First and foremost, I would like to thank Dr. Ammon B. Peck who has provided a
great deal of support and advice. I am so grateful to have worked under someone who is
extremely knowledgeable but is also willing to listen to new ideas. I would like to thank
Dr. Michael Humphreys-Beher for allowing me to enter his lab and giving me my very
first project. I am very greatful for the help I received from my committee members, Dr.
Sally Litherland and Dr. David Ostrov, who opened their labs and offices to me.
I would like to thank my entire lab who has provided an immense amount of
support. I would like to thank Janet Cornelius and Dr. Seunghee Cha who aided me with
their years of experience and lots of their time. I also thank Lori Boggs, Daniel Saban,
Eric Sing-Son and Smruti Killedar who have both provided lots of technical assistance
I am very grateful for all of the support I received outside of the Peck lab. I thank
Dr. Clare-Salzler's laboratory, Dr. Petersen's laboratory, Dr. Burne's laboratory, Dr. Oh's
laboratory, and Dr. Khan's laboratory for all of the assistance.
I would also like to thank my friends who provided much needed moral support
and a great deal of advice, specifically, Grace Kim and Dr. Kenyon Meadows.
TABLE OF CONTENTS
L IST O F T A B L E S ...... .. .. ....... ... ......... ... .................................................... .. vii
LIST OF FIGURES ............. ............. ........ ....... .......................... viii
ABSTRACT .............. .......................................... ix
1 IN TR OD U CTION ............................................... .. ......................... ..
B background of Sj gren's Syndrom e..................................... ......................... ...........
A nim al M models ................................................ .......................... 5
A adoptive Transfer Studies .......................................................... ............... 13
2 M ATERIALS AND M ETHOD S ........................................ ......................... 16
A n im a ls ........................................................................ 1 6
A adoptive Transfer .................. ................................... .. .............. ... 16
S aliv a C collection ................................................. ................ 17
Protein Concentration ............................................... .. ..... ................. 18
A m ylase A activity A analysis ................................................. ............. ............... 18
A p opto sis D election ................ .. .. ...... ................ .......................... .. ................... 2 0
Apoptosis Detection via Flow Cytom etry ................................ ..................... 20
Akt Expression via Polymerase Chain Reaction..............................................22
A garose G el E lectrophoresis ........................................................... .....................23
Caspase-3 A activity D election .................................... ............................. ....... 24
In Situ Apoptosis Detection Kit. .............................................. ............... 24
D election of Infiltration ................................................................... .....................2 5
3 R E S U L T S .......................................................................... 2 7
A adoptive Transfer .................. ...................................... .......... .. ..27
S aliv a C collection ................................................. ................ 2 8
A m ylase A activity A analysis .................................................. ............ ............... 29
Amylase Activity Detected in Saliva. ...................................... ............... 29
Detection of Salivary Gland Amylase Activity .............................................29
Protein Concentration ........ ............................ ........ ...... .. ................30
A poptosis D election ......... .............................................. .............. .. .... ...... 32
Apoptosis Detection via Flow Cytom etry ................................ ..................... 32
Caspase-3 A activity D election ......... ................. ........................ ............... 34
Akt Expression via Polymerase Chain Reaction.............................................34
In Situ Apoptosis Detection Kit. .............................................. ............... 36
D election of Infi ltration ...................... .. .. ......... .. ......................... ...................... 36
4 D ISC U S SIO N ...................... .. .. ......... .. .. ......... ...................................40
LIST OF REFEREN CES ................................................................... ............... 49
B IO G R A PH IC A L SK E TCH ..................................................................... ..................52
LIST OF TABLES
3-1 Adoptive transfer, combinations of donor splenocytes transferred to the scid. .......28
3-2 The saliva from each adoptively transferred scid was collected and quantified...... 31
3-3 The submandibular glands for the adoptively transferred scid mice and the
controls were collected and homogenized....................... ..................... 32
3-4 Apoptosis detection using Apo-Direct kit (BD Pharmingen) ................................33
3-5 A kt ex pression ratio s ...................................................................... .............. 3 5
3-6 Focus score derived from H+E stained slides of smg glands..............................39
LIST OF FIGURES
1-1 Apoptosis pathway. Notice that AKT is an inhibitor early in the pathway and
Caspase-3 one of the last activated caspases (7). ...................................................9
3-2 Immunohistochemical staining of submandibular glands for lymphocyte-
in fi ltratio n s ...............................................................................................................3 7
3-3 Hematoxylin/eosin-stained tissue sections of submandibular glands ....................38
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
ADOPTIVE TRANSFER STUDIES TO ESTABLISH A
MODEL OF PHASE II EXOCRINE GLAND DYSFUNCTION IN THE NOD MODEL
OF SJOGREN'S SYNDROME
Vinette B. Brown
Chair: Ammon B. Peck
Major Department: Medical Sciences
Sjogren's Syndrome (Sj S) is an autoimmune exocrinopathy that results in the
development of xerostomia (dry mouth) and keratoconjunctivitis (dry eyes). There are
two distinct phases of Sj S. Phase I which is lymphocyte independent and phase II which
is lymphocyte-dependent. The non-obese diabetic (NOD/LtJ) mouse displays symptoms
associated with both phases of the disease and, therefore, is a useful model for studying
Sj S. The insertion of the scid mutation into the NOD/LtJ background has led to the
development of the NOD.CB 17-PrkdcScid/J (scid). The scid mouse develops phase I of
Sj S but as a result of the absent lymphocytes, development of phase II does not occur.
The lack of mature lymphocyte development in the scid allows it to be used as a recipient
in adoptive transfer studies to determine the combination of lymphocytes required to
induce phase II of Sj S.
The work described in this thesis involves the transfer of lymphocyte combinations
derived from specific strains, all of which have the same NOD/LtJ genetic background,
into the scid mouse. Adoptively transferred scid mice were analyzed for the progression
of disease. Observation of the salivary flow, saliva proteins, presence of apoptosis and
infiltration provided information on progression of disease.
Results indicate the adoptive transfer of functional lymphocytes can lead to
development of phase II. However, the time required for disease progression may vary
depending on the origin of the lymphocytes. The development of this adoptive transfer
model has provided parameters for future experiments which may clarify the
immunopathology important in progression of phase II and onset of Sj S-like disease.
Many illnesses decrease the quality of life, often making normal daily tasks
extremely difficult. Sj gren's syndrome (Sj S) is one such disease, which often causes the
patient great discomfort. Patients afflicted with SjS have difficulties eating and speaking,
due to an inability to secrete saliva. These patients also have dry eyes; therefore, they
have a constant feeling of irritation in the eyes. The etiology of Sj S is not known and a
cure is not available. The current treatment for Sj S is salivary and tear stimulants (1).
These stimulants are sometimes effective, providing a limited amount of relief, but they
can also have undesirable side effects. By understanding the underlying causes and
mechanism of Sj S, a more complete treatment can be devised.
Background of Sj6gren's Syndrome
In 1932, Henrik Sjogren reported the triad of keratoconjunctivitis sicca (dry eyes),
xerostomia (dry mouth), and rheumatoid arthritis (2). This triad of symptoms became
known as Sjogren's Syndrome, a chronic autoimmune disorder in which the secretary
functions of various exocrine glands are disrupted. The major areas affected are the
lacrimal and salivary glands, as well as the skin, upper respiratory, gastrointestinal tract
and the genitals. The resulting dryness of mucosal surfaces results in various
complications such as gastrointestinal discomfort, difficulty speaking, fatigue and
musculoskeletal complaints (3). The disease is not usually fatal, but can be quite
distressful for those affected. Furthermore, due to the immunological basis of the disease,
patients with Sj S are at an increased risk of developing a non-Hodgkin's B-cell
lymphoma. At this time, the exact etiology is not known, so patients remain uncured.
Today, patients are usually treated with secretagogues, which induce lacrimal and
salivary secretion, along with other glandular secretion.
While secretagogues are readily available, a patient may not receive this treatment
due to the difficulties diagnosing Sj S. As with many systemic diseases, Sj S appears with
a wide range of clinical manifestations. These variations, may lead clinicians to
misdiagnose symptoms. There are numerous reports describing lung, renal and central
nervous system (CNS) involvement (4). Over the years, a number of disease
classifications (San Francisco, San Diego, California, Japanese, European original and
European revised), each with their own methods and standards for diagnosing Sjogren's
syndrome, have been proposed. Among the different classification systems, xerostomia
(dry mouth) is usually diagnosed by a lip biopsy. The demonstration of focal lymphocytic
infiltrates, on a minor salivary gland (SG) biopsy, has remained the gold standard for the
oral component of Sj S (2). A salivary gland is considered infiltrated when there are
clusters of fifty or more lymphocytes, known as foci, present. For most classification
systems the presence of two foci per 4 mm2 is required to identify the gland as being
infiltrated. A less invasive measure of xerostomia is the use of scintigraphy and
sialography. Scintigraphy is a technique where an appropriate, short-lived gamma-
emitting radioisotope is introduced. Through radiographic imagery the uptake,
concentration and excretion of the radioisotope by the major salivary gland
submandibularr) is measured. This can be a very sensitive test for glandular function.
Sialography is a similar method in which the release of saliva by the salivary glands is
evaluated via nuclear imaging.
Keratoconjunctivitis (dry eyes) is typically diagnosed by tear volume measured by
what has become known as the Schirmer test. This test measures tear stability by the non-
invasive break up time and usage of a rose bengal dye to stain the ocular surface, which
results in identification of epithelial cell destruction (5). Serologic tests for the detection
of autoantibodies, specifically SS-A (anti-Ro), SS-B (anti-La), and anti-nRNP, are also
used for correct diagnosis of SjS. In eighty-six Sjogren's syndrome patient sera, more
than 96% had SS-A, and 87% had SS-B, compared to 95% of patients with anti-nRNP
(6). Serology not only facilitates diagnosis, but also can be useful in predicting the
subsequent outcome and complications in patients with primary Sjogren's syndrome (2).
The presence of anti-SS-A antibodies may identify patients with systemic disease (7), and
in anti-SS-A/ anti-SS-B positive patients, the relative risk of developing non-Hodgkin
lymphoma has been reported as high as 49.7%, within 10 years of diagnosis (4).
There are two types of Sjogren's syndrome. Individuals that exhibit the classic
symptoms (dry eyes and dry mouth), in the absence of another autoimmune disease, are
diagnosed as having primary Sj S. Secondary Sj S includes the presence of the
aforementioned symptoms, in addition to another autoimmune rheumatic disease, such as
rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Due to the variability
in classification systems, the exact percentage of patients with Sj S is not known, but it is
estimated to have a prevalence not exceeding 0.6% of the general population (2). As with
most connective tissue autoimmune diseases, there is a sexual dimorphism present in
Sjogren's syndrome. There is a 9:1 ratio of women to men in SjS, with most women
presenting symptoms of the disease between the ages of 40 and 60 years of age.
A prominent feature of Sjogren's syndrome is the genetic predisposition (2). It is
not uncommon for two or more cases of Sj S to occur within a family. Polymorphisms in
the major histocompatibility complex (MHC) genes are the best documented genetic risk
factors for the development of autoimmune diseases overall (3). In Sjogren's syndrome,
the most relevant MHC complex genes are the class II genes, specifically the HLA-DR
and DQ alleles (8). However, variations in SjS associated haplotypes amongst different
ethnicities, makes it difficult to establish which of the genes confers risk. The severity of
the disease also seems to be dependent on the combination of the risk-associated alleles.
