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Regulation of murine interferon gamma production by epidermal growth factor

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Regulation of murine interferon gamma production by epidermal growth factor
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Abdullah, Na'eem Adib, 1956-
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viii, 63 leaves : ill. ; 29 cm.

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Antibodies ( jstor )
Cell growth ( jstor )
Cells ( jstor )
Fibroblasts ( jstor )
Human growth ( jstor )
Interferons ( jstor )
Lymphocytes ( jstor )
Molecules ( jstor )
Receptors ( jstor )
Signals ( jstor )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( mesh )
Growth Substances ( mesh )
Immunology and Medical Microbiology thesis Ph.D ( mesh )
Interferon Type II -- biosynthesis ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1988.
Bibliography:
Bibliography: leaves 52-62.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Na'eem Adib Abdullah.

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University of Florida
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REGULATION OF MURINE INTERFERON GAMMA PRODUCTION BY EPIDERMAL GROWTH FACTOR










By

Na'eem Adib Abdullah











A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1988
















ACKNOWLEDGEMENTS

Allah, God the Creator, has asked people to study creation and to

find and use its hidden treasures to help mankind. This dissertation is dedicated to that end. The last revelation of Allah, the Holy Qur' an, is unparalleled in its prophetic scientific description of natural phenomena. Allah is the greatest (Al Kabir), the most wise (Al Hakim), and the most kind (Al Latif).

I give a special thanks to Howard M. Johnson, Ph.D., as my mentor

and guide through the "real world" of scientific research. His piercing insights have helped me enormously in maturing as a scientist. I thank the supervisory committee members Donna H. Duckworth, Ph.D., Lindsey M. Hutt-Fletcher, Ph.D., and Arthur K. Kimura, Ph.D., for their many suggestions and scientific insights in the preparation of this manuscript. Another titan that helped me at the bench, in writing and through discussions was Barbara A. Torres, M.S. She took precious time away from running the lab to instruct me on many new techniques and the nuances and quirks of each of them. The two postdocotral scientists Carol Hanlon-Pontzer, Ph.D., and Jef fry K. Russell, Ph.D., and the graduate students Myron 0. Downs, D.V.M., Michael A. Jarpe, B.S., and Harold I. Magazine, B.S., were great peers and counsel to interact with in many areas of research and allowed me to look at a given problem from different prospectives. Miss Sarah A. Hunt was most helpful in ordering



ii









and tracking materials, reagents and scientific correspondences required for my experiments as well as helping to set up actual experiments.

I wish to thank my parents, Marvin Moore Anderson, M.D., and

Dorothy-Jean Barbee Anderson and brothers Mansur Abdullah, Clarence H. Anderson, Christopher A. Anderson, Eric Anderson, M.D., and his wife Ellen, who gave me tremendous love, support and encouragement throughout my graduate studies. A special thanks to Juanita Maria Maxwell whose encouragement and companionship were appreciated. They will never know how much their support meant to me.

I must thank my many other relatives, friends, colleagues, and associates who are too numerous to mention but whose support and encouragement were deeply appreciated and spurred me on to obtain the Doctorate of Philosophy. Finally, thankyou Jennifer (Maynard) for preparing and retyping this manuscript through its numerous revisions and impending deadlines.
















TABLE OF CONTENTS



ACKNOWLEDGEMENTS......................................................1ii

ABBREVIATIONS.......................................................... v

ABSTRACT............................................................. vii

CHAPTERS

1 INTRODUCTION................................................... 1

Interferon Gamma...............................................1I
The Interferon Gamma Molecule.............................1I
Regulation of IFNY Production............................. 2
Immunoregulatory Functions of IFN Y....................... 3
Epidermal Growth Factor........................................ 5
The EGF Molecule.......................................... 5
Functional Roles of EGF................................... 6
EGF Related Peptides TGFa and VOF......................... 7
The EGF Receptor.......................................... 8
Specific Aims............................................. 11

2 MATERIALS AND METHODS.......................................... 17

Mice........................................................... 17
Reagents....................................................... 17
IFN YProduction................................................ 18
Balb/c 3T3 Fibroblast Mitogenic Assay......................... 18
Anti-EGF Antibody Functional Studies.......................... 19
Radioiodination of EGF......................................... 19
Baib/c 3T3 Fibroblast Growth Factor Binding Assays ............20
Murine Spleen Lymphocyte Binding Assays....................... 20
Reverse Phase High Performance Liquid Chromatography
of Radioiodinated EGF...................................... 21

3 RESULTS........................................................ 22

4 DISCUSSION..................................................... 41

APPENDIX............................................................. 46

REFERENCES............................................................ 52

BIOGRAPHICAL SKETCH................................................... 63

iv
















ABBREVIATIONS



BSA, bovine serum albumin, radioimmunoassay grade from Sigma CPM (cpm), counts per minute EBSS, Earle's balanced salt solution EGF, epidermal growth factor FBS, fetal bovine serum FGF, fibroblast growth factor Hepes, N-2-Hydroxyethyl piperazine-N'-2-ethanesulfonic acid HMEM, minimal essential medium modified with Earle's salts, penicillin,

streptomycin and 1 mM glutamine

HPLC, high performance liquid chromatography hr, hours

IFN, interferon M, molar

MAF, macrophage activation factor pM, micromolar

mM, millimolar

nM, nanamolar

min, minutes

NaN3, sodium azide PBS, phosphate buffered saline PDGF, platelet-derived growth factor Pen, penicillin 100 U/ml final concentration

v









rpm, revolutions per minute SDMEM, Dulbecco's minimal essential medium supplemented with 15 mM Hepes,

lx nonessential amino acids, 1 mM sodium pyruvate, 0.075% sodium

bicarbonate, penicillin, streptomycin, and 1 mM glutamine SEA, staphylococcal enterotoxin A sec, seconds

Strep, streptomycin 20 pg/ml final concentration TGFa, transforming growth factor a TH, T helper cell TS, T suppressor cell VGF, vaccinia virus growth factor



































vi















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

REGULATION OF MURINE INTERFERON GAMMA PRODUCTION BY EPIDERMAL GROWTH FACTOR

By

Na'eem Adib Abdullah

December, 1988

Chairman: Howard M. Johnson
Major Department: Immunology and Medical Microbiology

It has recently been shown that the epidermal growth factor (EGF)

molecule is capable of positive regulation of immune function by inducing the production of gamma interferon (IFNY), establishing a functional relationship between nonhematopoietic growth factors and the immune system. In order to study this relationship further EGF and EGF-related growth factors, transforming growth factor a (TGFa), and vaccinia virus growth factor (VGF), which all exert their effects on cells through the EGF receptor on cells, were studied for their functional and physicochemical effects on IFNY production at the receptor level.

IFN yproduction requires T helper (TH) cell function. Both purified murine EGF and recombinant human EGF were capable of restoring competence for IFNy production by murine spleen cell cultures depleted of TH cells. In agreement with this observation, anti-EGF antibodies were capable of specifically neutralizing the EGF effect. Interestingly, EGF receptors are not constitutively expressed on normal or primary lymphocytes. We observed that treatment of lymphocytes with T-cell mitogens induced the vii









expression of the EGF receptor, analogous to the induction of the high affinity receptor on lymphocytes for interleukin-2. Neither TGFa nor VGF were capable of restoring competence for IFNy production by TH cell depleted spleen cell cultures. Furthermore, neither TGFa nor VGF could block the ability of EGF to restore competence for IFN production, suggesting that the induced EGF receptor on lymphocytes is different from that which is expressed on nonhematopoietic cells such as mouse 3T3 fibroblasts.

Consistent with the functional data for a novel inducible EGF receptor on lymphocytes, the receptor was also detected by binding studies with 1251-labeled EGF. The receptor has a dissociation constant of about 5 nanomolar. The receptor is optimally expressed after 48 hr treatment of lymphocytes by T-cell mitogen. TGFa did not compete with 1251-EGF for the lymphocyte EGF receptor. Both TGFa and VGF induced proliferation of mouse 3T3 cells. Furthermore, consistent with previous studies, both mitogens competed with EGF in receptor binding studies on 3T3 cells. Thus, the failure of TGFa and VGF to functionally mimic the EGF help in IFNy production, and the failure of TGFa to compete for receptor on lymphocytes, is compatible with the hypothesis that lymphocytes express a novel inducible EGF receptor that differs from the classic receptor expressed on cells such as 3T3 fibroblasts.













viii
















CHAPTER 1

INTRODUCTION

Interferon Gamma

The Interferon Gamma Molecule

In 1957 an antiviral substance was discovered by Isaacs and

Lindenmann (1). This substance, which they termed interferon (IFN), plays an important role in host defense against viral infection. There is a large body of evidence that IFNs, particularly IFNy, play a central role in the regulation of a variety of immune functions.

The human and murine IFNs are classified into three broad groups

which are based on antigenic properties, analogous to the classification of immunoglobulin isotypes (2). They are designated IFNa, IFN, and IFN Induction of IFNa and IFN in appropriate cell types is achieved by the addition of virus or polyribonucleotides, while IFNy is induced in T lymphocytes or natural killer (NK) cells by specific antigens or T cell mitogens (Table 1-1) (3).

The genes for all of the known IFNs have been cloned. The cloning data has revealed 23 IFNa genes, at least 15 of which code for full length proteins (4). IFNa is apparently glycosylated. The IFNa genes, none of which contain introns, are clustered near each other on the same chromosome. IFNa induces rearrangement of the T cell antigen receptor achain and matures T lymphocyte clones to cytotoxicity in vitro (5,6). There are at least 3 IFN genes, one of which does not contain introns, while the other two appear to contain 4 introns (7-12). The intron1









2

containing IFNB genes are called IFNB2 (7). IFNB2 has a number of interesting properties in addition to its antiviral properties (8-13). While IFNB2 was initially identified as an antiviral protein isolated from fibroblasts, others have found that IFN02 from T cells acted as a Bcell differentiation factor (BSF-2) that can enhance antibody secretion and induce B-cell proliferation (8,10). IFNB2 is also called interleukin-6 (IL-6) (13).