Sjogren's syndrome patients with DQ1/DQ2 alleles have a much more severe
autoimmune disease then patients with any other allelic combination at HLA-DQ (6).
Even with specific gene combinations, environmental factors may play a role in the
disease onset. Among the possible etiologic factors, viral infections are the most often
proposed as a possible trigger of autoimmune disease. Potential viral triggers include
Epstein-Barr virus and Hepatitis C. Furthermore, a possible relationship between
Sjogren's Syndrome and Helicobacter pylori infection has been suspected (2). With these
concurrent infections, the risk of mucosa-associated lymphoid tissue lymphoma increases
in patients with Sj S (2).
Dentists and the ophthalmologists encounter patients afflicted by Sj S most often,
although a few dermatologists are faced with treating patients presenting cutaneous
lesions, in addition to the classic symptoms. A dentist may encounter a Sj S patient who
complains of dryness in the oral cavity, difficulty swallowing, and a burning sensation in
the throat. A patient in the ophthalmologist's office would complain of dryness in the
eye, which could lead to infections, keratitis, and sometimes, melting of the cornea or
ocular perforations (5). The cutaneous lesions in patients with Sj S are described by
dermatologists as palpable and nonpalpable eruptions, pruritic or non-pruritic, as well as
symmetric or nonsymmetric in distribution (2). The dermatologist may not recognize
these lesions as being associated with SjS and thus, misdiagnose it as Wegener's
granuloma. There is still much to be learned about this disease, and once the information
is attained it will aid in recognition and treatment of SjS.
The current classification systems provide standards to aid clinicians in diagnosing
SjS. However, clinicians are inhibited in their ability to make a diagnosis because the
exact onset of the disease is difficult to determine. The onset is usually around forty years
of age; however, there are cases of juvenile Sj S. Research of Sj S within the human
population is impeded by the ethical and legal limitations of collecting patients' salivary
glands. To study the pathology and etiology of Sj S, a variety of mouse models have been
developed, such as the New Zealand Black (NZB) and the MRL/n substrains. Today, the
mouse used most often is the non-obese diabetic (NOD/LtJ) mouse. The NOD/LtJ (NOD)
is characterized has having insulitis, a leukocytic infiltrate of the pancreatic islets. NOD
mice have marked decreases in insulin production by the age of 12 weeks. The NOD/LtJ
is considered diabetic when there is moderate glycosuria, and the non-fasting plasma
glucose is higher than 250 mg/dl (9). The NOD mouse developed early out of breeding
experiments with the Cataract Shionogi (CTS) strain (9). The CTS mice were selected
for a high fasting glucose level. From those selected mice a few spontaneously developed
overt insulin-dependent diabetes mellitus with insulitis (IDDM), which is now the
NOD/LtJ used today. In the NOD mouse, leukocytic infiltrates and antibodies destroy the
pancreatic islets, with exocrine glands infiltrated as well. Of particular interest to Sj S
researchers is the observation of lymphocytic foci present in the major salivary gland
submandibularr) (10), tear-producing gland (lacrimal) and thymus (11). NOD mice have
destruction of the submandibular gland, a marked decreased in salivary flow, and changes
in the salivary protein composition. Sera from NOD mice is positive for antinuclear
antibodies (SS-A and SS-B), which are detectable by nuclear staining. These symptoms
are similar to those seen in humans with secondary Sjogren's Syndrome, thus making the
NOD a good mouse model for Sj S research.
The pathogenesis of the Sj S-like disease seen in the NOD mouse can be divided
into two phases. Phase I is a lymphocyte-independent phase where there are
abnormalities intrinsic to the submandibular and lacrimal glands of the NOD. Phase II is
marked by the autoimmune response via lymphocytic infiltrates in the submandibular
(sialoadenitis) and lacrimal (dacryoadenitis) glands, followed by a decrease in tear and
saliva production. The lymphocytic infiltrates appear as periductal foci within the
glandular architecture of the salivary and lacrimal glands (12). The cause of the
autoimmune reaction may reside in the target organ of the autoimmune response, in the
immune system, or in both (13). It is theorized that the loss of secretary function is due to
lymphocyte-directed destruction. Increased numbers of apoptotic epithelial cells have
been detected in the minor salivary glands of patients with Sj S (13).
The phase II occurs in the NOD mouse around 10 weeks of age. Current evidence
suggests the existence of genetically programmed abnormalities in the exocrine glands of
the NOD mouse that may contribute to initiation of the autoimmune reaction (11).
Antigen presenting cells, such as dendritic cells, are important mediators of lymphocyte
activation, and may respond to organ abnormalities. There is evidence of accumulation of
dendritic cells in the submandibular gland (smg) before the development of lymphocytic
infiltrates (13). When these lymphocytic infiltrates form in phase II, dendritic cells
interact closely with T-cells, possibly serving to locally activate autoreactive T-cells (11).
These organized lymphocytic infiltrates seen in the NOD are also described in the
biopsies of Sj S patient (14).
As stated before, phase II of Sj S is lymphocyte-dependent in humans and the NOD.
Research indicates that there is an inappropriate response of dendritic cells to
abnormalities of the smg and thus an activation of naive autoreactive T-cells. These
autoreactive T-cells are then capable of stimulating an inflammatory response and
autoantibody production. In addition, the dendritic cells of the NOD were found to have a
decreased ability to stimulate T suppressor cells (11). T suppressor cells are important
regulators of the immune system and have been shown to posses the capacity to alter the
stimulation of autoreactive T-cells (15). Infiltrates of exocrine tissues primarily consist of
CD4+ T-cells with a minority of CD8 T-cell and B-cell populations (12). Studies
indicate that activated T-cells, predominantly CD4+ T-cells, are necessary for the
initiation of autoimmune disease, as indicated by T-cell transfer studies (12). The
infiltrates also exhibit aberrant production of the pro-inflammatory cytokines IL-P, IL-2,
IL-6, IL-7 IL-10, IL-12, IFN-y, and TNF-a. Cytokines are important mediators in the
immune response, and their altered production can lead to an inappropriate stimulation of
In addition to the changes in proteins seen in phase I of Sj S, there are increases in
apoptosis of the submandibular gland acinii (13). Apoptosis, otherwise known as
programmed cell death, is an important inhibitory mechanism of cell growth, aiding in
organogenesis, maintaining tissue morphology, and deletion of autoreactive lymphocytes
(16). As seen in Fig. 1-1, apoptosis is an complex pathway with many stimulators, such
as Caspase-3, and inhibitors, such as AKT. Apoptosis is highly regulated due its
destructive potential. Evidence shows that apoptotic cells can induce inflammation and/or
result in the formation of cryptic epitopes (13). Previous studies show increase levels of
apoptosis in the scid mouse and the NOD, compared to the BalB/c and young NOD (17).
The increased levels of apoptosis in the scid mouse, indicates that apoptosis may be one
of the regulatory abnormalities of the submandibular gland present in phase I (18). The
increased cysteine proteases in the scid mouse implies that apoptosis is lymphocyte-
independent in phase I. However, cysteine proteases have been shown to increase in
activity as the NOD aged from 8 weeks to 20 weeks, with the scid mouse showing the
highest level of activity (17).
The humoral response plays an important role in phase II of Sj S. In addition to the
SLE antibodies (SS-A and SS-B), anti-M3 muscarinic acetylcholine receptor
autoantibodies have been identified in humans (19) and the NOD mouse (17). SS-A and
SS-B are antibodies targeted to antigens that are usually confined to the nucleus (20);
however, due to certain events in phase I of Sj S these antigens may be presented to the
immune system. Muscarinic acetylcholine receptors are important in the secretary
function of mammalian exocrine glands. The muscarinic-cholinergic receptor is
stimulated by neurotransmitters, resulting in a signal transduction that allows fluid
secretion from the salivary gland acinar cells (17). The interaction between an anti-M3
antibody and the M3 receptor may result in down-regulation of receptor density, as well
as postreceptor second messenger pathway signaling events necessary for proper
activation of fluid movement through epithelial cells (20). In studies where the anti-M3
antibody and other autoantibodies were introduced into mice lacking the adaptive
immune response (scid mutation), the anti-M3 antibody was the only antibody capable of
inhibiting secretary function (20). The development of autoantibodies could be a primary
immunological response or a secondary effect. In the case of Sj S, it is most plausible that
the presence of autoantibodies is a secondary effect of the immune system activation
(17). Interestingly, many of the autoantibodies are targeted to cell surface proteins
important in the secretary response (20), possibly explains why other areas (skin, lungs,
GI tract and vaginal tissues), besides the smg and lacrimal glands, are affected in Sj S.
S Survival ,
PP2C AM \
C. .spi- '
Figure 1-1. Apoptosis pathway. Notice that AKT is an inhibitor early in the pathway and
Caspase-3 one of the last activated caspases (16).
The NOD mouse has allowed researchers insight into the phase I of Sj S, plus the
subsequent development of clinical manifestations. However, a multitude of factors
important in the initiation and progression of the disease are not known. Through the
development of congenic NOD strains, specific aspects of Sj S can be studied. The
NOD.CB 17-PrkdcScid/J (scid) mouse is a congenic mouse that was developed by
backcrossing the immunodeficiency locus (scid) onto the NOD genetic background. The
scid locus confers a functional loss of the B and T lymphocytes, otherwise the NOD-scid
mouse retains the genetic profile of the NOD/LtJ. The NOD-scid mouse clarifies the
significance of the genetic background in the initiation of Sj S, as well as the role of
lymphocytes in the clinical manifestations. In addition, the scid mouse is capable of
accepting allogenic and xenogenic grafts, making it a good mouse model for adoptive cell
There is evidence of intrinsic factors present in the salivary glands of the NOD
mouse that are capable of triggering an autoimmune response (14). Analysis of the NOD-
scid mouse can clarify the role genetics has in the onset and progression of the disease.
During phase I, the NOD-scid mouse shows a morphological change in the salivary
glands, involving a loss of acinar cells within the submandibular glands. The loss of
acinar cells increases with age and may be due to either hyperproliferation of the
submandibular ductal cells, or apoptotic events (21). However, there are dramatic
increases in cysteine protease activity (enzymes important in programmed cell death) in
the submandibular glands of older NOD mice and NOD-scid mice (12). While the scid
mouse depicts the same glandular changes as the NOD mouse, the salivary flow rate does
not decrease in the scid. It is possible; therefore, that there is compensation by the minor
salivary glands parotidd, sublingual, and those in the oral mucosa), resulting in a stable
saliva production (21). Nevertheless, data strongly suggest that lymphocytes are
necessary for disease progression and the loss of secretary function. Although salivary
flow remains stable, there are changes in its composition. The composition of salivary
proteins, as shown by epidermal growth factor (EGF) and amylase concentrations in
NOD-scid saliva, show significant changes with age (21). There is also a novel
expression of PRP and an internally cleaved PSP isoform (27 kDa), prominent in 15-
week-old NOD-scid mouse saliva, but not detected in normal BALB/c mice or younger
NOD-scid mice (21). These protein changes are similar to those observed in the
NOD/LtJ. Interestingly, the time at which this new isoform of PSP appears in the saliva
and submandibular glands of NOD-scid mice coincides with the appearance of
lymphocytic infiltrates in the salivary glands of NOD mice (21). It is possible that the
new isoform of PSP, intrinsic to the NOD mouse and its progenitors, may present as a
"foreign antigen" to the NOD immune system, thus initiating the autoimmune response.