IFNyhas been cloned from the human (14,15), mouse (16), and bovine

(17) genomes. Only one gene has been found in all 3 species and it contains 3 introns. The precursor polypeptide is processed and glycosylated. Based on the cDNA structure of the human and mouse genes the mature IFNYmolecule is composed of 133 amino acids and has a mass of 17,000 daltons (14,16). The molecular weight of natural IFNy varies from 20,000 to 25,000 daltons by SDS-PAGE due to glycosylation of the mature molecule (18). Gel filtration studies have assigned IFNya mass of 40,000 to 50,000 daltons suggesting a natural multimer formed by the mature molecule (19).

Regulation of IFNy Production

The cellular requirements for IFNY production have been studied in detail in both the human and mouse systems (20-22). IFNY production in the C57BL/6 mouse spleen cell system is regulated by a dynamic interaction between helper cells (Lyt 1+,2-), suppressor cells (Lyt 1+,2+), and IFNy-producing cells (Lyt 1-,2+) (Figure 1-1) (3). Helper cells aid IFNyproduction via production of interleukin-2 (IL-2), which acts in concert with a T-cell mitogen, staphylococcal enterotoxin A (SEA), to induce IFNY. Suppressor cells inhibit or block IFNY production by









3

adsorption or sequestration of IL-2. The IL-2 helper signal for inducrion of IFN yis dissociated from its ability to stimulate T-cell proliferation (3).

The question arose as to whether nonhematopoietic growth factors were also able to stimulate IFNY production. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), were shown not to stimulate lymphocyte proliferation (23,24). It was demonstrated that these growth factors induce the production of IFNYin conjunction with a T-cell mitogen signal (23,24). By comparison, neither rat growth hormone nor human growth hormone are capable of inducing IFNY production. Immunoregulatory Functions of IFNy

Interest in the immunoregulatory functions of IFNs began with the observation that murine IFNa and IFN suppressed antibody production in in vitro assays (25,26). Concurrent studies showed that IFN could also regulate cellular immunity and delayed type hypersensivity (27,28). Once it was esLablished that IFN enhanced natural killer (NK) cytotoxic activity, the immunology community began a more rigorous study of the immunomodulatory effects of the IFNs. It became apparent that the scope of the immunoregulatory effects of the IFNs went far beyond the initial observations, as shown in Table 1-2 (25-50).

Some of the immunoregulatory properties of IFNy are described herein. Class I MHC molecules are up-regulated upon the addition of IFNy (44,45). This surface antigen is important for the efficient killing of virally infected cells. Class II MHC antigens are also up-regulated by IFNy

(43). The class II molecules are required for the cellular interactions important for immune function such as antigen presentation. The expres-











sion of class II molecules on the surface of macrophages is modulated by iFNy as shown by their induction upon the addition of recombinant IFNY (43,51). Indeed there are other surface molecules which are also up regulated by IFNY, including the IL-2 receptor and Fc receptors (48,52).

Another important function of IFNY is its activity as a macrophage activating factor (MAF) which augments various functions of macrophages, including increased secretion of proteinases, hydrolases, oxygen metabolites, and increased killing capacity for tumor or virally infected cells. A ivmphokine MAF activity was identified when it was observed that activated T lymphocytes or their soluble products could prime or activate macrophages for increased tumor cell killing (53). After activation, macrophages are triggered to kill tumor cells by the addition of a small amount of lipopolysaccharide which acts as a second signal. Studies using purified IFN Y, recombinant IFN y, and IFN yneutralizing antibodies have shown that the MAF activity is predominantly, if not exclusively, due to the IFNY molecule. This data does not preclude there being other structurally distinct MAFs. For example, lFNa and IFNB are also MAFs, though not as potent as IFNy (54). As mentioned previously, T killer lymphocyte and natural killer (NK) cytotoxic activity against tumor cells and virally infected cells is also enhanced by the addition of IFNs, especially IFNy (3).

Finally, IFNy has been shown to modulate the differentiation of myeloid and B lymphocyte cells (50,55). In the latter case the differentiation signal provided by recombinant IFNY, was shown to be sufficient to induce the secretion of antibodies (50).








5

Thus, IFNy is an immunoregulatory molecule which can modulate a wide range of physiological states in a variety of cell types; its biological functions augment both cellular and humoral defense systems as well as identify potential targets for immune responses. An understanding of the biological signals which regulate the production of IFNy will help in understanding the initiation of the complex immune response. The work presented here addresses the regulation of production of IFNy by EGF with particular emphasis on receptor recognition.

Epidermal Growth Factor

The EGF Molecule

EGF is mitogenic for a wide variety of cell types. Studies on EGF and its receptor have helped to increase our understanding of cell growth, ligand-receptor interactions, receptor activation, and oncogenes (56,57).

EGF was discovered in 1959 when it was observed that murine submaxillary gland extracts, when injected into newborn mice, induced early eyelid opening and incisor eruption (56,58). Purification of the responsible substance showed that the active factor was EGF and not nerve growth factor which is also present in extracts of murine submaxillary glands (56). The purified EGF was also shown to stimulate proliferation of epidermal cells and keratinocytes (59,60).

Mature murine submaxillary gland EGF is composed of 53 amino acids ,,7ith a molecular mass of 6,000 daltons (61). It has three disulfide bridges which are required for biological activity. EGF is not glycosylated. There is 70% homology between mouse and human EGF and the two molecules behave identically in cell and biochemical assays (56,57,62).









6

The EGF gene encodes a large precursor molecule containing EGF and 7 related peptides (63,64). The mature EGF molecule is contained in the carboxyl terminal region of the precursor molecule and is obtained by processing of the polyprotein. The functions of the other EGF-related peptides in the polyprotein are currently unknown.

As indicated above, the submaxillary gland is a major source of EGF in the mouse, whereas platelets are a major source of EGF in humans (65,66). The submandibular and Brunner's glands are also thought to be sources of EGF in humans (67). Studies are in progress to further determine the roles of various hematopoietic cells as sources of EGF and related growth factors (68,69).

Functional Roles of EGF

Purified EGF has a wide range of biological effects. These include

(1) augmentation of ectodermal and mesodermal cell (e.g. fibroblast) proliferation (56); (2) regulation of spermatogenesis (70,71); (3) Inhibition of gastric acid secretion (72); and (4) regulation of immune functions such as IFNy induction (22,23).

With regard to the first point, the proliferative effect of EGF on ectodermal and mesodermal cells types in vitro suggests that it may play a role in wound healing. in controlled experiments EGF does indeed aid in the wound healing process (73). With regard to the second point, EGF apparently plays a major role in spermatogenesis. Male mice which have been treated so as to not produce EGF (by submaxillary gland removal) have low sperm and spermatid counts, while the addition of exogenous EGF to such mice reverses this condition. Humans also have an EGF-like molecule in their seminal plasma. Thus, EGF is probably a differenti-









7

ation signal for spermatogenesis in mammals. The third point, addressing EGF's ability to inhibit gastric acid secretion, suggests that EGF can inhibit gastric acid secretion without stimulating cell growth. Thus, EGF may exert its effects in the alimentary canal not just as a growth factor but also as a regulator of gastric acid secretion.

Finally, EGF has recently been shown to modulate the production of the immunoregulatory molecule IFNy. The Observation is unique because nonhematopoietic growth factors were heretofore thought not to play a role in the immune system. Also, the literature suggests that hematopoietic cells do not constitutively express EGF receptors (57). Thus, the observation implies that under certain conditions hematopoietic T cells do express EGF receptors which, when bound by the EGF ligand, initiate the production of an important biological response modifier, IFNY.

EGF Related Peptides Transforming Growth Factor a and Vaccinia Virus
Growth Factor

Transforming growth factor a (TGFa) and vaccinia virus growth factor (VGF) are structural and functional relatives of EGF. Both molecules, or their relevant functional sequences, have (1) approximately 30% homology with the EGF molecule (74-76); (2) have the same number of cysteines and disulfide linkage alignment (77); (3) compete with EGF for its receptor and use the EGF receptor solely for their known functions (69,75,76); and

(4) are mitogenic for the same tissues as EGF (57). The structural and functional similarities imply that the three growth factors evolved from a common ancestral gene (78,79).

TGFa was initially found in the supernatants of transformed cells and was thought to be related to oncogenesis (80). But it is now known









8

to be a growth factor encoded in the genome of several species. The TGFa gene encodes a messenger RNA (mRNA) that synthesizes a precursor polypeptide, but unlike EGF there is no evidence of TGFa related peptides in the precursor molecule. The mature TGFa molecule is a 6,000 dalton protein of 50 amino acids (74,75) and like EGF it is not glycosylated. Biological properties that TGF has demonstrated are mitogenic effects on fibroblasts (57,69), and wound healing by inducing keratinocyte proliferation and migration (81). TGFa is synthesized by both macrophages and keratinocytes and thus may contribute directly to wound healing in vivo as both cells are found in such lesions (68,82,83).

VGF, like EGF and TGFa, is synthesized as a precursor polypeptide. The mature VGF molecule has 77 amino acids, is glycosylated, and has a molecular mass of 23,000 daltons as determined by SDS polyacryiamide gel electrophoresis (76). The structural relationship of EGF, TGFa, and VGF is shown in Figure 1-2.

VGF has several EGF-like and other biological activities which

include: (1) mitogenic activity for fibroblast cells in vitro (57); (2) initiation of mitogenesis and migratory activities for keratinocytes to aid in wound healing (81); (3) augmentation of production of viral progeny from infected cells (84). This latter property suggests that VGF aids the lifecycle of Lhe vaccinia virus through its cellular proliferative properties.

Again, it should be emphasized that all of the known effects of TGFa and VGF are mediated through the EGF receptor. The EGF Receptor

The EGF receptor, also called c-erb B-1 (57,85), has been found on almost all tissues but has not been shown to be constitutively produced









9

in hematopoietic cells. Studies have not been carried out to determine if the receptor is inducible in such cells as primary lymphocytes in a manner analogous to the receptor for IL-2, where the high affinity IL-2 receptor is not constitutively expressed but is induced by lymphocyte mitogens (86). The EGF receptor is a 170,000 dalton single chain glycoprotein with intrinsic tyrosine kinase activity (87). The mature receptor is composed of three major domains, which are: (1) a large glycosylated extracellular domain, (2) a transmembrane hydrophilic region of 23 amino acids, and (3) a cytoplasmic region containing the tyrosine kinase domain (57) that is composed of residues that are characteristic of the tyrosine kinase family (88). Binding of EGF to its receptor results in activation of the protein tyrosine kinase and thus triggers subsequent intracellular events (57,89). The binding site of EGF on its receptor is between residues 294 to 543 (90). Signal transduction is mediated through the autophosphorylation of tyrosine residue 1173 (91).