In the NOD mouse, there is stimulation of autoreactive T-cells and autoantibodies. The
stimulation of both branches of adaptive immunity may result in the progression of the
disease to phase II of Sj S.
The NOD-scid aids in clarifying the roles of genetics and lymphocytes in the
initiation and progression of Sj S. It is been made clear, by observing the scid mouse, that
lymphocytes are important in the loss of saliva secretion. However, the mechanism by
which lymphocytes affect the salivary gland function is unclear. There are a few factors
of the immune system that may separately, or collectively, inhibit saliva secretion. There
is evidence of apoptotic activity in the salivary glands in the NOD mouse (13). The NOD
shows the presence of antibodies specific to exocrine gland receptors, which are capable
of reducing salivary flow (20). As stated previously, there are a variety of aberrantly
expressed cytokines. The possible pathological effects of these immune responses,
provide a range of areas to be researched. In order to study the different immunological
aspects, we examined congenic, NOD-derived strains. Two interesting strains used in this
study are the NOD.Igh6tm Cg, commonly known as NOD.Ignt"l and for convenience in
this thesis, Ig~null, and NOD. 129P2(B6)-I/4 tmtCgnDvsJ, commonly know as NOD.IL-4--
and for convenience in this thesis, IL-4-.
The NOD.Igng"l mouse is a congenic partner strain of the NOD mouse that is
deficient in B-cells. This mouse was developed by disrupting one of the membrane exons
of the gene encoding the p-chain constant region by gene targeting in mouse embryonic
stem cells (13). NOD.Ignuit1 mice exhibit glandular abnormalities of the NOD. Aberrant
PSP production is present, but exocrine gland dysfunction (xerostomia,
keratoconjunctivitis) does not occur (21). These data imply that B-cells have a significant
role in the appearance of xerostomia and keratoconjunctivitis sicca. The B-cell may be an
important antigen presenting cell, capable of stimulating autoreactive T-cells. The B-cell
also produces autoantibodies present in the NOD mouse and patients with Sj S. Transfer
of serum from human patients into the Iggnul mice results in a decrease in salivary flow,
thus providing evidence that the B-cell is important as an antibody producer, rather than
as an antigen presenter (22). This also implies a role for autoantibodies as the effector
mechanism for secretary inhibition.
The NOD mouse has a cytokine profile that is specific to the appearance of
infiltrates in the salivary glands. The cytokines present consist of interferon (IFN)-y,
tumor necrosis factor (TNF)-P, IL-10, IL-2, IL-6, IL-12 and IL-18, but usually lacking
detection of IL-4 (23). Due to its absence, IL-4 has been disregarded as important in
progression of the disease. However, the increasing evidence of autoantibodies as an
effector mechanism of secretary loss supports a possible role for IL-4 in development of
phase II of Sj S. The IL-4 cytokine regulates the B-cell maturation and the switch from the
IgM to IgGi antibody. NOD.IL 4-/' mice exhibit aberrant glandular formations, the novel
PSP isoforms, and glandular infiltrations. However, the IL-4-- mouse does not exhibit the
loss of salivary flow. The B-cells, unable to class switch, can not produce autoantibodies
specific to exocrine gland receptor, such as the M3R. This findings support the theory that
the humoral response is the mechanism by which exocrine gland secretion is disrupted.
Adoptive Transfer Studies
The use of various gene knockout NOD strains allow different aspects of the
disease to be investigated. The NOD-scid clarifies the roles of genetics and the immune
response. In addition, the roles of B-cells and the IL-4 cytokine are elucidated by
observing the NOD.Ign""l and NOD.IL-4-/-. All of these strains have the glandular
abnormalities and protein production characteristic of phase I of Sj S, but development of
xerostomia or keratoconjunctivitis does not occur. These observations, as well as
serologic analysis of the NOD mouse and patients with Sj S, suggest that the humoral
response is a pathologic mediator of Sj S. By utilizing the adoptive transfer model, the
introduction of a combination of lymphocytes into the NOD-scid mouse, we hope to
identify immune cells important in the role of autoantibody production in the
immunopathology of Sj S.
The adoptive transfer model, using the NOD-scid mouse, was originally developed
to study the etiology of diabetes in the NOD/LtJ. Islet-infiltrating lymphocytes, as well as
splenic lymphocytes from NOD mice, can initiate diabetes when transferred into NOD-
scid mice (24). In those studies, lymphocytes were collected from pre-diabetic and
diabetic mice. The pre-diabetic and diabetic lymphocytes were transferred separately, and
also as a heterogeneous pool, into NOD-scid recipients. The incidence of diabetes in the
recipient NOD-scid was approximately 70%, similar to the NOD/LtJ control, when
splenic lymphocytes from diabetic NOD mice were transferred into NOD neonate
recipients (pre-diabetic and pre-SjS) (18). At 12 weeks of age, 50% of the recipient NOD
mice showed submandibulary gland infiltrations (18). Lymphocytes from the NOD/LtJ
also initiate sialoadenitis in the scid mouse, but these findings do not identify the role of
the humoral response in the cessation of salivary secretion.
The congenic NOD.IL-4-- mouse does not develop Sj S. T-cells from the IL-4--
mouse are not capable of stimulating isotype switching in B-cells and consequently the
IgGi, anti-M3R antibody is absent from its sera. In a preliminary study, NOD.IL-4/- mice
were intraperitoneally injected with splenic T lymphocytes from NOD.Igtnulor NOD.IL
4-/- mice (23). This resulted in a decrease of salivary flow in 66% of the Ig"lU -ll T cell
recipients, in contrast to the stable salivary flow of the IL 4-" T cell recipient (23). The
inherent inability of IL 4-- mice to develop Sj S implies that the T-cell's main role in
disease progression is as an activator of isotype-switching in B-cells. This theory is
further supported by the progression of xerostomia elicited by adoptively transferred
splenic T-cells. As shown in a previously mentioned study, NOD.Ig"U[11 T-cells are
capable of stimulating autoantibody production. However, the Igt"11 mouse does not
develop xerostomia. The absence of xerostomia was reversed by transfer of human serum
IgG, from Sjogren's Syndrome patients, into the Igat" mouse (22). Therefore, a logical
conclusion from these data, is that the humoral response appears to be a necessary and
sufficient mediator of xerostomia. In order to clarify the causal effect of B-cells and T-
cells in Sjogren's Syndrome, the research of this thesis seeks to fulfill the following
1. Adoptively transfer different combinations of splenic lymphocytes from the
NOD/LtJ mouse and its congenic strains, NOD.Ignal" and NOD.IL 4-/- into the
NOD-scid mouse in order to establish the adoptive transfer model for phase II
of Sj S.
To verify the establishment of Sj S in these adoptive transfer mice, I have
The salivary flow and enzyme activity within the saliva of the treated
The submandibular gland of the treated scid mice to detect the presence
of apoptotic events, using flow cytometry, cysteine protease activity, in
situ staining, and protein expression via polymerase chain reaction.
MATERIALS AND METHODS
Animals used in this study were NOD-scid (experimental recipient), NOD/LtJ
(experimental donor), NOD.IL 4-/- (experimental donor), NOD.Ign""l (experimental -
donor), and NOD-scid (negative control), and NOD/LtJ (positive control). Female mice
were used in all experiments. The donor mice were 16 weeks of age, while the recipient
and control mice were 12 weeks old at the time of adoptive transfer. Each treated set
includes 4 recipient mice (NOD-scid), 2 positive control mice (NOD/LtJ), and 2 negative
control mice (NOD-scid). Most of the NOD-scid mice were purchased from Jackson
Laboratory (Bar Harbor, Maine). The other NOD-scid mice and the NOD/LtJ,
NOD.IL 4--, NOD.Ig""ul were purchased from the University of Florida, Department of
Pathology Mouse Colony (Gainesville, FL) and were housed in the Department of
Pathology Mouse Colony under specific, pathogen free conditions.
The donor mice were euthanized by cervical dislocation and their spleens extracted.
The spleens were then pressed through a wire mesh in order to separate the splenic
lymphocytes. The lymphocytes were collected and the erythrocytes lysed with 0.84%
NaC1. The cells were then washed and incubated for 30 min with the appropriate
antibody. Antibodies used were anti CD19 FITC, anti CD3 APC and anti CD25 PE.
All antibodies were from BD Pharmingen. After incubation the cells were washed and
diluted to a concentration of 8 x 106 cells/ml in 2% FBS in PBS. The lymphocytes were
then sorted by Douglas Smith at the University of Florida, Flow Cytometry Core
Laboratory. CD19 B-cells and CD3+CD25- T-cells were collected. The fractionated
splenic lymphocytes were then washed and placed in 1001p IX Phosphate Buffered
Saline. The fractionated splenic lymphocytes were intravenously injected into the
recipient scid mice in a 1 to 1 ratio (B-cells to T-cells). Each recipient mouse received
approximately 3 x 106 cells. The experimental and control mice were divided into two
sets of 8 mice (4 experimental scid mice, 2 positive control NOD mice, and 2 negative
control scid mice). One set was observed for 4 weeks, post-transfer (housed until 16
weeks of age), before being euthanized. The other set was observed for 8 weeks, post-
transfer (housed until 20 weeks of age) and then euthanized. The submandibular glands,
spleens and pancreas of the euthanized mice were collected. The submandibular glands
were sectioned into 3 fractions. One section was placed in 2 ml microcentrifuge tubes on
ice, for flow cytometry analysis. The next section was placed in cassettes, stored in 4%
Formalin overnight, and then placed in 70% EtOH to be formed into paraffin-embedded
blocks. The last section was placed in 2 ml microcentrifuge tubes and frozen with dry ice.
The frozen tubes were then stored in at -800C for future analysis.
Saliva was collected from the experimental (NOD-scid), positive control
(NOD/LtJ), and negative control (NOD.IL 4-/-). The salivary glands were stimulated with
secretagogues to induce salivation. The secretagogue solution is composed of
Isoproterenol (Img/ml) and Pilocarpine (2mg/ml) (Sigma) in IX Phosphate Buffered
Saline. Each mouse received 100 .il of the secretagogue via an intraperitoneally injection.
The saliva was stimulated for 1 minute and then collected by pipetting for 10 minutes.
The collected saliva was placed in 1.5 ml microcentrifuge tubes. The volume of saliva
was quantified using pipettors and recorded. The saliva was then stored in a -80C freezer
for future protein analysis.
The protein concentration of the collected saliva and submandibular gland lysate
was determined, using the Bradford assay. 1 mg of Bovine Serum Albumin (Sigma) was
diluted in 1 ml deionized H20. The Bradford protein assay dye was then diluted 1:5 in
deionizd H20. The standards and samples were made and later read in in 2 ml cuvettes.
The 1 mg/ml BSA was diluted with H20, to a total volume of 25 il, to make the
following standards: 0 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml. For each
sample, 5 ll of sample and 20 pl of H20 was added to each cuvette. 1 ml of the diluted
Bradford dye was added to each cuvette, and mixed with a pipette. The standards and
samples were allowed to incubate for 5 minutes at room temperature. The protein
concentration was detected using the protein concentration program available in the
spectrophotometer (Bradford). The standards were used to make a standard curve and
then the protein concentrations of each sample extrapolated from the standard curve.
Amylase Activity Analysis
Detection of amylase present in collected saliva and submandibular gland lysate
was accomplished by using the Infinity Amylase Reagent (Thermotrace). Amylase
activity is detected via utilization of Ethylidene-pNP-G7 (EPS) as the substrate. The
cleaved EPS reacts with a-glucosidase, resulting in release of a chromophore. The color
change is then detected at 405nm in a spectrophotometer (BioRad). Amylase activity is
defined as the rate of formation of EPS fragments per liter of sample (U/L). The saliva
samples were diluted 1:200 in deionized H20. The submandibular gland lysate were
analyzed undiluted However a few gland lysate samples had an amylase activity that
was unmeasurable when not diluted. These samples were diluted 1:100 in H20. 1 ml of
Infinity Amylase reagent was added to a cuvette and incubated in a waterbath set at 37C
for 1 min. 25 il of diluted sample was then added to the heated reagent and incubated for
1 min. After the 1 min incubation was completed, the timer for the next incubation
minute is started. The reagent/sample was mixed with a pipette and read in the
spectrophotometer to obtain the initial reading. Afterwards the reagent/sample was placed
in the waterbath until the incubation time is complete. The timer was started for the final
incubation time of 1 min, immediately after the second incubation time was completed.
The reagent/sample was read in the spectrophotometer to obtain the second reading. The
reagent/sample was placed in the waterbath. At the end of the final incubation time, the
reagent/sample was read. The spectrophotometer was blanked with H20 in the Infinity
Reagent. The positive and negative controls were obtained from the provider of the
amylase reagent (Thermotrace) and treated the same as the samples. From this assay,
three O.D. readings are obtained. The change in amylase activity is obtained by
subtracting the initial O.D. reading from the final O.D. reading and dividing by the
amount of incubation time between the initial and the final reading (2 min). The obtained
value is then multiplied by the conversion factor (5140).The conversion factor is
calculated based on manufacturer instructions. The amylase activity of each sample (U/L)
is then divided by the known protein concentration (pg/L), and then multiplied by 1 x 104
to standardize. The resulting amylase activity is measured as the rate of formation of EPS
per gram of protein (U/gram of protein). The assay was performed three times in order to
obtain statistical analysis.
Apoptosis Detection via Flow Cytometry
The day the collected submandibular glands are retrieved from the experimental
and control mice, the glands are digested. The submandibular glands are minced and
placed in a vial with 5 ml of Collagenase Solution I (2 mg/ml of collagenase IV (Sigma)
in 1X Hanks Balanced Salt Solution (HBSS) and 200 Cl of Dnase). The vials were then
placed in a shaking waterbath, set at 370C, for 15 minutes. The partially-digested glands
were passed through a series of syringes with needles increasing in gauge, from 18 to 23.
The digested glands were then placed in a vial with Collagenase Solution II (1 mg/ml of
collagenase in IX HBSS with 200 Cl of Dnase). The glands were incubated in a shaking
waterbath for 5 min. The digested gland mixture was passed through a series of syringes
again. At this point the glands should be fully digested. The submandibular cells were
placed in 50 ml conical tubes containing 2% FBS in IX HBSS and placed on ice. The
submandibular cells were collected after centrifugation and counted. The submandibular
cells were diluted with 1X PBS to a concentration of 1X 106 cells/ml. In sterile 12 ml
plastic tubes, 5 mls of 55% Percol was added and 2 mls of the diluted, digested glands
were carefully layered on top with siliconized pipettes. The tubes were centrifuged for 30
min at a speed of 2,000 rpms. After the gradient separation the acinar cells and infiltrates
were collected with siliconized pipettes and placed in 12 ml plastic tubes. The cells were
washed and counted. The acinar and infiltrates were then placed in a sterile culture plate
with a solution containing 2% FBS in RPMI (Sigma) and IX antibiotics. The cells were
cultured overnight to allow the cells to recuperate. The following day, the acinar and
infiltrate cells were fixed in 1 % paraformaldehyde in 1X PBS for 15 min on ice. The
cells were washed and placed in 5 ml flow cytometry tubes with 70% ETOH. The tubes
were placed in a -20C freezer to be stored for 24 hrs. The fixed cells were washed in
preparation of staining by reagents provided in the APO-Direct kit (BD Pharmingen).
This assay utilizes a Tunel labeling system, where terminal deoxynucleotidyl transferase
enzyme (TdT) enables the template independent addition of FITC labeled deoxyuridine
triphosphate nucleotides (FITC-dUTP) to dna strand breaks present in apoptotic cells.
Propidium Iodide (PI)/RNase solution was used to stain total DNA. Apoptotic cells were
provided by the kit as a positive control, as well as non-apoptotic cells as a negative
control. After staining, following the procedure outlined by manufacturer specifications,
the cells were incubated in Propidium Iodide and analyzed by flow cytometry.
Manufacturer suggestions were used as a guide to determine appropriate parameters for
apoptosis detection. Cells were identified as apoptotic if they expressed FITC-dUTP. The
parameters for positive apoptosis was based on the level of positivity determined by the
positive and negative control. A window was selected that included positive control cells
that express dUTP, but excluded negative control cells. In order to analyze and
standardized the results, the following formula was used: (%pos. cells sample x % pos.
cells in the neg. control)/% pos. cells in pos.control. Using the aforementioned formula,
substituting the %pos. cell sample x with the %pos.cells positive control (provided in the
kit), will standardize the positive control as 100% positive. The same formula is applied
to the negative control (provided in the kit) so that the level of positivity is 0%. The
equation is applied to each sample and the positivity determined. For comparison, the
relative positivity in each treated set is assigned a representative value on a scale from 0
to 10 (0 is equal to 0% positive and 10 is equal to100% positive).
Akt Expression via Polymerase Chain Reaction
The stored experimental and control submandibular glands were removed from the
freezer and 25 mg of tissue removed. The sectioned piece of submandibular gland was
then treated according to manufacturer specifications to extract total RNA (Qiagen). The
extracted total RNA was then quantified in the spectrophotometer. A quartz cuvette was
used and the spectrophotometer was blanked with deionized water. The total RNA was
diluted 1: 50 in deionized water and read in the spectrophotometer. Only total RNA with
at least a concentration of 800 .g/ml were used to produce cdna. To a .02 ml nuclease-
free centrifuge tube the reagents were added in the following order; 1 tl of pd(T)12-18, 1
pl of 10 mM dNTP, 1 pg of total RNA, and enough water so that the total volume in the
tubes equal 10 pl. After mixing, the tubes were heated in a waterbath for 5 min at 65C
and quick chilled on ice. The tubes were then centrifuged and the reagents added in the
following order; 4 pl of 5X First-Strand buffer, 2 p0l 0.1M DTT, 1 pl RNasin, and 2 pl
acetylated BSA. After gentle mixing, the tubes were incubated in a waterbath, and set at
42 C for 2 minutes. To each tube 1 p1 of SuperScript II was added and mixed gently. The
tubes were incubated at 420 C for 50 minutes and then 700 C for 15 minutes in the
thermocycler. The cdna concentration was quantified by the spectrophotometer. A quartz
cuvette was used and the spectrophotometer blanked with deionized water. The cdna was
diluted 1 : 50 in deionized water and read in the spectrophotometer. A master mix was
made by adding the following reagents to a 2 ml nuclease-free centrifuge tube; 5.1 pl
10X Buffer, 1.1 pl 10 mM dNTPs, and 1.6 pl of MgC12 (formula is for one per reaction).
7.5l of the master mix was added to a .02ml nuclease-free centrifuge tube. To each .02
ml per tube the following reagents were added; 1.5 pl of the forward primer, 1,5 pl of
the reverse primer, 2 pg of cdna, and enough water so that the volume of the
microcentrifuge tube equals 49.75 [il. The primer sets used were 18S (housekeeping
gene) and AKT. Both primer sets were individually developed by Invitrogen. The tubes
were mixed, centrifuged and incubated in a thermocycler, following the standard program
for polymerase chain reaction. The annealing temperature was set at 57.20C. After the
dentaure step, but before the 1st annealing temperature is reached, .25 tl of Taq DNA
Polymerase (Invitrogen) is added to each tube. The per reaction is then continued in the
Agarose Gel Electrophoresis
A 2% TBE, agarose gel was made and 5 itl of ethidium bromide added to the gel
while it was still warm. After the gel set, the previously made per products were
defrosted. 12 pl of 18s amplicons and 3 pl of 6X Blue/Orange Dye (Promega) were
added per well. In the lane above, 24 pl of akt amplicons and 4 pl of 6X Blue/Orange
Dye (Promega) were loaded into each well. 1 tl of the PGEM DNA marker (Promega),
4 tl of dye and 23 pl of 1X TBE was loaded into a well. The TBE gel was then run at 70
volts for 1.5 hr. Afterwards, the gel was visualized with a gel document system, and
quantitative analysis done using the AlphaEase FC program (Alpha Innotech). The level
of akt expression per sample is based on the samples corresponding level of 18s
expression. 18s is ubiquitous in all cells and should; therefore, all samples should have
the same amount of expression. However slight variations in the level of expression
occurs. Within each lane of a gel are many samples. To standardized the values for 18s
expression, a ratio is formed by dividing highest 18s value within that lane by the 18s
value for each sample within the same lane. The inverse of the calculated 18s ratio
(highest 18s/18s of sample x) is then multiplied by the corresponding akt expression
value (inverse of 18s ratio of sample x, multiplied by akt of sample x). From these
calculations a relative, qualitative expression value is obtained.
Caspase-3 Activity Detection
Stored submandibular glands were defrosted on ice and homogenized with 600 ptl
of Tris-HCL, ph 8.0 in 5 ml plastic tubes. The gland lysate was transferred to 2.0 ml
microcentrifuge tubes. The gland lysate was then assayed following manufacturer
specifications (Calbiochem). The activity assay was read in a microplate reader for 20
minutes and the values calculated in Excel. The data points were plotted on a graph,
activity (x axis) vs. time (y axis), and a trendline generated. The slope of the trendline
was then multiplied by the conversion factor (conversion factor tabulated as suggested by
In Situ Apoptosis Detection Kit.
The previously paraffinized submandibular glands were sectioned and placed on
slides by Histology Technical Services. The slides were then deparaffinized by
incubation in a series of solutions in the following order: two incubations in Xylene for 5
min, two incubations in 100% EtOH for 2 min and two incubations in 95% EtOH for 2
min, two incubations in 70% EtOH for 2 min, two incubations in H20 for 2 min. The
slides were then stained, following manufacturer specifications (Trevigen), in a humidity
chamber. After the slides were dehydrated, the tissues were covered with a non-aqueous
mount and a coverslip added. The slides were allowed to dry overnight and then viewed
under brightfield microscopy.