Binding experiments with radiolabeled EGF to its receptor demonstrate a stoichiometric ligand-receptor interaction of one-to-one (92). Scatchard analysis of EGF binding to intact cells, with high receptor numbers, suggests the presence of different receptor classes with distinct affinities toward EGF. High affinity receptors comprise 5 to 10% of the total, while the remaining receptors are of low affinity (93). In EGF receptor negative 3T3 cells the introduction of wild type EGF receptor showed both high and low affinity binding (94). Thus, the basis for the same EGF receptor showing different affinities is not known.

Cells which are treated with the tumor promoter phorbol myristate acetate (PMA) or platelet-derived growth factor (PDGF) abolish the high-








10

affinity receptor state (93,95-99). This "receptor transmodulation" process also reduces the tyrosine kinase activity of the EGF receptor. Receptor transmodulators bind to their own distinct receptors (100) and activate protein kinase C (and/or other kinases) which phosphorylates EGF receptor threonine (Thr) 654. Phosphorylation of Thr 654 on the EGF receptor by PMA has been shown to block the mitogenic effects of EGF, but this phosphorylation event occurs before phosphorylation by the tyrosine kinase phosphorylase (101).

The EGF receptor has a half-life of greater than 5 hr (89). After ligand binding the half-life decreases to approximately 1 hr. Receptor degradation is mediated by coated pit internalization and subsequent lysosomal enzyme degradation (56). EGF receptor mutation studies have shown that the kinase activity is also essential for degradation of the wild type receptor (89,100).

The EGF receptor molecule has several relatives. The c-erb B-2 gene product is a 185,000 dalton glycosylated protein that shares about 50% homology with the EGF receptor (102). It has an extensive extracellular domain like its c-erb B-1 counterpart yet it does not bind EGF or any known ligand. C-erb B-2 transcripts have been detected in placenta, kidney, embryonic tumors, stomach adenocarcinomas, and neuroblastomas. Greater than 80% of the tyrosine kinase domain in c-erb B-2 is homologous with its EGF receptor counterpart. The c-erb B-2 receptor is phosphorylated via interaction of EGF with the c-erb B-1 receptor and is believed to be a substrate for the c-erb B-1 phosphorylating activity (103,104). Four independent cell lines have the identical mutation from valine 664 to glutamic acid in c-erb B-2 in the transmembrane region,









11

which subsequently converts it to the oncogenic form called neu (105,106). The neu oncogene product of c-erb B-2 differs from its wild type counterpart by this single point mutation which drastically alters the function of the gene product. Another related oncogene is the v-erb B gene product from the avian erythroblastosis virus (107). It is 68,000 daltons and has approximately 95% homology to the EGF receptor within a stretch of 400 cytoplasmic domain residues (57). The amino terminal of this molecule is truncated and thus does not bind EGF and there is also a small truncation at the carboxyl terminus. The v-erb B oncogene has autophosphorylation activity (108,109).

Thus, the interaction of EGF with its receptor initiates a complex series of events which are responsible for EGF induced physiological cell functions.

Specific Aims

The objective of the work outlined in this dissertation is to

characterize the EGF helper signal for induction of IFNy in a C57B1/6 murine splenic T cell system. We will focus on the functional and receptor binding properties of EGF on mouse splenic lymphocytes as compared to the interactions of EGF with its classic receptor on murine 3T3 fibroblast cells. We propose to achieve the objective through the following specific aims: (1) definitively determine if EGF can provide the helper signal for IFNy induction by using both EGF purified to homogeneity and recombinant EGF in IFNy production studies; (2) determine if the EGF related growth factors TGFa and VGF have the ability to provide the helper signal for IFNY production and/or modulate the EGF helper signal through competition for the receptor. Such studies should









12

provide insight into the relationship of the EGF receptor on lymphocytes to the well characterized EGF receptor on 3T3 fibroblasts to which all three mitogens bind and induce cellular proliferation; (3) determine if the EGF receptor on splenic lymphocytes is constitutively produced or is induced upon stimulation with SEA, and determine the binding characteristics of EGF with this receptor; (4) determine if TGFa and/or VGF can compete with EGF for the EGF receptor on splenic lymphocytes.










13











A


Ti Ts


Lyt I ,2D / Lyt 1 ,2



IL2


A



I F Ni y
Producer Lytl-,2+







Figure 1-1. IFNY production is regulated by the dynamic interaction between helper (TH), suppressor (TS), and IFNy producer cells. Helper cells provide IL-2. Suppressor cells absorb IL-2. Arrows: (- ),
positive signal; (----. negative signal (3).









14








0 50 60
VGF P D PA R L C P 0 s G Y C H G C I H A R 0

TGFa V V S H N K C P 0 S H T Q Y CF H T C R F L V Q

MEG NS YPC-CP S Y 0 G Y CLN G G VCM H I E S

h EGF NS D S ECPL 5 H D G Y CL H 0G V C I Y EA




70 80 90
VGF I D G YAC RC S H G Y T G 1 R C Q H V V L V D Y Q R

TGF E:E K PACVCH 5 G Y V G VR RCEHADLLA

mEG F L Y T CNC G Y G RC T R D LR W W E L R

hEGF .L 0 K -Y A C N C V V G Y IG E R C Q Y R 0 L K W W E L R











Figure 1-2. Alignment of the VGF, rTGFa (rat), mEGF (mouse), and hEGF (human). The sequences of the mature peptide growth factors are shown in their entirety, numbered from the N-terminus of the precursor VGF. Residues conserved in all four sequences are boxed. The VGF sequence represents the recombinant subcloned VGF used in the present studies and was provided by Oncogen Inc., Seattle, WA. Adapted from Brown et al. Nature 313:491 (1985) Reprinted from Nature, Vol. 313, No. 6002, pp. 491-492. Copyright (c) 1985 by Macmillan Journals Limited (77).









i5




Table 1-i
Classification of IFN's


Former
Interferon Cellular Source Inducer Nomenclature


IFN-a Lymphocytes (B, null, Viruses, polyribonucleo- Leukocyte,
and T), macrophage? tides, tumors, chemicals Type I
IFN-3 Fibroblast, epithelial, Viruses. polyribonucleo- Fibroblast,
macrophage Lides, chemicals Type I
IFN-y T lymphocyte Antigens, T cell mitogens Immune,
Type TI

Reprinted from Lymphokines 11:33, 1985. Copyright (c) 1985 by Academic Press, Inc. (3)









16





Table 1-2
Some Immunoregulatory Effects of IFN Effects References

Suppression and enhancement of antibody production 25,26,29-31

Suppression of antigen- and mitogen-induced
lymphocyte proliferation 27,28,32

Enhancement of specific cytotoxicity of T lymphocytes 33

Enhancement of NK cell cytotoxicity 34-37

Enhancement of antibody-dependent cell-mediated
cytotoxicity 38

Activation of macrophages for enhanced tumor cell
killing 39-41

Modulation of expression of products of the major histocompatibility complex on the cell membrane 42-45

Modulation of expression of Fc receptors on the
cell membrane 46-48

Modulation of expression of interleukin 2 receptors
on the cell membrane 49

Maturation of B cells for immunoglobulin production
and secretion 50

Reprinted from Lymphokines 11:33, 1985. Copyright (c) 1985 by Academic Press, Inc. (3)















CHAPTER 2

MATERIALS AND METHODS



Mice

C57B1/6 female mice, 8 to 12 week old, are obtained from the Jackson Laboratory, Bar Harbor, ME.

Reagents

The T cell mitogen SEA is purchased from Toxin Technology, Madison, WI. Ultrapure mouse submaxillary gland EGF is obtained from Toyobo Co., Ltd., New York, NY. Recombinant human EGF is obtained from Scott Laboratories, Fiskeville, RI. Highly purified synthetic rat TGFa is obtained from Peninsula Laboratories, Inc., Belmont, CA. Recombinant VGF is a gift from Dr. Gregory Bruce of Oncogen, Inc., Seattle, WA. Recombinant c-sis PDGF (PDGF B) is from AMGen Biologicals, Thousand Oaks, CA. 1251odide as a sodium salt (15 mCi/pg) and tritiated thymidine (21 mCi/mg) are obtained from Amersham Corp., Arlington Heights, IL. Monoclonal anti-Lyt 1.2 antibody and anti-Lyt 2.2 antibody are obtained from New England Nuclear, Boston, MA. The source of complement is serum from New Zealand white rabbits. Endotoxin-free fetal bovine serum (FBS) is obtained from HyClone Laboratories, Logan, UT. Trifluoroacetic acid and radioimmunoassay grade bovine serum albumin (BSA) are obtained from Sigma, St. Louis, MO. Dibutyl phthalate (gold label) and dioctyl phthalate are obtained from Aldrich Chemical Co., Milwaukee, WI. Rabbit anti-mouse EGF antibody (IgG fraction) is obtained from Collaborative 17









18

Research Inc., Bedford, MA. HPLC grade acetonitrile is obtained from Fisher Scientific, Orlando, FL.

IFN-y production

Spleen cells (1 ml at 1.25 x 107 cell/ml), 0.1 ml of a 10-2 dilution of monoclonal anti-Lyt 1.2 antibody and rabbit complement (0.1 ml of serum) are mixed and incubated at 37C for 1.5 hr in RPMI 1640 containing 10% FBS. The helper cell-depleted cultures are then washed twice, suspended in the RPMI 1640 media containing 10% FBS, and cultured in duplicate at 1.25 x 107 viable cells/ml. Whole spleen cell- and helper cell-depleted cultures are stimulated with 0.5 pg/ml of SEA for 1 to 3 days in the presence of growth factors, and the supernatants are assayed for IFNy activity on mouse L cells by using vesicular stomatitis virus as described (110). The IFN produced is identified as IFNY by neutralization with specific antisera.