Detection of Infiltration
The paraffinized submandibular slides were obtained as stated previously. They
were deparaffinized (as stated previously) and placed in a heated coplin jar, containing
IX antigen unmasking solution (Trevigen). The coplin jar was then incubated in a
waterbath for 30 min at 950C. The slides were then washed with H20 and placed in a
humidity chamber. The slides were equilibrated with the buffer, 1X TBST (Dako) for 5
min. 200 pl of diluted (1:67) goat serum (Sigma) was pipetted onto each slide and
incubated for 20 min at room temperature. The goat serum was carefully wiped off the
slide, so that the tissue remained undisturbed. The slides were then incubated for 1 hr
with 200 pl diluted (1:25) B2-20 antibody (BD Pharmingen), at room temperature. The
slides were then washed with IX TBST for 5 min and incubated with 200 pl of diluted
(1:200) anti-rat antibody (Sigma), for 30 min. Before the diluted anti-rat antibody is
added to the slides approximately 5 pl of mouse serum is added to the 2 antibody
solution. The slides are washed with IX TBST and treated with an ABC alkaline
phosphates kit from Vector Labs. The slides were treated with reagents as specified by
the manufacturer (Vector Labs). The substrate used in the alkaline phosphates system
was Vector Red (Vector Labs). The substrate was mixed following manufacturer
specifications and incubated on the slides for 20 min. The slides were then washed in
deionized H20 for 1 min and then washed with 1X TBST for 5 min. The slides were
incubated with 200 pl of diluted (1:67) rabbit serum (Sigma) for 20 min. The slides were
then incubated with 200 1l, diluted (1:125) CD3 antibody (Santa Cruz), overnight at 40
C. The slides were then washed with 1X TBST for 5 min, and incubated with 200 Cl of
diluted (1:200) a-goat antibody (Sigma) for 30 min. The slides were then treated with the
reagents provided in the ABC immunoperoxidase system (Vector Labs). The slides
were treated as specified by the manufacturer. DAB (Vector Labs) was used as the
substrate, and was mixed as specified by the manufacturer. The slides were incubated
with the DAB for 5 min. The slides are then washed in H20 for 1 min. The slides are
immersed in a working solution of the Light Green Counterstain (Sigma) for 1 min. The
slides are then immersed twice in H20 for 1 min. The slides are then dehydrated by being
immersed in a series of solutions for 2 min; 70% EtOH, 95% EtOH, 2 times in 100%
EtOH and followed by immersion in Xylene for 5 min, 2 times. Afterwards the slides are
coverslipped and visualized by brightfield microscopy. The presence of lymphocytes is
determined as cells that positively stained with Vector Red (T-cells) and DAB (B-cells)
Purified splenic lymphocytes from certain donor mice were labeled with the
appropriate fluorescent conjugated antibody (except for the unfractionated donor
NOD/LtJ splenocytes) and sorted by flow cytometry. The collected splenocytes were
selected based on the level of binding to the conjugated antibody. The splenocytes were
collected in two separated fractions. Selected B-cells depicted a high level of CD19
FITC expression. Selected T-cells depicted a high level of CD3+APC while showing no
CD25+ PE expression. CD25+ T-cells have shown to have a protective quality in adoptive
transferred of diabetes studies (25). To circumvent inhibition of disease transfer, the
CD25+ phenotype was set as a negative selection parameter. The purity of the collected
splenocytes was analyzed by flow cytometry. Each fraction had a purity of approximately
93%. The collected splenocytes were then washed and intravenously injected into the
recipient NOD-scid. Each scid received an average of 3 x 106 B and/or T-cells.
The donor splenocytes were selected based on their expected contribution to the
development of Sj S. The T-cells and B-cells from the NOD/LtJ were selected because
they should transfer the disease to the scid. The T-cells of the Ig n"ul was selected due to
its shown ability to stimulate an autoreactive B-cell, thus transferring the disease. The B-
cells from the NOD.IL 4-- should interact with a normal T-cell, resulting in transfer of the
disease. Those scid mice that received T-cells or B-cells, but not both, are expected to
exhibit no disease transfer.
Table 3-1. Adoptive transfer, combinations of donor splenocytes transferred to the scid.
Recipient Donor T-cells Donor B-cells Pos. Control Neg. Control
NOD-scid Total spleen Total spleen NOD/LtJ NOD-scid
NOD-scid Total spleen Total spleen NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD/LtJ NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD/LtJ NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD.IL4' NOD/LtJ NOD-scid
NOD-scid NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD.Igjn1l NOD.IL4' NOD/LtJ NOD-scid
NOD-scid NOD.Igtn l NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD.Igynn1 NOD/LtJ NOD-scid
NOD-scid NOD.Igynn1 NOD/LtJ NOD-scid
NOD-scid NOD.Igjn1l NOD/LtJ NOD/LtJ NOD-scid
NOD-scid NOD.Igtn l NOD/LtJ NOD/LtJ NOD-scid
A significant manifestation of Sj S in the NOD/LtJ mouse is the presence of
xerostomia. Xerostomia in the recipient scid was detected by quantifying the volume of
saliva produced in 10 minutes. Salivation was stimulated by secretagogues and the saliva
collected from 4 treated scid mice, 2 positive control NOD mice and 2 negative control
scids. The collected saliva from the 4 treated mice, per group, was pooled. The collected
saliva from the pos. control was pooled, and the neg. control saliva was pooled as well.
Saliva was collected once a week for 7 wks, but many mice were euthanized early.
Diabetes, which is present in the NOD, was transferred along with the Sj S. The mice that
became diabetic were treated with insulin, but many expired early. To circumvent the
loss of all experimental mice; many diabetic scids were euthanized early.
The collected saliva from 4 week post transfer was compared to the volume of
saliva from the last week of collection. As seen in Table 3-2, the scid (Unfractionated
NOD) did not show a change in salivary flow. The scid (B and T NOD) expressed a
decrease of 46%, which is comparable to previous data on the NOD. The
scid (B IL4-- and T NOD) showed a 68% decrease in flow. The scid (B IL4 )
showed a small decrease of 13%. The scid (T Igniull and B IL4 -) and
scid (T Igni"l") showed a increase of about 35%. The scid (T Ign"ull and B NOD)
had a small increase of 9%. The scid mice that were recipients of either T or B cells, but
not both, had a stable flow, similar to the NOD-scid. Surprisingly, none of the NOD mice
depicted xerostomia. The NOD, in previous studies, has proven to be a good positive
control for Sj S, so this was certainly an unexpected occurrence.
Amylase Activity Analysis
Amylase Activity Detected in Saliva.
An important aspect of Sj S is the aberrant production of salivary components such
as amylase. The collected saliva was analyzed to determine the amylase activity of the
saliva. As shown in Table 3-2, the amylase activity of mice 4 weeks post-transfer (16
week old mice) was compared to the activity of mice 8 weeks post-transfer (20 week old
mice). The calculated readings showed that the scid (Unfractionated NOD) had an
increase of 62%, the scid (T and B NOD) amylase activity decreased by 8% and the
scid (T NOD and B IL4- -) had an increase of 46%. The scid (B IL4 -) had a
decrease in activity of 57%. The scid recipients of T-cells from the Igu""ll all showed a
decrease in amylase activity that ranged from 5% in the scid(T Ignf"ll and B IL4-'-) to
33% in the scid(T Ign"ull and B NOD). The NOD control shows a 21% decrease and a
43% decrease in the scid.
Detection of Salivary Gland Amylase Activity
An important aspect of the initiator phase of Sj S is the change in composition of the
submandibular glands. In order to determine if there are changes, the amount of amylase
present was analyzed. The submandibular glands were homogenized and the gland lysate
incubated with Infinity Amylase Reagent (Thermotrace) for the appropriate time. The
activity was detected in spectrophotometer. As seen in Table 3-3, the scid
(Unfractionated NOD) and scid (T NOD and B IL4--) exhibited an increase in activity
or 300% and 970%, respectively. The scid (T and B NOD) had a decrease of 80%. The
scid (B IL4-/-) showed an increase of 500%. The scid (T Ig"ull" and B IL4- -) and the
scid (T Ig"ull and B NOD) had a decrease in amylase activity of 90% and 34%,
respectively. The scid (T IgnL"ll) showed a less than 6% increase. The NOD and scid
showed a decrease in amylase activity of 33% and 25%.
A significant aspect of Sj S is the aberrant protein production, specifically the
production of two novel forms of parotid secretary protein (PSP). The deviant PSP
production allows the protein concentration to remain stable, even as other major proteins
decrease. Using the Bradford protein assay the protein concentration of saliva and
submandibular gland lysate was determined. The protein concentration of the saliva did
not show a change in concentration in any of the adoptively transferred scid mice or the
controls. The adoptively transferred scid mice and the controls was had an average
protein concentration of 3.6 mg/ml, 4 weeks post-transfer. The average protein
concentration at 8 weeks post-transfer was 3.8 mg/ml. The submandibular gland lysate
depicted a decrease in protein concentration for the scid mice that received a combination
of T and B-cells. This decrease ranged from 78% in the scid (T NOD and B IL4-/-) to
22% in the scid (T Igni"u and B I14--). The scid (T Ign""ll) depicted a decrease of
55%. The scid (B IL4-/-), scid (Unfractionated NOD) and the controls did not show a
change in protein concentration.
Table 3-2. The saliva from each adoptively transferred scid was collected and quantified.
The saliva was then assayed to determine the amount of amylase present and
the concentration of proteins.
Age of treated mouse Total Amylase activity Total protein
salivary (U/gram of protein) (mg/ml)
4 wks NOD (n = 10) 185 5881 671d 3.72 .77
>8 wks NOD (n = 7) 236 4643 541 3.48 .3
4wks-scid (n=12) 180 7082 547 3.01 .8
>8 wks scid (n= 12) 216 4051 879 3.21 .1
4 wks scid (Unfractionated NOD) (n = 4) 168 2786 + 173 3.92 + .02
>8 wks scid (Unfractionated NOD) (n= 4) 170 4509 + 410 4.85 .03
4 wks scid (T and B NOD) (n = 4) 168 4881 + 446 3.72 .02
>8 wks scid (T and B NOD) (n = 2) 90a e* 4486 + 107 4.60 + .02
4 wks scid (T NOD and B IL-4-) (n = 4) 125 6055 + 106 3.63 + .003
>8 wks scid (T NOD and B IL-4- ) (n = 2) 40b 8846 + 288e** 2.55 + .02
4 wks scid (B IL-4-) (n = 4) 200 4282 + 126 5.02 + .04
>8 wks scid (B IL-4-) (n = 4) 175 1824 + 450 5.25 + .03
4 wks scid(T- Iggnul and B IL-4-) (n= 4) 63e* 7202 + 443e* 2.93 + .01
>8wks scid(T- Iggnu andB IL-4--)(n= 2) 100 6788 + 136 3.42 + .01
4 wks scid (T- Iggn") (n= 4) 113 4154 + 667 3.51+ .03
>8 wks scid (T- Iggnl) (n= 4) 150 3160 + 519 3.06 + .02
4 wks scid (T- Iggnl" and B NOD) (n= 4) 110 7091 + 442 3.02 + .02
>8 wks scid (T- Iggn aand B NOD) (n = 3) 120 4735 + 397 3.44 + .01
a Due to the transfer of diabetes, this set euthanized at week 5.
b Due to the transfer of diabetes, this set euthanized at week 6.
c Due to the transfer of diabetes, this set euthanized at week 5.
d Values are given as the mean standard error.
e Statistical comparison of adopotively transferred NOD-scid mouse groups to the age-matched NOD-scid parental control by the one
way ANOVA test: (*P < 0.05, **P < .001).
Table 3-3. The submandibular glands for the adoptively transferred scid mice and the
controls were collected and homogenized. The level of caspase activity was
determined using the Caspase-3 activity kit. The gland lysate was assayed to
determine the amylase present and protein concentration.
Age of treated mouse
(U/gram of protein)
4 wks NOD (n = 10) 3.4 .08 101.2 94 39.8 1
>8 wks NOD (n = 7) 4.3 .41 67.9 .4 31 5
4wks-scid (n=12) 3.4 .02 159.4 30.8 28.4 3
>8 wks scid (n = 12) 4.4 .02 119.1 + 10.2 30.1 6
4 wks scid (Unfractionated NOD) (n = 4) 4.0 + .04 194.7 + 94 59.6 3.3
>8 wks scid (Unfractionated NOD) (n = 4) 4.2 .01 863 433b** 26.5 C
4 wks scid (T and B NOD) (n= 4) 5.2 .02 b* 415 53 29.1
>8 wks scid (T and B NOD) (n = 2) 1.6 .02b** 77 4 40.5 (
4 wks scid (T NOD and B IL-4-) (n= 4) 5.4 .Olb* 34 13 34.6
>8 wks scid (T NOD and B IL-4--) (n = 2) 1.2 .Olb** 365 84 265.8 23.:
4 wks scid (B IL-4--) (n = 4) 2.9 + .03 137 + 29 38.6
>8 wks scid (B IL-4--) (n = 4) 4.6 .02 955 257 44.1
4wks scid(T-Ignull andB IL-4--)(n= 4) 4.3 .Olb** 409 148 36.5
>8wks scid(T- Ignu llandB IL-4--)(n = 2) 1.6 .01 43 14 46.8
4 wks scid (T- Ign"11) (n = 4) 3.4 + .02 161 30 28.6
>8 wks scid (T- Iggn") (n = 4) 1.5 + .Olb** 170 114 62.8 11
4wks scid(T-Ignull andB -NOD) (n = 4) 5.1 + .02b* 862 + 153b* 31.0 -
>8wks scid(T- IgInullandB -NOD)(n= 3) 4.0 + .01 573 129b* 48.3 +
aValues are given as the mean standard error.
b Statistical comparison of adopotively transferred NOD-scid mouse groups to the age-matched NOD-scid parental
control by the one way ANOVA test: (*P < 0.05, **P < .001).
Apoptosis Detection via Flow Cytometry
The presence of apoptosis is a significant aspect of xerostomia and is a noted event
in disease progression. Apoptosis in the submandibulary acinii cells was detected
following the protocol provided in the Apo-Direct kit (BD Pharmingen). Apoptotic and
non-apoptotic cells were provided as a positive and negative control. The level of
positivity for apoptosis was based on the controls. As seen in Table 3-4, apoptosis was
not detected in the scid (B IL4--) at 4 weeks and 8 weeks post-transfer. Apoptosis was
also detected in the scid (T Ign""ll) at 8 weeks. The NOD mice exhibited a low level of
apoptosis at 4 weeks post-transfer with no apoptosis present at 8 weeks. There was no
apoptotic events detected in the scid negative control or the experimental scid mice at 4
weeks or 8 weeks.
Table 3-4. Apoptosis detection using Apo-Direct kit (BD Pharmingen). The positivity of
each sample is based on the positive and negative controls provided in the kit.
The samples were standardized by the following formula: (% Pos of sample X
% Pos. of Neg. control)/ (% Pos. of Pos. Control). Numerical values are
assigned so that 0/10 is equal to 0% positive for apoptosis and 10/10 is equal
to 100% positive.
Recipient NOD-scid (age) Donor cells Apoptosis Activity (AU)
T- NOD and B- I14-/-
T- NOD and B- 114-/-
T and B NOD
T and B NOD
T Ig "ull
T- Ig[ nul and B I14-
T- Ig[ nul and B- I14/
T Igiunl and B NOD
T Ignunl and B NOD
Caspase-3 Activity Detection
Apoptosis, otherwise known as programmed cell death, is an important inhibitory
mechanism of cell growth. Apoptosis aids in organogenesis, maintenance of tissue
morphology, and deletion of autoreactive lymphocytes. As seen in Fig. 3-1, there are a
series of events that occur in the apoptotic pathway. One of the last events is the
activation of caspases. Caspase-3 is a member of the interleukin 11 converting enzyme
(ICE) family of cysteine proteases. It is activated by a series of upstream proteases and is
one of the last signaling events before apoptosis occurs. The submandibular gland lysate
samples were analyzed in an enzymatic assay. For each treated set caspase activity was
compared at 4 weeks post-transfer to 8 weeks post-transfer. As seen in Table 3-3, the
scid (Unfractionated NOD) depicted a 55% decrease in caspase activity. The
scid (T and B NOD) showed an increase in activity of 40%. The
scid (T-NOD and B-IL 4- -) depicted an increase of over 200%. The scid (B-IL 4/-) had an
increase of 14% in caspase activity. The scid (T-Ignu"ll) and the
scid (T-Ignull" and B-NOD) both depicted an increase in activity of about 50%. The
scid (T-Ignull and B-IL 4- -) depicted a slight decrease of 18%. The NOD had a decrease
in activity of 22% and the scid had a less than 6% increase in caspase activity.
Akt Expression via Polymerase Chain Reaction
As a critical mechanism of homeostasis maintenance, apoptosis also has a large
destructive potential, if not closely controlled. As seen in Fig 3-1, there are many proteins
capable of inhibiting apoptosis. Akt, also known as protein kinase B, is the protein of
interest for this thesis. Akt inhibits apoptosis by phosphorylating, and thus inactivating
procaspases, Bad, and other transcription factors. The level of akt expression is
determined by amplifying cdna, from each sample, by polymerase chain reaction. The
polymerase chain reaction products are visualized by gel electrophoresis. The level of
expression is tabulated for 18s per products (housekeeping gene) and the akt per
products, using the AlphaEase FC program. The relative expression of akt of each treated
sample is then compared to the NOD (pos. control) and the scid (neg. control), to form
ratios. The sample/scid ratio is then compared to the sample/NOD ratio to determine the
relative increase in expression from 4 weeks to 8 weeks post-transfer. As shown in Table
3-5, the scid (Unfractionated NOD) depicted an increase in akt expression when
compared to the NOD and the scid. The scid (T and B NOD),
scid (T NOD and B IL4--) showed a decrease in akt expression compared to the NOD
and the scid. The scid (T Igfnull and B IL4--) decreased in akt expression compared to
the scid, and remained stable compared to the NOD. The scid (B IL4--) depicted an
increase in akt expression compared to the scid but remained stable compared to the
NOD. The scid consistently had an increased level of akt expression from 4 weeks to 8
weeks, when compared to the NOD.
Table 3-5. Akt expression ratios.
Age of treated mouse
4 wks scid (Unfractionated NOD) (n = 4)
>8 wks scid (Unfractionated NOD) (n = 4)
4 wks scid (T and B NOD) (n = 4)
>8 wks scid (T and B NOD) (n = 2)
4 wks scid (T NOD and B IL4-) (n = 4)
>8 wks scid (T NOD and B IL4--) (n = 2)
4 wks scid (B IL4--) (n = 4)
>8 wks scid (B IL4-) (n = 4)
4 wks scid (T- Iggn1l and B IL4-) (n = 4)
>8 wks scid (T- Ignl"" and B IL4-) (n = 2)
4 wks scid (T- Ign"l1) (n = 4)
>8 wks scid (T- Ignul") (n = 4)
4 wks scid (T- Ignull and B NOD) (n = 4)
>8 wks scid (T- Iggn"" and B NOD) (n = 3)
compared to NOD)
aThe fold increase ratio based on the akt expression detected by gel electrophoresis.
bThe amount of akt expressed is 2x or more than the scid control when compared to the adoptively-
In Situ Apoptosis Detection Kit.
The presence of apoptotic cells was detected by TUNEL staining using fixed
section of smg. As seen in Fig. 3-3, apoptosis has detected in untreated scid mice at 4 wks
pos-transfer and 8 wks post-transfer. Apoptotic cells were also detected in the
scid (T and B NOD) mouse, scid (T Ig"ull and B IL-4- -) mouse and the
scid (T Ign"ull and B NOD) mouse. The average number of apoptotic cells per five
fields was determined. As seen in Table 3-6, the adoptively transferred scid mice showed
a higher number of apoptotic cells compared to the scid parental controls.
Detection of Infiltration
Many classification systems use the presence of infiltration in the salivary glands as
a definitive marker of Sj S. Lymphocytic infiltrates are seen in patients with Sj S and the
NOD/LtJ. After euthanization the submandibular glands of the adoptively-transferred and
control mice were removed. A fraction of those submandibular glands were fixed and set
in paraffin blocks. Afterwards, sections of paraffinized tissue were placed onto glass
slides. The tissue was then treated with immunohistochemistry reagents to visualize
infiltrates present in the submandibular gland. These slides were stained with Vector
RedTM and DAB to visualize T and B-cells, respectively. A stained T-cell appears red and
the B-cell appears as brown. A light green counterstain was used as well. As seen in Figs.
3-3 A and 3-3 A, there is no infiltrates present in the NOD-scid. Figs. 3-3 B and 3-3 B
show infiltration of NOD/LtJ submandibular glands. There is a predominance of B-cells
in the population. Figs. 3-3 C and 3-3 C show infiltration is present in the
scid (T Ign"1ul + B IL4-/-), with T-cells being the predominate cell.
Sections of paraffinized tissue were also made into hematoxylin and eosin stained
slide. The slides were visualized by brightfield microscopy in order to detect the presence
of infiltrates. Infiltrated lymphocytes were counted to determine the focus score. The
NOD at both 4 weeks and 8 weeks post-transfer had a focus score of >2 (Table 3-6). The
scid positive control did not show any infiltration at week 4 or week 8.
0 3,- lip .
Figure 3-2: Immunohistochemical staining of submandibular glands for lymphocyte-
infiltrations. Staining was performed on paraffin-embedded sections of
submandibular glands. B-cells stained with DAB (brown), T-cells stained with
VectorRed (red) and counterstained with light green. A) 20 wk old NOD-scid
smg gland; focus score = 0. B) 20 wk old NOD/LtJ smg gland; focus score =
1. C) 16 wk old (4 wks post-transfer) NOD-scid (T Ig""ul + B IL4 -);
focus score = 1. Magnification = 10x.
Figure 3-3. Hematoxylin/eosin-stained tissue sections of submandibular glands. Staining
was performed on paraffin-embedded sections of submandibular glands. A) 20
wk old NOD-scid smg gland; focus score = 0. B) 20 wk old NOD/LtJ smg
gland; focus score = 1. C) 16 wk old (4 wks post-transfer) NOD-scid (T -
Igg"ll + B IL4--); focus score = 1. Magnification = 10x.
The scid (T and B NOD) had a focus score of >2 at 4 weeks with no infiltration
seen by week 8.The scid (T Igg"ull and B IL4-/-) at 4 weeks post transfer had a focus
score of 1 (moderate infiltration), but by 8 weeks no infiltration was detected. The
scid (T Iggnull and B NOD) showed no infiltration at 4 weeks with a focus score of >2
at 8 weeks. The other adoptively transferred mouse groups did not show infiltration at 4
weeks or 8 weeks post-transfer.
* S --: .
S t I *
16*.. ;, ^
I.' -' ,* ;.* *
-. .*4 I 4
'. *? *
-. ,' a' *'* *, *u t
r -, **
4 ~ 7
t '" -: *" -' '
S ..," -- .. ,,
, ,;t t. .
.... ._, -. .. ; ,
I.'#, .'. H. -. .,
:. : .' ,.- .. .
S -* .. : ,, o ,.
: C" -^ -*^ *:"^ '^ ^
~ ~ ~ ~ '. I :.^^l;''l L.^^/ : -_ '-.' ^
Table 3-6. Focus score derived from H+E stained slides of smg glands. A focus score of 1
is equivalent to 50 lymphocytes in a view. 50 lymphocytes in a view is given a
score of 1; 100 lymphocytes is given a score of 2; and over 100 lymphocytes
is given a score of >2.