Balb/c 3T3 Fibroblast Mitogenic Assay

The 3T3 growth factor mitogenic assay is performed using a

modification of Kobayashi et al. (111). The 3T3 fibroblasts are grown to subconfluent monolayers in supplemented Dulbecco's minimal essential medium (SDMEM) containing 10% FBS. Note, 3T3 fibroblasts are not used beyond passage number 40. The cells are harvested, washed and seeded in 96 well plates (Falcon) at 5 x 104 cells/well in 0.2 ml. All peripheral wells receive 0.2 ml of medium only to keep the humidity of the target wells constant. After 24 hrs in 5% CO2, 37'C the spent medium is replaced with 0.1 ml of SDMEM containing either 10% FBS or 0.2% FBS with or without dilutions of growth factors in duplicate. After 22 hrs at 5% CO2, 37C, 0.1 PCi of 3H-thymidine in 10 pl is added to each well. The samples are incubated another 2 hrs at 37'C, 5% CO2. After washing twice









19

with phosphate buffered saline (PBS), cells are solublized by treatment with 0.1 M NaOH (0.1 ml/well). The contents of the wells are then harvested and counted on a LKB beta liquid scintillation counter (Gaithersburg, MD).

Anti-EGF Antibody Functional Studies

Rabbit anti-mouse EGF antibodies are used to block EGF functional

interactions with EGF receptors on Balb/c 3T3 cells and on C57B1/6 mouse spleen cells. Mouse EGF and anti-EGF are mixed and incubated for I hr at 37C prior to addition to either 3T3 cells or spleen cells. 3T3 proliferation and IFNy production assays are carried out as previously described. Recombinant human c-sis PDGF is also incubated with antimouse EGF as described above and is added to spleen cell cultures.

Radioiodination of EGF

EGF is labeled with 1251 using a modification of Das et al. (112).

Briefly, 2 pg in 10 pl of EGF (200 pg/ml in 0.4 M KPO4, pH 7.5) is added to a 1.5 ml Eppendorf test tube. Five p1 of Na 1251odide (500 PCi) and 10 pl of freshly dissolved chloramine-T (6.25 mg/ml) are added. After 1 min the reaction is quenched by the addition of 10 pl of Na metabisulfide (12.5 mg/ml) and 10 pl of KI (80 mg/ml). Then 10 pl of BSA (Sigma) (20 mg/ml) is added and the sample is run over a 7 ml Sephadex G10 column (Pharmacia) equilibrated with 50 mM KPO4 (pH 7.5) containing 0.15 M NaCl and 2 mg BSA/ml. Fractions are counted and those with the highest counts are pooled. The specific activity of the pooled sample is generally from 100-125 pCi/pg. 1251-EGF is aliquoted and stored at -70'C for no more than 3 weeks prior to use.









20

Balb/c 3T3 Fibroblast Growth Factor Binding Assays

The 3T3 growth factor binding assay is performed using a

modification of Kobayashi et al. (111). 3T3 fibroblasts are grown as above and seeded at 6 x 104 cells/well in 0.2 ml volume. All the peripheral wells are treated as above. After 24 hrs at 37C, 5% CO2 confluent monolayers are washed 3 times with Earle's balanced salt solution (EBSS) lacking calcium (57) containing Pen/Strep and 20 mM NaN3, 2 mg BSA/ml and aspirated to dryness. Then 25 4i of EBSS or dilutions of growth factors are added to wells in triplicate. After 15 min, 25 pi of 1251-EGF is added. After 1 hr at room temperature the wells are washed 3 times with EBSS and NaOH (50 4i of 0.1 M) is added to each well to solubilize the cells. The liquid is absorbed by cotton-tipped applicators and counted on an LKB gamma counter.

Murine Spleen Lymphocyte Binding Assays

Spleen cells are resuspended at 5 x 106 cells/ml in SDMEM containing 10% FBS in the presence or absence of 0.5 pg SEA/ml. The cells are seeded at 25 ml per 150 cm2 Corning flask and incubated at 37C, 5% CO2 for 48 hr. Nonadherent cells are then collected and centrifuged at 1,000 rpm for 10 min. Red blood cells are lysed by the addition of cold distilled water (1.5 ml per 3 spleens) to the completely dispersed pellet. After 10 seconds, cold EBSS (5.5 ml per 3 spleens) lacking calcium and containing 0.15 M NaCl, 15% FBS, 20 mM NaN3, and 2 mg/ml RIABSA is added to the spleen cells. After 10 minutes on ice the cells are placed into a 15 ml Corning test tube and centrifuged at 133 X g for 40 sec (adding 30 sec for each additional 5 ml). The supernatant is aspirated and the cells are washed twice in cold EBSS containing 20 mM









21

NaN3 and 2 mg/ml RIA-BSA (7 ml) exactly as above. The cells are resuspended in 0.1 ml of the above buffer and the debris is allowed to settle for 2 min. The cells are counted and resuspended in the above buffer at a final concentration of 4 x 106 cells/ml final concentration. Cells are incubated on ice or at room temperature for 15 min in the presence of cold growth factors or buffer prior to the addition of 125I EGF. The reaction volume is 40 4i/tube and all experiments are performed in triplicate. Binding is carried out on ice or at room temperature for

1 hr at which time samples are carefully layered into 0.4 ml polypropylene tubes over a phthalate oil mixture (1:1.27 ratio of dioctyl phthalate to dibutyl phthalate, v/v). Samples are centrifuged at 12,000 rpm for 30 sec to separate free 1251-EGF from bound 125I-EGF. Test tubes are then cut and cell pellets are counted on an LKB gamma counter. Reverse Phase High Performance Liquid Chromatography of Radioiodinated EGF

The HPLC apparatus uses a Perkin-Elmer Series 400 pump (Norwalk, CT), a Rheodyne model 7125 injector (Cotati, CA) with a 50 4i loop, a 25 cm x 4.6 mm Pecosil C18 10 pm particle size reversed phase cartridge column and a Pellicular C18 packed guard column (Perkin-Elmer). Fractions are collected on a LKB 2111 Multirac fraction collector. Murine and human EGF are each radioiodinated as described and 106 cpm in 0.8 ml are loaded onto the column. Elution is accomplished by a linear gradient of 10% acetonitrile and 0.1% trifluoroacetic acid at pH 3.1 to 100% acetonitrile over 60 min (I min per fraction). Samples are collected and counted on a gamma counter. A salt peak of free 125I eluted early in each run and is not shown in the figures.















CHAPTER 3

RESULTS



It was previously shown that electrophoretically pure mouse submaxillary gland EGF (from Collaborative Research) could replace the IL-2 requirement for IFN yproduction in the mouse (C57B1/6) spleen cell system (23,24). To confirm the above, another source of mouse submaxillary gland EGF (Toyobo) that was purified to homogeneity was tested for its ability to replace the IL-2 requirement, and was observed to provide essentially maximal help for IFNy production at 1 nM (Table 3-1). Purified recombinant human EGF (Scott Labs) also provided the helper signal at 1 nM as shown in Table 3-1. Thus, both EGF preparations have equivalent potency for inducing IFNy. Moreover, specific anti-murine EGF antibodies (Collaborative Research) could completely block EGF-induced IFNY production while this same antibody preparation had no effect on the recombinant c-sis PDGF (AMGen) helper signal for IFNY production (Table 3-2). Purified murine submaxillary gland EGF and recombinant human EGF were radioiodinated and passed over a reverse phase high performance liquid chromatography column in order to confirm their indicated purity. In both cases these labeled EGFs eluted as a single peak as indicated in Figures 3-1 and 3-2. This provides further evidence that the helper signal provided by EGF for IFNy production is due to the EGF molecule and not to a contaminant in these preparations.



22









23

As indicated, the growth factors TGFa and VGF are related to EGF

(Figure 1-2). They are in the family of EGF-like peptides as determined by structural similarity, the placement of cysteine residues within the molecules, and, most importantly, they exert their mitogenic effects on fibroblasts via the EGF receptor (57,74-77). Thus, synthetic rat TGFa (Peninsula Labs) and recombinant VGF (Oncogen) were examined for their ability to provide the helper signal for IFN Yproduction. Unlike EGF, neither TGFa nor VGF could restore competence for IFNy production by mouse C57BI/6 spleen cell cultures that were depleted of helper cell function (Figure 3-3) (113). Both growth factors were as competent as EGF in stimulating proliferation of 3T3 fibroblasts (Figure 3-4), so their inability to provide the helper signal for IFNy production was not due to factors such as lack of biological activity. Rather, it is possible that the EGF receptor on lymphocytes is different from that on 3T3 cells and thus novel, or that TGFa and VGF can bind to the EGF receptor on lymphocytes, but cannot trigger the signal for IFNy induction. If the former were true, then TGFa and VGF should not functionally compete with EGF for the receptor and thus block its helper signal, whereas if the latter were true, then they should block the EGF helper signal for IFNy production. As shown in Table 3-3, neither TGFa nor VGF in molar excess blocked the helper signal of EGF for production of IFNY by lymphocytes (113). Additionally, neither growth factor blocked IFNy production by spleen cells that were not depleted of helper cell function (Table 3-4). Thus, the functional data suggest that EGF provides its helper signal for IFNy production by interaction with a novel receptor on lymphocytes.









24

Hematopoietic cells have been reported to lack EGF receptors (57), thus EGF receptor binding studies were performed with untreated splenic lymphocytes cultured for 48 hr and splenic lymphocytes that were stimulated for 48 hr with the T-cell mitogen SEA. Exogenous SEA is required for several aspects of IFNY production (3). First, SEA acts as a T cell mitogen by inducing IL-2 production by TH cells (3). Second, SEA is a required second signal, along with IL-2, to initiate T cell production of IFNY.

As shown in Table 3-5, specific binding of 1251-EGF (125 pCi/pg

protein) was observed only in cultures that were stimulated with SEA for 48 hr (113). Specific binding was generally 25 to 50 percent of total binding. The data indicate that the EGF receptor is induced on lymphocytes and not constitutively expressed as in 3T3 fibroblasts. Similar data have been obtained in at least 3 separate experiments. A saturation binding curve for !25I-EGF on splenic lymphocytes is presented in Figure 3-5, and indicates a KD of 5 to 10 n1M. Since the cultures were depleted of adherent cells, it is unlikely that the binding observed was due to macrophages, but it cannot be ruled out that small amounts of contaminating macrophages may play some role in EGF binding to lymphocytes. The question has arisen that the novel EGF receptor described above may actually be the IL-2 receptor on lymphocytes, since the high affinity IL-2 receptor is also induced by T-cell mitogens. It has previously been shown, however, that EGF did not compete with IL-2 for the IL-2 receptor on lymphocytes (86). Therefore, the novel EGF receptor on lymphocytes is different from the IL-2 receptor on lymphocytes.