Recipient NOD-scid (age) Donor cells Focus score
T and B NOD
T and B NOD
T- NOD and B- I14-/
T- NOD and B- 114-/-
T- Ig[ nu and B I14
T- Iggull and B- I14/
T- Ig nu""
T Ig 1ull
T Iggnl and B NOD
T Iggnl and B NOD
Studies using the NOD-scid mouse have led to advancement in the research of Sj S.
Observations on the scid mouse have provided clarification of the glandular and protein
changes that occur in the salivary gland in phase I of Sj S disease. Although the scid
mouse has these glandular changes, its lack of progression to phase II identifies the
critical role lymphocytes have in development of phase II of the disease. Providing the
scid mouse with appropriate lymphocytes was hypothesized to result in progression of
phase II. The results of this thesis provide details on the ability to adoptively transfer
various combinations of lymphocytes from the NOD/LtJ, NOD.IL-4--, and NOD.Iggn""
into the NOD-scid mouse, thereby, transferring phase II of Sj S to this non-autoimmune
The NOD mouse is considered a good mouse model for Sj S research and is the
positive control used in the research of this thesis. It is expected that lymphocytes from
the NOD, placed in the scid mouse, should result in Sj S development. The data show a
46% decrease in the salivary flow, of adoptively transferred scid mice from week 4 post-
transfer to week 8, receiving T and B-cells from the NOD. However, there is no change
in the salivary flow of scid recipients of unfractionated NOD splenocytes. The stable
salivary flow observed in the scid (Unfractionated NOD) mouse implies that there is a
population of cells in the splenic lymphocytes capable of inhibiting disease transfer. The
removal of this population allows for Sj S progression, as seen in the
scid (T and B NOD) mouse. Presumably, the T-cells from the NOD activates antibody
production by the B NOD, thus resulting in the observed salivary loss. The T NOD
also show an ability to transfer disease when combined with B-cells from the NOD.IL 4-/
. The NOD.IL-4-- mouse does not develop phase II disease, even though disease
precursors are present. The absence of IL 4 cytokine, which is necessary for class
switching, prevents disease progression. However, T-cells from NOD mice provide the
IL-4 cytokine, thus allowing antibody production and the resulting decrease in salivation.
The scid (B IL-4--) mouse had little change in saliva volume, possibly due to the
absence of an appropriate T-cell. B -cells from the NOD mouse, as seen in the
scid (T and B NOD) mouse, have an ability to transfer disease when activated by an
appropriate T-cell. The NOD.Ig""ul, which lacks B-cells, has functional T-cells that
produce IL-4 and are capable of activating B-cells. The recipients of T-cells from the
Ig"u"ll mouse and B-cells from the NOD mouse did not show a decrease in salivary flow
as might be predicted, yet transfer of phase II is possible as shown by the
scid (T and B NOD) mouse and the scid (T NOD and B IL-4 -) mouse. Thus,
transferred disease may be delayed in certain combinations, such as the
scid (T Ign""ul and B NOD) mouse. In the NOD environment, antigen presentation by
B-cells to T-cells, or other APC, may occur before the splenocytes are fractionated.
Under such conditions, T-cells are already producing the cytokines required for class
switching before transfer into the scid mouse. This would allow for rapid transfer of
disease. However the Ign"ull, without B-cells capable of presenting antigens, may have
"naive" T-cells. Transfer of these "naive" T-cells may then require a longer incubation
for development of phase II. Thus, it is possible that scid recipients of T Igj"ull and
B IL-4-- did not show a decrease in salivary flow at 8 weeks post-transfer due to the
"naive" nature T-cells from the Ig""1ul mouse. In a previous study, T-cells from the
Ig"ull" were transferred into the IL-4- mouse, resulting in development of phase II (23).
However, the researchers of this study allotted more incubation time for progression of
phase II (12 weeks compared to the 8 weeks allotted in this thesis). It is also possible that
there is a loss in salivary flow in the
scid (T Ign"ull and B NOD) mouse and the scid (T Ign"ull and B IL-4- -), but this
loss is belied by the method of collection. Pooling of saliva during collection and then
quantifying may mask any loss of salivary flow that is occurring in some of the treated
mice. In the scid (T and B NOD) mouse and the scid (T NOD and B IL-4 -) mouse
the loss of salivary flow for the 4 scid mice was not simultaneous. A variation in timing
of the appearance of phase II was also observed in the T Igni"ul transfer to NOD.IL-4--
study. The scid recipients of T-cells from the Ig"ull mouse had normal salivary flow,
indicating no disease progression.
The NOD has been shown previously to be a good positive control, typically
having a 45 75% loss in salivary flow by 20 weeks of age (17,22,25). However, a loss in
saliva volume was not detected in these experiments. Nevertheless, the parental NOD-
scid mouse had stable salivary flow. Diabetes, a disease present in the NOD, appeared in
all of the scid mice that received B and T-cells from the NOD, but not unfractionated
splenocytes. It is apparent that diabetes was co-transferred with phase II. Due to the
presence of diabetes, 0.1 cc of insulin was administered to each mouse, once a day.
Despite treatment, a few of the experimental and positive controls expired early. In order
to prevent the loss of all of the treated mice, some were euthanized prior to 8 weeks post
transfer. This early euthanization could have affected the results.
Concomitant with the loss of saliva secretion, there are significant changes in the
composition and activity of salivary proteins. Probably indicating multiple mechanisms
of action. The decline in amylase may be a result of glandular changes that results from
defects in glandular homeostasis (21). Amylase, an enzyme that hydrolyzes starch, is one
of the glandular proteins that have an age-related decline in NOD and scid mice, and
therefore, a marker of phase I disease. The NOD and scid controls, as well as the
adoptively transferred scid mice, all had a decline in salivary amylase activity with the
exception of the scid (Unfractionated NOD) mouse and scid (T NOD and B IL-4 )
mouse, which had increases of 46%. The decline in amylase activity indicates that there
are glandular changes occurring, to which the adoptively transferred lymphocytes may
respond. Although the scid (T NOD and B IL-4-/-) mouse has an increase in amylase
activity, the decrease in salivary flow suggests transfer of disease. It is not clear why
there is a discrepancy in this particular adoptive transfer combination. Observation of the
results shows variability in the amylase activity of the submandibular glands amongst the
treated scid mice. The NOD positive control and scid negative control both showed a
decline in amylase activity in the submandibular gland lysate. The scid (T and B NOD)
mouse, scid (T Ignu"ll and B IL-4--) mouse and scid (T Ign"ull and B NOD) mouse
showed variable levels of declining amylase activity in the submandibular gland.
However, the scid (Unfractionated NOD) mouse, scid (T NOD and B IL-4'-) mouse,
scid (B IL-4--) mouse and scid (T Igni"l) mouse showed an increase in amylase
activity. It is not certain what causes such variability in detection of amylase activity in
the submandibular gland, when this same method is useful for detection of salivary
amylase activity. There seemed to be significantly less amylase in the submandibular
gland lysate compared to the saliva. Many samples were analyzed undiluted (a few were
diluted 1:100). From a technical point of view, though, it is possible that this assay was
not sensitive enough to detect amylase activity in the smg lysate.
Phase I of Sj S is marked by aberrant production of salivary proteins. There is
production of two neoteric forms of PSP, which enables the protein concentration to
remain constant, despite declines in other major proteins (21). Results show that there is
no change in salivary protein concentration for the adoptively transferred mice or the
controls. However, there were decreases in submandibular gland lysate protein
concentration from scid mice that received a combination of T and B-cells, irrespective of
the origin of the lymphocytes. These decreases in protein concentration from 4 weeks to
8 weeks post-transfer may be indicative of degradation of the submandibular gland by
apoptosis. The scid (T Ign""ll) mouse also exhibited a decrease in protein concentration.
Apoptosis was measured to determine the effect the transferred lymphocytes may
have on inducing cell death in the submandibular gland. Apoptosis occurs in the NOD
and the scid during phase I of Sj S. This apoptosis is lymphocyte-independent and is most
likely a result of the aberrant glandular homeostasis (27,28,29). As Sj S progresses to the
effector stage, the NOD mouse exhibits increased levels of apoptosis in the
submandibular gland, which has been shown to be lymphocyte-dependent (13). The
results of the caspase-3 activity analysis revealed an increase of activity in all of the scid
recipients receiving combinations of T and B-cells, except for the
scid (Unfractionated NOD) mouse. The increase in Caspase-3 activity in scid recipients
of T and B-cell transfer, indicate that the transferred lymphocytes have an effect on
apoptosis. The scid (T Ign"ll) mouse had a large increase (120%) in caspase activity.
This may indicate that there is T-cell dependent role in apoptosis via cytokine production
or cytotoxic action of CD8+ T-cells.
Caspase-3 activation is one of the last required events before apoptosis is
completed, thus the observed increase in activity. However, AKT, as an inhibitor of
apoptosis, would exhibit a decrease of expression. The results indicate a decrease in AKT
expression in the scid (T and B NOD) mouse and scid (T NOD and B IL-4-/-) mouse
compared to the level of AKT expression in the NOD. These groups also had a decrease
of AKT expression when compared to the scid negative control. These findings suggest
that AKT production has been decreased and may be allowing activation of apoptosis in
the submandibular gland. This correlates with the increased level of caspase-3 activity
seen in these two groups. Interestingly, the scid (Unfractionated NOD) mouse depicted
an increase of AKT expression when compared to the NOD and scid, as well as a
decrease in capase-3 activity. This is possibly due to what is a still an unidentified,
inhibitory factor in the unfractionated NOD splenocyte population. The scid (B IL-4- )
mouse did not show a decrease in AKT expression when compared to the NOD mouse
and had a slight increase in expression, when compared to the scid mouse. The increase
of AKT expression in the scid (B IL-4-/-) mouse and concurrent decrease of caspase-3
activity, would indicate a cessation in apoptosis activity. The
scid (T Ign"ull and B IL-4-/-) mouse did not show a decrease in AKT expression
compared to the NOD, but had a decrease in AKT expression compared to the scid
mouse. This group also showed an increased level of caspase-3 activity, which is
indicative of apoptosis. The scid positive control had an average 2-fold increase of AKT
expression compared to the NOD mouse.
The presence of elevated capase-3 activity and decreased AKT expression suggests
the presence of apoptotic cells. Visualization of apoptotic cells by flow cytometry was
attempted. However, apoptosis was only detected in the scid (T Ign""ll) mouse and
scid (B IL-4 -) mouse. It is possible that apoptotic cells were lost in the preparation of
the acinar cells and unable to be detected by flow cytometry. The high level of apoptosis
detected in the scid (T Ig"ull) mouse by flow cytometry may be the result of cytokine
production. It is not clear why a high level of apoptosis was detectable in the
scid (B IL-4 -) mouse. The results of the TUNEL stain show apoptotic cells in the scid
mouse, parental control and the adoptively treated scid mice. Previous studies show that
apoptotic cells are present in phase II, thereby indicating the development of phase II in
the adoptively transferred scid mice. Previous findings show that the number of apoptotic
cells increases as the scid mouse ages (17,19). However the NOD-scid parental control
had a decrease in the number of apoptotic cells present.