25

EGF, TGFa, and VGF compete similarly for the EGF receptor on 3T3

cells (69,75,76). We confirmed this in competitive binding experiments with 1251-EGF and the EGF, TGFa, and VGF that were used in the functional studies above (Figure 3-6). PDGF does not bind to the EGF receptor, but does slightly down regulate the expression of high affinity receptors (97-99). Thus, 1251-EGF binding to 3T3 cells was reduced by PDGF treatment, but not nearly as much as by the ligands that compete for the receptor. Thus, the data in Figure 3-6 are consistent with and confirm previous observations on the properties of the EGF receptor on 3T3 fibroblasts.

As indicated, the functional data presented suggest that EGF exerts its effects on lymphocytes by binding to a novel receptor, since TGFa and VGF could not provide the helper signal for IFNy production, and could not functionally block the help of EGF. The binding competition data of Figure 3-7, where cold submaxillary gland EGF blocked 1251-EGF binding to lymphocytes, but TGFa at the same concentrations was without effect is further evidence that the EGF receptor on lymphocytes is novel. We currently do not know why relatively high concentrations of cold EGF are required in receptor competition experiments with lymphocytes, but the relatively low specific binding to total binding may play a role. Also, it is possible that the binding studies using submaxillary gland 1251-EGF could involve a contaminant in the EGF preparation, but this is unlikely since purified recombinant human EGF inhibited submaxillary gland 125IEGF binding as effectively as did cold submaxillary gland EGF (Table 3-6). Similar competitive binding patterns have been observed in other systems such as the adrenergic receptors (114).









2-)6

The above data raise questions about functional sites on the EGF, TGFoi, and VGF molecules that are involved in receptor interactions. It has been suggested that the third disulfide loop of these growth factors comprises the binding site for the EGF receptor on such cells as 3T3 fibroblasts (115). The data presented here raise questions about a common binding site for these 3 growth factors with respect to interactions with lymphocytes. In order to gain further insight into the structural relationship of these molecules we generated composite surface profile plots which are based on hydrophilicity, accessibility, and flexibility of secondary structure (116,117). Mouse and human EGF showed very similar Composite surface profiles (Figure 3-8a and 3-8b). Their profiles were different from those of TGFa and VGF (Figure 3-8c and 38d). The composite surface profile plots suggest structural differences between EGF and TGFa and VGF. A precise delineation of these differences could possibly provide insight into EGF interaction with lymphocytes in the absence of such an interaction by TGFa and VGF.

W~e feel that the data presented are quite interesting and allow us to draw the following conclusions: (1) EGF provides a helper signal for the induction of IFNy, (2) EGF provides its signal by interaction with a novel receptor on lymphocytes based on functional and receptor competition data with TGFa and VGF, (3) the novel EGF receptor on lymphocytes is not expressed constitutively but is induced by a T cell mitogen.

Related to our discovery of a novel EGF receptor on lymphocytes is a recent finding of a novel PDGF receptor on human fibroblast and baby hamster kidney cells (118-120).









27 8060








4020









20 40 60







Figure 3-i. HPLC profile of murine EGF. Murine submaxillary gland EGF (Toyobo) was radioiodinated and passed over a C018 reverse phase HPLC column. Fractions are eluted and counted in a gamma counter.









40- 28










30










20










10











20 40 60
Figure 3-2. HPLC profile of recombinant human EGF. Recombinant human EGF (Scott Labs) was radioiodinated and passed over a C18 reverse phase HPLC column. Fractions are eluted and counted in a gamma counter.










29




C













200















80







O. rv 10. 100

G.CWTH FACTOR (nr)
Figure 3-3. Ability of growth factors to restore competence for IFN y production by Lyt L-, 2* spleen cells. EGF (O), TGFa (A), and VGF (0) were incubated with Lyt 1-depleted spleen cells in the presence of the Tcell mitogen SEA. IFNytiters are from day 3 of culture. Reprinted from Progress in Leukocyte Biology 8:159, 1988. Copyright (c) by Alan R. Liss, Inc. (113).










30










i~








80
X



S60



~40




20





01001 0.01 0,1 1 10 30

GROWTH FACTOR (nM) Figure 3-4. Relative mitogenic activity of growth factors on Balb/c 3T3 cells. Cells were incubated for 24 hr in SDMEM containing 10% FBS, washed extensively, and growth factors were added at the indicated concentrations. After 24 hr at 37'C in the presence of 5% CO2. 'Hthymidine was added for 2 hr. Cells were then harvested and counted on a beta scintillation counter. Symbols: EGF (A); TGFa (0); VGF (0).









31














cooo

3,5002,0001.000- 0
0 5 10 1 20 25 30




Figure 3-5. Binding of 125T-EGF to spleen cells. Spleen cells were incubated with 3.3 pM EGF for 15 min prior to a 1 hr incubation with 1251-EGF at the indicated concentrations. Cells were then harvested and counted on a gamma counter. Data are plotted as bound 125I-EGF vs. free 1251-EGF.










32















2,400-,000




4 0

L


0 0.1 1 10 100 Z00
GROWTH FACTOR (nM)


Figure 3-6. Competitive binding of 125-EGF to Balb/c 3T3 cells in the presence of growth factors. Confluent monolayers were incubated for 15 min with cold EGF (0), TGFa (0), VGF (&), PDGF B (0), or buffer (A). 1251-EGF was then added at a final concentration of 5 nM. After 1 hr incubation at room temperature, cells were harvested and counted on a gamma counter.









33































800








0 100 0 1,000 3,330

GROWTH FACTOR (nM)





Figure 3-7. Competitive binding of i251I-EGF to spleen lymphocytes in the presence of growth factors. SEA-stimulated spleen lymphocytes were harvested as previously described and incubated for 15 min with EGF (0), TGFa (A), or buffer ([3). 125i-EGF was then added at a final concentration of 5 nM. After 1 hr incubation at room temperature, cells were harvested and counted on a gamma counter.









/ \t o! oo
00




50 / 50
Io I


0 Ii\/ ,L _ __ _,_ f _"_10 20 30 40 50 0 1too 100


I).

505




010 2TO4 5 1 10 20 30 40 50









Figure 3-3. Surface profile of EGF-related proteins. Composite surface profiles of mouse EGF (A), human EGF (B), rat TGFa (C), and VGF (D) were developed as described by using a computer program (118). This program takes into account HPLC mobility, accessibility, and segmental mobility (B values) of amino acids in model proteins and peptides. Residues with high composite values are most likely to reside on the surface of a protein molecule. The abscissa equals the surface value: the
ordinate equals the residue number.








35




Table 3-1

Relative abilities of mouse EGF and recombinant human EGF
to provide the helper signal for IFNy production
by Lyt 2+ spleen cells

GFa IFNy (U/ml + S.D.)
(nM) moEGF huEGF


0 <3 <3

0.1 <3 <3

1 65 7 55 7

10 60 14 85 21

100 95 7 200 141


aSamples were from day 3 of culture. Whole spleen cells had 95 35 IFN Yunits/ml.








36




Table 3-2

Ability of anti-EGF antibodies to block EGF
helper signal for IFNy production by Lyt 2+ spleen cells


Anti-EGF Antibodiesa IFNy(U/ml + S.D.)
(pg/ml) EGF 5nM PDGF 5nM



0 120 28 105 21

0.5 80 14 220 28

5 <3 85 21

50 <3 135 21



aSamples were from day 3 of culture. Whole spleen cells had 80 28
IFNYunits. Anti-Lyt 1.2 + C' control cells had <3 IFN yunits.








37




Table 3-3

Failure of TGFa and VGF to block helper signal for
[FNY production by mouse spleen cells



SEA-stimulated culturesa IFNY (U/ml SD)



Lyt 1-, 2+ cells <7

Lyt 1-, 2+ cells + EGF 270 42

Lyt 1-, 2+ cells + EGF + TGFa 290 t 14

Lyt 1-, 2+ cells + EGF + VGF 295 7

Whole spleen cells 215 21



aSamples are from day 3 of culture. Concentration of factors: EGF, 5 nM; TGFa, 14 nm; VGF, 17 nM. Reprinted from Progress in Leukocyte Biology 8:159, 1988. Copyright (c) 1988 by Alan R. Liss, Inc. (113)









38




Table 3-4


Failure of TGFa and VGF to block IFNY production in spleen cells



SEA-stimulated culturesa IFNY (U/ml SD)



TGFa 275 35

VGF 350 71

Control 275 106



aSamples are from day 3 of culture. Concentration of factors: TGFa,, 14 nM; VGF. 17 nM.









39




Table 3-5

Induction of EGF receptor by SEA on mouse spleen lymphocytesa



SEA Cold CPM p-value
treatment, EGF,
0.5 pg/ml 3 iiM



1115 256

+ 1009 49 N.S.


+ 1539 64

+ + 1158 54 <0.005



aSpleen cells were incubated with SEA for 48 hr, at which time cultures were depleted of macrophages and red blood cells. CPM (counts per minute) are from lymphocyte bound 1251-EGF. Reprinted from Progress in Leukocyte Biology 8:159, 1988. Copyright (c) by Alan R. Liss, Inc. (113).








40




Table 3-6

Ability of recombinant human EGF to compete with murine purified submaxillary 1251-EGF for the
EGF spleen cell receptora


rHuEGF MuEGF CPM SD
(nM) (nM)


1488 269

10 1424 197

100 1463 235

330 1189 183

1000 1184 97

3300 1100 108

3300 1152 78


aSpleen cells were stimulated with SEA for 48 hr and washed extensively prior to binding with 125f-EGF at 5 nM final concentration.















CHAPTER 4

DISCUSSION



EGF is a molecule with a wide variety of biological functions.

These include: (1) the proliferation of ectodermal and mesodermal cells

(56) and thus wound healing (73); (2) regulation of spermatogenesis (70,71); (3) inhibition of gastric acid secretion (72); and (4) regulation of IFNY production (22,23). The first three functions are believed to be mediated through the well characterized EGF receptor (57,100).

The EGF receptor is constitutively found on almost every tissue

studied except for the hematopoietic cell types (57). It has been cloned and sequenced (87). It is a 170,000 dalton glycoprotein which has been extensively characterized in its structure and its functions. The EGF receptor has three major domains: (1) an extracellular domain which is responsible for the signal transduction after ligand binding; (2) a transmembrane region; and (3) the intracellular region which contains the tyrosine kinase domain responsible for the signal transduction after ligand binding onto the receptor (57,100). Signal transduction is mediated through autophosphoralation of the EGF receptor at tyrosine 1173.

The EGF-related peptides TGFa and VGF are structurally related to EGF and compete with this ligand for its receptor (57,74-76). They also share known biological functions that are mediated through the EGF 41









42

receptor. These include mitogenic effects on fibroblasts (57,69,76) and wound healing (81). VGF can also augment the production of viral progeny from infected cells (84). As indicated in the Results EGF, TGFa, and VGF molecules are structurally and functionally related to each other, suggesting evolution from a common gene (78,79). The third disulfide loop, a region that is most common to all three ligands in sequence and structure, is believed to be the ligand binding region which is recognized by the classic EGF receptor (115). In the data presented here we have confirmed that TGFa and VGF, like EGF, can stimulate the proliferation of 3T3 fibroblasts as well as compete for binding to the EGF receptor on these cells using 125I-labeled EGF. The question arises as to whether purified and recombinant EGF from other sources and whether TGFa and VGF can similarly provide the helper signal for induction of IFN Y.

In the course of studies on the regulation of murine IFNy production by various growth factors it was found that EGF was capable of inducing IFNy production in spleen cell cultures depleted of TH cells (23,24). The mouse submaxillary gland EGF preparation used in these studies (from Collaborative Research) was electrophoretically pure. This preparation was found, however, to restore the production of IFN Yat 17 nM. This concentration is higher than that required for the proliferative functions of EGF for fibroblasts. Thus, the question was raised that a possible contaminant of the EGF preparation was responsible for the helper signal for IFNY production. To address this question and to further extend our knowledge of the effects of EGF in the production of IFN Y, we obtained EGF from several other sources. We have shown here









43

that purified mouse submaxillary gland EGF (Toyobo) and recombinant human EGF (Scott Labs) were both capable of inducing the production of IFNy at

1 nM. Thus, unlike the relatively high EGF concentrations required to induce IFNY in the previous study, the IFNY inducing concentrations of these EGF preparations approximated those concentrations required to detect mitogenic effects of EGF on fibroblasts. The initial observation that EGF can provide the helper signal for IFNY production, then, apparently involved the use of EGF that was not fully active. The EGF preparations that we used in these studies were radioiodinated, eluted from a reverse phase column and only one peak was found in each sample, indicating only one major species in each sample. These data excluded the possibility of there being a contaminant in these preparations that was responsible for the induction of IFNy. Thus, the data presented here show EGF to provide the help for IFNy production at concentrations similar to those for its other biological effects. Also anti-EGF antibodies inhibited EGF induction of IFNY in a specific manner.

The above data demonstrate that EGF can regulate lymphokine

production. As indicated earlier, lymphocytes do not constitutively express an EGF receptor. Although EGF receptors are generally thought not to be expressed on hematopoietic cells there is one preliminary report of induction of an EGF receptor on B lymphoblastoid cells (121). The B lymphoblastoid lines RPMI 1788 and RPMI 8392 were both shown to bind 1251-EGF after treatment with the B cell mitogen staphylococcal B cell mitogen (STM). RPMI 1788 cells expressed between 500 to 1,000 EGF receptors while RPMI 8392 expressed between 2,000 to 4,000 EGF receptors. Normal lymphocytes from blood or spleen could also be induced to express









44

EGF receptors after treatment with B cell mitogens. Induction of receptor was not observed when the cells were treated with the T-ceil mitogen Concanavalin A. Other T-cell mitogens were not tested. This EGF receptor was not further characterized. Interestingly, in our studies we found an EGF receptor on splenic lymphocytes that required induction by the T-cell mitogen SEA. The splenic lymphocyte EGF receptor was detectable after a 48 hr incubation in the presence of SEA. Approximately 25% of total binding was specific, which would suggest the presence of low receptor numbers. The splenic lymphocyte EGF receptor has an apparent dissociation constant of 7 nM.

The IL-2 receptor is another example of an inducible lymphocyte receptor (86). This receptor is induced by a T-cell mitogen and takes several days before it can be detected on the surface of lymphocytes. The induced IL-2 receptor does not recognize the EGF molecule as a competitive ligand in the presence of labeled IL-2. Therefore, the induced EGF receptor is different from the IL-2 receptor on lymphocytes.

Since we had shown that the EGF molecule itself, and not a

contaminant, regulated the production of the IFNY lymphokine, we then determined if the EGF-related growth factors TGFa (synthetic) and VGF (recombinant) also induced IFNy production. We were surprised to find that, unlike EGF, neither TGFa nor VGF were able to induce the production of IFNY(Figure 3-3). Moreover, neither peptide functionally blocked the ability of EGF to induce IFNY; nor were these peptides able to suppress the production of IFNY in whole spleen cells. Thus while TGFa and VGF were biologically active in induction of 3T3 fibroblast proliferation (Figure 3-4), unlike EGF. they were not capable of inducing IFNy in









45

lymphocytes. Finally, in competitive binding studies we demonstrated that TGFa did not compete with 125I-EGF for the induced lymphocyte EGF receptor while it did compete for receptor on 3T3 cells. We conclude that there is a novel EGF receptor on lymphocytes which, unlike the classic EGF receptor, does not functionally or physicochemically (in binding studies) recognize the EGF-related peptides TGFa and VGF.

The induction of IFNy by EGF is a newly described function for this nonhematopoietic growth factor. Its effect is mediated through a novel EGF receptor. This EGF receptor differs from the classic EGF receptor in that it is inducible by a T-cell mitogen, and fails to recognize the EGFrelated growth factors TGFa and VGF.















APPENDIX



PRELIMINARY DATA



Previous studies have shown that rabbit polyclonal antibodies to the classic EGF receptor blocks EGF induced proliferation of mouse 3T3 fibroblasts (122). We compared the effects of these antibodies on both EGF induced 3T3 proliferation and EGF helper signal for IFNy production. Table A-I shows partial suppression of 3H-thymidine incorporation into 3T3 cells. Normal rabbit serum was not inhibitory and at higher concentrations had an enhancing effect. These data are similar to that of Adamson and colleagues (122). Anti-EGF receptor antibodies had no effect on EGF helper signal for IFNy production (Table A-2). The antiEGF receptor antibody data supports our previous conclusion that the lymphocyte EGF receptor is different fron the classic EGF receptor.

In order to determine the size of the lymphocyte EGF receptor, we

labeled EGF with 1251 and used this as a probe. We confirmed the size of the classic EGF receptor by cross-linking the 1251-EGF onto Balb/c 3T3 fibroblasts in the absence and presence of cold EGF. The preparations were prepared and run on an 8% SDS-PAGE system. After autoradiographic exposure the dominant 170,000 dalton EGF receptor was seen as expected (Figure A-i). The minor bands at 120,000 and 78,000 dalton bands are known EGF receptor breakdown products (58). As expected there was no radiolabeled signal in the preparation containing cold EGF. The same 46








47

procedure using radiolabeled EGF was carried out with 48 hr SEAstimulated lymphocytes. The detected bands migrated at 130,000 daltons, 100,000 daltons, and 50,000 daltons upon autoradiographic exposure (Figure A-2). The 100,000 dalton and 50,000 dalton bands could represent degradation products of the 130,000 dalton band or the 3 bands could represent a complex EGF receptor on lymphocytes. Further studies need to be conducted to better delineate the nature of the induced lymphocyte EGF receptor.










48

1 2



205

180



116




.84

58




48


















Figure A-!. SDS-PAGE in 8% acrylamide/bis with 1251-EGF cross-linked onto 3T3 fibroblasts in the absence (lane 1) and presence (lane 2) of 1 PM cold EGF. Confluent 3T3 cell monolayers were incubated in the absence and presence of 1 pM cold EGF for 15 min prior to the addition of
5 nM 125I-EGF. After 1 hr at 0C, cells were washed and cell-bound EGF was covalently cross-linked to the cellular receptor by the addition of Bis (sulfosuccinimidyl) suberate (BS3 After i hr at 00C, the BS3 was removed. Cells were suspended in SDS-PAGE sample buffer. Samples were electrophoresed under reducing conditions on an 8% separating gel. Molecular weight markers were also run on the gel.









49

1 2







205


180





11 6

84

58



48"




36









Figure A-2. SDS-PAGE in 8% acrylamide/bis with 1251-EGF cross-linked onto 48 hr SEA stimulated splenic lymphocytes in the presence (lane 1) and absence (lane 2) of 3.2 pM cold EGF. Samples are incubated for 15 min at 00C in the presence or absence of 3.2 pM final concentration cold EGF prior to the addition of 5 nM 1251-EGF. 'Cells are washed without BSA and cell-bound EGF is covalently cross-linked to the cellular receptor by the addition of freshly prepared BS3. After 1 hr at 00C the BS3 was removed and the spun sample pellets treated with SDS-PAGE sample buffer and sonicated. Samples were centrifuged to remove debris and supernatants were boiled prior to being electrophoresed. Samples were electrophoresed on an 8% separating gel as in the 3T3 EGF receptor SDS-PAGE protocol. The gel was autoradiographed at -70'C for 6 weeks with intensifying screens prior to film development.