A definitive aspect of Sj S is the appearance of infiltrates in the submandibular
gland. The results from the stained histology slides show the presence of infiltrating
lymphocytes in the scid (T and B NOD) mouse, scid (T Ig null and B IL-4'-) mouse,
and the scid (T Igf"ull and B IL-4-/-) mouse. This localization of lymphocytes in the
submandibular gland is a precursor to the loss of salivary flow, thus indicating a transfer
of Sj S-like disease. Interestingly, the scid (T and B NOD) mouse depict a high level of
infiltration (focus score >2) at 4 weeks, and also exhibited a decrease in salivation.
However, the scid (T Ig ~"ll and B NOD) mouse did not have infiltration until 8 weeks
post-transfer, and did not lose salivary flow within that time. This supports the theory that
adoptive transfer of T-cells from the Ign""ul mouse into the scid recipient may require a
longer incubation for Sj S to develop. A similar situation is observed in the
scid (T Ign""ul and B IL-4--) mouse, which had a lesser amount of infiltration then the
scid (T and B NOD) mouse and did not have a decrease in salivation at 8 weeks post-
transfer. Infiltration in the scid (T NOD and B IL-4 -) mouse was not observed at 4 or
8 weeks post-transfer; however, there was a loss of salivary flow. This raises the
possibility of a great effect by cytokine or other soluble factors.
In conclusion, transfer of a clinical Sjogren's Syndrome-like disease into the scid
mouse by adoptive transfer is possible under certain conditions. The results of this thesis
show that a transfer of T-cells from the NOD with functional B-cells will allow transfer
of phase II to the scid recipient. Seemingly, the transfer of entire NOD splenocyte
population may allow for a delay in Sj S or even disease prevention. The results of the
different analysis show that the scid (Unfractionated NOD) mouse consistently had
results that were contradictory to the results observed in the scid (T and B NOD)
mouse. This provides more evidence of a possible inhibitory factor present in the
unfractionated NOD splenocytes. There is a population of T-cells that can inhibit
development of diabetes suggesting an important role of regulatory cells within the NOD
mouse. This population may be present in the unfractionated population, and capable of
preventing disease progression. Although the loss of salivary flow was not detected in
adoptive transfer of T-cells from the Ig"nul mouse together with functional B-cells,
adoptive transfer of phase II may be possible. It is possible that T-cells from the Ign"ull
may not be presented with the necessary antigens until interacting with a functional B-
cell. In this situation, the development of disease would require a longer period of time. It
is also probable that pooling of saliva during collection may mask individual changes in
flow rates and protein content, especially since the loss of salivation may occur at
different times in individual mice. Future directions of this research would be to use the
NOD.B10.H-2b mouse as one of the donor mice. The NOD.B10.H-2b develops SjS, but
does not develop diabetes as per the NOD (30), thus preventing diabetes complications.
Utilization of the NOD.B 10.H-2b should allow for longer observation of the treated
scid mice. In addition to the analysis performed in this study, detection of autoantibodies
in the adoptively transferred mouse sera would be beneficial. Completion of future
experiments may lead to a clarification of the etiopathology of Sj gren's Syndrome,
which may result in better treatment for human patients.
LIST OF REFERENCES
1. Vivino F. The treatment of Sjogren's syndrome patients with Pilocarpine-tablets.
Scand J Rheumatol 2001;115:1-13.
2. Jonsson R, Moen K, Vestrheim D, Szodoray P. Current issues in Sjogren's
syndrome. Oral Diseases 2002;8:130-140.
3. Nepom GT. MHC and autoimmune diseases. Immunol Ser. 1993;59:143-64.
4. Davidson BK, Kelly CA, Griffiths ID. Primary Sjogren's syndrome in the North
East of England: a long-term follow-up study. Rheumatology (Oxford). 1999
5. Rolando M. Sjogren's syndrome as seen by an ophthalmologist. Scand J Rheumatol
Suppl. 2001;(115):27-31; discussion 31-3.
6. Harley JB, Alexander EL, Bias WB, Fox OF, Provost TT, Reichlin M, Yamagata
H, Arnett FC. Anti-Ro (SS-A) and anti-La (SS-B) in patients with Sjogren's
syndrome. Arthritis Rheum. 1986 Feb;29(2):196-206.
7. Kelly CA, Foster H, Pal B, Gardiner P, Malcolm AJ, Charles P, Blair GS, Howe J,
Dick WC, Griffiths ID. Primary Sjogren's syndrome in north east England--a
longitudinal study. Br J Rheumatol. 1991 Dec;30(6):437-42.
8. Reveille JD. The molecular genetics of systemic lupus erythematosus and Sjogren's
syndrome. Curr Opin Rheumatol. 1992 Oct;4(5):644-56.
9. The Jackson Laboratory. http://jaxmice.jax.org/jaxmice-
cgi/jaxmicedb.cgi?objtype=pricedetail&stock=001976 (Accessed 24 March 2003).
10. Humphreys-Beher MG, Brinkley L, Purushotham KR, Wang PL, Nakagawa Y,
Dusek D, Kerr M, Chegini N, Chan EK. Characterization of antinuclear
autoantibodies present in the serum from nonobese diabetic (NOD) mice. Clin
Immunol Immunopathol. 1993 Sep;68(3):350-6.
11. van Blokland SC, Versnel MA.Pathogenesis of Sjogren's syndrome: characteristics
of different mouse models for autoimmune exocrinopathy. Clin Immunol. 2002
May; 103(2): 111-24.
12. Humphreys-Beher MG, Yamachika S, Yamamoto H, Maeda N, Nakagawa Y, Peck
AB, Robinson CP. Salivary gland changes in the NOD mouse model for Sjogren's
syndrome: is there a non-immune genetic trigger? Eur J Morphol. 1998 Aug;36
13. van Blokland SC, van Helden-Meeuwsen CG, Wierenga-Wolf AF, Drexhage HA,
Hooijkaas H, van de Merwe JP, Versnel MA. Two different types of sialoadenitis
in the NOD- and MRL/lpr mouse models for Sjogren's syndrome: a differential role
for dendritic cells in the initiation of sialoadenitis? Lab Invest. 2000 Apr;80(4):575-
14. Stott DI, Hiepe F, Hummel M, Steinhauser G, Berek C. Antigen-driven clonal
proliferation of B-cells within the target tissue of an autoimmune disease. The
salivary glands of patients with Sjogren's syndrome. J Clin Invest. 1998 Sep
15. Crispin JC, Vargas MI, Alcocer-Varela J. Immunoregulatory T-cells in
autoimmunity. Autoimmun Rev. 2004 Feb;3(2):45-51.
16. Rudin CM and Thompson CB. Apoptosis and disease; Regulation and clinical
revelance of programmed cell death. Annu Rev Med. 1997;48:267-81.
17. Kong L, Robinson CP, Peck AB, Vela-Roch N, Sakata KM, Dang H, Talal N,
Humphreys-Beher MG. Inappropriate apoptosis of salivary and lacrimal gland
epithelium of immunodeficient NOD-scid mice. Clin Exp Rheumatol. 1998 Nov-
18. Goillot E, Mutin M, Touraine JL. Sialadenitis in nonobese diabetic mice: transfer
into syngeneic healthy neonates by splenic T lymphocytes. Clin Immunol
Immunopathol. 1991 Jun;59(3):462-73.
19. Bacman S, Sterin-Borda L, Camusso JJ, Arana R, Hubscher O, Borda E.
Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary
Sjogren's syndrome. Clin Exp Immunol. 1996 Jun;104(3):454-9.
20. Nguyen KH, Brayer J, Cha S, Diggs S, Yasunari U, Hilal G, Peck AB, Humphreys-
Beher MG. Evidence for antimuscarinic acetylcholine receptor antibody-mediated
secretary dysfunction in nod mice. Arthritis Rheum. 2000 Oct;43(10):2297-306.
21. Robinson CP, Yamamoto H, Peck AB, Humphreys-Beher MG. Genetically
programmed development of salivary gland abnormalities in the NOD (nonobese
diabetic)-scid mouse in the absence of detectable lymphocytic infiltration: a
potential trigger for sialoadenitis of NOD mice. Clin Immunol Immunopathol. 1996
22. Robinson CP, Brayer J, Yamachika S, Esch TR, Peck AB, Stewart CA, Peen E,
Jonsson R, Humphreys-Beher MG. Transfer of human serum IgG to nonobese
diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretary
function of exocrine tissues in Sjogren's syndrome. Proc Natl Acad Sci U S A.
1998 Jun 23;95(13):7538-43.
23. Brayer JB, Cha S, Nagashima H, Yasunari U, Lindberg A, Diggs S, Martinez J,
Goa J, Humphreys-Beher MG, Peck AB. IL-4-dependent effector phase in
autoimmune exocrinopathy as defined by the NOD.IL-4-gene knockout mouse
model of Sjogren's syndrome. Scand J Immunol. 2001 Jul-Aug;54(1-2): 133-40.
24. Rohane PW, Shimada A, Kim DT, Edwards CT, Charlton B, Shultz LD, Fathman
CG. Islet-infiltrating lymphocytes from prediabetic NOD mice rapidly transfer
diabetes to NOD-scid/scid mice. Diabetes. 1995 May;44(5):550-4.
25. Szanya V, Ermann J, Taylor C, Holness C, Fathman CG. The subpopulation of
CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-
selectin and high levels of CCR7. J Immunol. 2002 Sep 1;169(5):2461-5.
26. Serreze DV, Chapman HD, Varnum DS, Hanson MS, Reifsnyder PC, Richard SD,
Fleming SA, Leiter EH, Shultz LD. B lymphocytes are essential for the initiation of
T cell-mediated autoimmune diabetes: analysis of a new "speed congenic" stock of
NOD.Ig mu null mice. J Exp Med. 1996 Nov 1;184(5):2049-53.
27. van Blokland SC, Van Helden-Meeuwsen CG, Wierenga-Wolf AF, Tielemans D,
Drexhage HA, Van De Merwe JP, Homo-Delarche F, Versnel MA. Apoptosis and
apoptosis-related molecules in the submandibular gland of the nonobese diabetic
mouse model for Sjogren's syndrome: limited role for apoptosis in the development
of sialoadenitis. Lab Invest. 2003 Jan;83(1):3-11.
28. Humphreys-Beher MG, Peck AB, Dang H, Talal N. The role of apoptosis in the
initiation of the autoimmune response in Sjogren's Syndrome. Clin Exp Immunol.
1999 Jan; 116: 383-387.
29. Yamamoto H, Sims NE, Macauley SP, Nguyen KH, Nakagawa Y, Humphreys-
Beher MG. Alterations in the secretary response of non-obese diabetic (NOD) mice
to muscarinic receptor stimulation. Clin Immunol Immunopathol. 1996
30. Cha S, van Blockland SC, Versnel MA, Homo-Delarche F, Nagashima H, Brayer J,
Peck AB, Humphreys-Beher MG. Abnormal organogenesis in salivary gland
development may initiate adult onset of autoimmune exocrinopathy. Exp Clin
Vinette B. Brown was born in Brooklyn, New York, in 1979. She comes from a
very large extended Jamaican family. When she was 10 years old, she and her parents
moved to Hollywood, Florida, where she graduated from Hollywood Hills High School
in 1997. Afterwards, she received a Bachelor of Science in zoology, with a minor in
business from the University of Florida in 2001. She went on to receive a Master of
Science in molecular genetics and microbiology in August 2004 from the University of
Florida. Vinette will work towards earning a Doctor of Dental Science degree at the
School of Dental and Oral Surgery at Columbia University.