50




Table A-1

Ability of anti-EGF receptor antibodies to block EGF-induced
3H-thymidine incorporation by 3T3 fibroblastsa


Immunoglobulin Percent of control
protein (ug/ml)b 3H-thymidine incorporation
Anti-EGF Normal
receptor antibody rabbit serum


0 100 100

30 64 98

100 64 126

300 45 221

1000 42 263



aBalb/c 3T3 fibroblasts were incubated in duplicate with either rabbit anti-EGF receptor antibodies or control normal rabbit serum for 1 hr prior to the addition of EGF (2 nM). After 24 h at 370C in the presence of 5% CO2, 3H-thymidine was added for 2 hr. Cells were then harvested and counted on a beta scintillation counter. bRabbit polyclonal anti-EGF receptor antibody was provided by Dr. E. Adamson (122). The antibody and normal rabbit serum were ammonium sulfate precipitated, dialysed in PBS, aliquoted, and frozen.








51




Table A-2

Effect of anti-EGF receptor antibodies on IFNy production by
Mitomycin C-treated spleen cells in the presence of EGF


EGF Anti-EGF receptor NRS IFNya
5nM antibodies (pg/ml) (jg/ml) (U/mi SD)


<3



35 7



+ 30 25 7

+ 100 28 4

+ 300 33 4

+ 1,000 28 4



+ 30 45 21

+ 100 65 21

+ 300 50 t 28

+ 1,000 28 4



aLFNY titers are from Day 3 of culture. Untreated spleen cells stimulated with SEA produced 85 21 units IFNY/mi on Day 3 of culture. Normal rabbit serum (NRS) was used as a control.















REFERENCES

1. Isaacs, A. and Lindenmann, J. Virus interference. I. The
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BIOGRAPHICAL SKETCH



On September 15, 1956, in Lorain, Ohio, Keith Anderson was born to Dr. Marvin Moore Anderson and Mrs. Dorothy-Jean Barbee Anderson. He has

4 brothers who are Mansur Abdullah, Clarence, Christopher and Eric Anderson. As a child and young adult he had a strong inclination toward natural sciences. His parents encouraged such activities. In 1971 he joined the Nation of Islam. In 1975 he graduated from Admiral King High School and became a Muslim. That year he enrolled in the Biology Department of Bradley University in Peoria, Illinois. lie graduated with a Bachelor of Science degree from Bradley University in 1979.

In 1979 he enrolled in the Department of Immunology and Medical Microbiology, College of Medicine at the University of Florida, Gainesville, Florida. In 1981 he began research in the laboratory of Dr. George E. Gifford. In July 1984 Keith Anderson legally changed his name to Na'eem Adib Abduiiah. He joined the laboratory of Dr. Howard 14. Johnson in 1986 to work on the regulation of interferon gamma production in the marine system. He received his Doctorate of Philosophy degree in December 1988.












63
















I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.


Donna H. Duckworth
Professor of Immunology and Medical Microbiology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.


Lindsey J/'< Hutt-Fletcher Associate Professor of Pathology and Laboratory Medicine


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.


Arthur K. Kimura
Associate Professor of Pathology and Laboratory Medicine















This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

December, 1988 Dean, College of Medicine



Dean, Graduate School




Full Text
10
affinity receptor state (93,95-99). This "receptor transmodulation"
process also reduces the tyrosine kinase activity of the EGF receptor.
Receptor transmodu'lators bind to their own distinct receptors (100) and
activate protein kinase C (and/or other kinases) which phosphorylates EGF
receptor threonine (Thr) 654. Phosphorylation of Thr 654 on the EGF
receptor by PMA has been shown to block the mitogenic effects of EGF, but
this phosphorylation event occurs before phosphorylation by the tyrosine
kinase phosphorylase (101).
The EGF receptor has a half-life of greater than 5 hr (89). After
ligand binding the half-life decreases to approximately 1 hr. Receptor
degradation is mediated by coated pit internalization and subsequent
lysosomal enzyme degradation (56). EGF receptor mutation studies have
shown that the kinase activity is also essential for degradation of the
wild type receptor (89,100).
The EGF receptor molecule has several relatives. The c-erb B-2 gene
product is a 185,000 dalton glycosylated protein that shares about 50%
homology with the EGF receptor (102). It has an extensive extracellular
domain like its c-erb B-l counterpart yet it does not bind EGF or any
known ligand. C-erb B-2 transcripts have been detected in placenta,
kidney, embryonic tumors, stomach adenocarcinomas, and neuroblastomas.
Greater than 80% of the tyrosine kinase domain in c-erb B-2 is homologous
with its EGF receptor counterpart. The c-erb B-2 receptor is phos-
phorylated via interaction of EGF with the c-erb B-l receptor and is
believed to be a substrate for the c-erb B-l phosphorylating activity
(103,104). Four independent cell lines have the identical mutation from
valine 664 to glutamic acid in c-erb B-2 in the transmembrane region,


59
84. Buller, R.M.L., Chakrabarti, S., Moss, B. and Fredrickson, T. Cell
proliferative response to vaccinia virus is mediated by VGF.
Virology 164:182, 1988.
85. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N.,
Stockwell, P., Ullrich, A., Schlessinger, J. and Waterfield, M.D.
Close similarity of epidermal growth factor receptor and v-erb-B
oncogene protein sequences. Nature 307:521, 1984.
86. Robb, R.J., Munck, A. and Smith, K.A. T cell growth factor
receptors. J. Exp. Med. 154:1455, 1981.
87. Ullrich, A., Coussens, L., Hayflick, J.S., Dull, T.J., Gray, A.,
Tam, A.W., Lee, J., Yarden, Y., Libermann, T.A., Schlessinger, J.,
Downward, J., Mayes, E.L.V., Whittle, N., Waterfield, M.D. and
Seeburg, P.H. Human epidermal growth factor receptor cDNA sequence
and aberrant expression of the amplified gene in A431 epidermoid
carcinoma cells. Nature 309:418, 1984.
88. Hanks, S.K., Quinn, A.M. and Hunter, T. The protein kinase family:
Conserved features and deduced phytogeny of the catalytic domains.
Science 241:42, 1988.
89. Honegger, A.M., Dull, T.J., Felder, S., Van Obberghen, E., Bellot,
F., Szapary, D., Schmidt, A., Ullrich, A. and Schlessinger, J.
Point mutation at the ATP binding site of EGF receptor abolishes
protein-tyrosine kinase activity and alters cellular routing. Cell
51:199, 1987.
90. Lax, I., Burgess, W.H., Bellot, F., Ullrich, A., Schlessinger, J.
and Givol, D. Localization of a major receptor-binding domain for
epidermal growth factor by affinity labeling. Molec. Cell. Biol.
8:1831, 1988.
91. Downward, J., Parker, P. and Waterfield, M.D. Autophosphorylation
sites on the epidermal growth factor receptor. Nature 311:483,
1984.
92. Weber, W., Bertics, P.J. and Gill, G.N. Immunoaffinity
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epidermal growth factor receptors. J. Biol. Chem. 257:3053, 1982.
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An insertional mutant of epidermal growth factor receptor allows
dissection of diverse receptor functions. The EMBO J. 6:2669,
1987.


48
1 2
Figure A-L. SDS-PAGE in 8% acrylamide/bis with L^^I-EGF cross-linked
onto 3T3 fibroblasts in the absence (lane 1) and presence (lane 2) of 1
mM cold EGF. Confluent 3T3 cell monolayers were incubated in the
absence and presence of 1 uM cold EGF for 15 min prior to the addition of
5 nM 1-251-egf. After 1 hr at 0C, cells were washed and cell-bound EGF
was covalently cross-linked to the cellular receptor by the addition of
Bis (suifosuccinimidyl) suberate (BSJK After I hr at 0C, the BSJ was
removed. Cells were suspended in SDS-PAGE sample buffer. Samples were
electrophoresed under reducing conditions on an 8% separating gel.
Molecular weight markers were also run on the gel.


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Howard M. Johnson,
Graduate Research Professor of
Immunology and Medical Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Ac QC /
/L b",/ ..1
Donna H. Duckworth
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
LindseyHutt-Fletcher
Associate Professor of Pathology and
Laboratory Medicine
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Arthur K. Kimura
Associate Professor of Pathology and
Laboratory Medicine


21
NaNg and 2 mg./ml RIA-BSA (7 ml) exactly as above. The cells are
resuspended in 0.1 ml of the above buffer and the debris is allowed to
settle for 2 min. The cells are counted and resuspended in the above
buffer at a final concentration of 4 x 10^ cells/ml final concentration.
Cells are incubated on ice or at room temperature for 15 min in the
presence of cold growth factors or buffer prior to the addition of I-
EGF. The reaction volume is 40 pl/tube and all experiments are performed
in triplicate. Binding is carried out on ice or at room temperature for
1 hr at which time samples are carefully layered into 0.4 ml
polypropylene tubes over a phthalate oil mixture (1:1.27 ratio of dioctyl
phthalate to dibutyl phthalate, v/v). Samples are centrifuged at 12,000
rpm for 30 sec to separate free ^-*I-EGF from bound ^~I~EGF. Test tubes
are then cut and cell pellets are counted on an LKB gamma counter.
Reverse Phase High Performance Liquid Chromatography of Radioiodinated
EGF
The HPLC apparatus uses a Perkin-Elmer Series 400 pump (Norwalk,
CT), a Rheodvne model 7125 injector (Cotati, CA) with a 50 pi
loop, a 25 cm x 4.6 mm Pecosil C-^g 10 pm particle size reversed phase
cartridge column and a Pellicular C^g packed guard column (Perkin-Elmer).
Fractions are collected on a LKB 2111 Multirac fraction collector.
Murine and human EGF are each radioiodinated as described and 10^ cpm in
0.8 ml are loaded onto the column. Elution is accomplished by a linear
gradient of 10% acetonitrile and 0.1% trifluoroacetic acid at pH 3.1 to
100% acetonitrile over 60 min (1 min per fraction). Samples are
collected and counted on a gamma counter. A salt peak of free LZ-JI
eluted early in each run and is not shown in the figures.


APPENDIX
PRELIMINARY DATA
Previous studies have shown that rabbit polyclonal antibodies to the
classic EGE receptor blocks EGP induced proliferation of mouse 3T3
fibroblasts (122). We compared the effects of these antibodies on both
EGF induced 3T3 proliferation and EGF helper signal for IFNy production.
Table A-l shows partial suppression of %-thymidine incorporation into
3T3 cells. Normal rabbit serum was not inhibitory and at higher
concentrations had an enhancing effect. These data are similar to that
of Adamson and colleagues (122). Anti-EGF receptor antibodies had no
effect on EGF helper signal for IFNy production (Table A-2). The anti-
EGF receptor antibody data supports our previous conclusion that the
lymphocyte EGF receptor is different iron the classic EGF receptor.
In order to determine the size of the lymphocyte EGF receptor, we
labeled EGF with and used this as a probe. We. confirmed the size of
the classic EGF receptor by cross-linking the -^^I-EGF onto Balb/c 3T3
fibroblasts in the absence and presence of cold EGF. The preparations
were prepared and run on an 8% SDS-PAGE system. After autoradiographic
exposure the dominant 170,000 dalton EGF receptor was seen as expected
(Figure A-l). The minor bands at 120,000 and 78,000 dalton bands are
known EGF receptor breakdown products (58). As expected there was no
radiolabeled signal in the preparation containing cold EGF. The same
46


39
Table 3-5
Induction of
EGE receptor
by SEA on mouse spleen
lymphocytes3
SEA
Cold
CPM
p-value
treatment,
EGF,
0.5 Mg/ml
3 i_iM
-
-
11.15 256
-
+
1009 49
N.S.
+
-
1539 64
+
+
1158 54
<0.005
aSpleen cells were incubated with SEA for 48 hr, at which
time cultures were depleted of macrophages and red blood
cells. CPM (counts per minute) are from lymphocyte bound
^^I-EGF. Reprinted from Progress in Leukocyte Biology
8:159, 1988. Copyright (c) by Alan R. Liss, Inc. (113).


BIOGRAPHICAL SKETCH
On September 15, 1956, in Lorain, Ohio, Keith Anderson was born to
Dr. Marvin Moore Anderson and Mrs. Dorothy-Jean Barbee Anderson. He has
4 brothers who are Mansur Abdullah, Clarence, Christopher and Eric
Anderson. As a child and young adult he had a strong inclination toward
natural sciences. His parents encouraged such activities. In 1971 he
joined the Nation of Islam. In 1975 he graduated from Admiral King High
School and became a Muslim. That year he enrolled in the Biology
Department of Bradley University in Peoria, Illinois. He graduated with
a Bachelor of Science degree from Bradley University in 1979.
In 1979 he enrolled in the Department of Immunology and Medical
Microbiology, College of Medicine at the University of Florida,
Gainesville, Florida. In 1981 he began research in the laboratory of Dr
George E. Gifford. In July 1984 Keith Anderson legally changed his name
to Naeem Adib Abdullah. He joined the laboratory of Dr. Howard M.
Johnson in 1986 to work on the regulation of interferon gamma production
in the murine system. He received his Doctorate of Philosophy degree in
December 1988.
63


60
95.Lee, L.-S. and Weinstein, I.B. Tumor promoting phorbol esters
inhibit binding of epidermal growth factor to cellular receptors.
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phorbol esters specifically alter affinity of epidermal growth
factor membrane receptors. Nature 279: 387 1979.
97. Collins, M.K.L., Sinnett-Smith, J.W. and Rozengurt, E. Platelet-
derived growth factor treatment decreases the affinity of the
epidermal growth factor receptors of Swiss 3T3 cells. J. Biol.
Chem. 258:11689, 1983.
98. Bowen-Pope, D.F., Dicorleto, P.E. and Ross, R. Interactions
between the receptors for platelet-derived growth factor and
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99. Olashaw, N.E., O'Keefe, E.J. and Pledger, W.J. Platelet-derived
growth factor modulates epidermal growth factor receptors by a
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Sci. USA 83:3834, 1986.
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multifunctional allosteric protein. Biochemistry 27:3119, 1988.
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Schlessinger, J. Release of a phorbol ester-induced mitogenic
block by mutation at Thr-654 of the epidermal growth factor
receptor. Molec. Cell. Biol. 8:2302, 1988.
102. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N.,
Miyajima, N., Saito, T. and Toyoshima, K. Similarity of protein
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103. King, C.R., Borrello, I., Bellot, F., Comoglio, P. and
Schlessinger, J. EGF binding to its receptor triggers a rapid
tyrosine phosphorylation of the erbB-2 protein in the mammary tumor
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independent activations of the neu oncogene by a point mutation
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Yang-Feng, T.L., Francke, U., Ullrich, A. and Coussens, L. The neu
gene: An erbB-homologous gene distinct from and unlinked to the
gene encoding the EGF receptor. Science 229:976, 1985.


61
107. Yamamoto, T., Nishida, T., Miyajima, N., Kawai, S., Ooi, T. and
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Fridkin, M. and Schlessinger, J. Antibodies against a synthetic
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58
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Analysis by mRNA phenotyping. Science 241:708, 1988.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF MURINE INTERFERON GAMMA PRODUCTION
BY EPIDERMAL GROWTH FACTOR
By
Na'eem Adib Abdullah
December, 1988
Chairman: Howard M. Johnson
Major Department: Immunology and Medical Microbiology
It has recently been shown that the epidermal growth factor (EGF)
molecule is capable of positive regulation of immune function by inducing
the production of gamma interferon (IFNY), establishing a functional
relationship between nonhematopoietic growth factors and the immune
system. In order to study this relationship further EGF and EGF-related
growth factors, transforming growth factor a (TGFa), and vaccinia virus
growth factor (VGF), which all exert their effects on cells through the
EGF receptor on cells, were studied for their functional and physicochem
ical effects on IFNY production at the receptor level.
IFN yproduction requires T helper (Tg) cell function. Both purified
murine EGF and recombinant human EGF were capable of restoring competence
for IFNY production by murine spleen cell cultures depleted of Tjj cells.
In agreement with this observation, anti-EGF antibodies were capable of
specifically neutralizing the EGF effect. Interestingly, EGF receptors
are not constitutively expressed on normal or primary lymphocytes. We
observed that treatment of lymphocytes with T-cell mitogens induced the
vii


REGULATION OE MURINE INTERFERON GAMMA PRODUCTION
BY EPIDERMAL GROWTH FACTOR
By
Na'eem Adib Abdullah
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988

ACKNOWLEDGEMENTS
Allah, God the Creator, has asked people to study creation and to
find and use its hidden treasures to help mankind. This dissertation is
dedicated to that end. The last revelation of Allah, the Holy Qur'an,
is unparalleled in its prophetic scientific description of natural
phenomena. Allah is the greatest (A1 Kabir), the most wise (A1 Hakim),
and the most kind (A1 Latif).
I give a special thanks to Howard M. Johnson, Ph.D., as my mentor
and guide through the "real world of scientific research. His piercing
insights have helped me enormously in maturing as a scientist. I thank
the supervisory committee members Donna H. Duckworth, Ph.D., Lindsey M.
Hutt-Fletcher, Ph.D., and Arthur K. Kimura, Ph.D., for their many
suggestions and scientific insights in the preparation of this manu
script. Another titan that helped me at the bench, in writing and
through discussions was Barbara A. Torres, M.S. She took precious time
away from running the lab to instruct me on many new techniques and the
nuances and quirks of each of them. The two postdocotral scientists
Carol Hanlon-Pontzer, Ph.D., and Jeffry K. Russell, Ph.D., and the
graduate students Myron 0. Downs, D.V.M., Michael A. Jarpe, B.S., and
Harold I. Magazine, B.S., were great peers and counsel to interact with
in many areas of research and allowed me to look at a given problem from
different prospectives. Miss Sarah A. Hunt was most helpful in ordering
ii

and tracking materials, reagents and scientific correspondences required
for my experiments as well as helping to set up actual experiments.
I wish to thank my parents, Marvin Moore Anderson, M.D., and
Dorothy-Jean Barbee Anderson and brothers Mansur Abdullah, Clarence H.
Anderson, Christopher A. Anderson, Eric Anderson, M.D., and his wife
Ellen, who gave me tremendous love, support and encouragement throughout
my graduate studies. A special thanks to Juanita Maria Maxwell whose
encouragement and companionship were appreciated. They will never know
how much their support meant to me.
I must thank my many other relatives, friends, colleagues, and
associates who are too numerous to mention but whose support and
encouragement were deeply appreciated and spurred me on to obtain the
Doctorate of Philosophy. Finally, thank you Jennifer (Maynard) for
preparing and retyping this manuscript through its numerous revisions and
impending deadlines.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABBREVIATIONS v
ABSTRACT vii
CHAPTERS
1 INTRODUCTION 1
Interferon Gamma 1
The Interferon Gamma Molecule 1
Regulation of IFNy Production 2
Immunoregulatory Functions of IFN Y. 3
Epidermal Growth Factor. 5
The EGF Molecule 5
Functional Roles of EGF 6
EGF Related Peptides TGFa and VGF 7
The EGF Receptor 8
Specific Aims 11
2 MATERIALS AND METHODS 17
Mice 17
Reagents 17
IFN Y Production 18
Balb/c 3T3 Fibroblast Mitogenic Assay 18
Anti-EGF Antibody Functional Studies 19
Radioiodination of EGF 19
Balb/c 3T3 Fibroblast Growth Factor Binding Assays 20
Murine Spleen Lymphocyte Binding Assays 20
Reverse Phase High Performance Liquid Chromatography
of Radioiodinated EGF 21
3 RESULTS 22
A DISCUSSION 41
APPENDIX 46
REFERENCES 52
BIOGRAPHICAL SKETCH. 63
iv

ABBREVIATIONS
BSA, bovine serum albumin, radioimmunoassay grade from Sigma
CPM (cpm), counts per minute
EBSS, Earle's balanced salt solution
EGF, epidermal growth factor
FBS, fetal bovine serum
FGF, fibroblast growth factor
Hepes, N-2-Hydroxyethyl piperazine-N'-2-ethanesulfonic acid
HMEM, minimal essential medium modified with Earle's salts, penicillin,
streptomycin and 1 mM glutamine
HPLC, high performance liquid chromatography
hr, hours
IFN, interferon
M, molar
MAF, macrophage activation factor
pM, micromolar
mM, millimolar
nM, nanamolar
min, minutes
NaN^, sodium azide
PBS, phosphate buffered saline
PDGF, platelet-derived growth factor
Pen, penicillin 100 U/ml final concentration
v

rpm, revolutions per minute
SDMEM, Dulbecco's minimal essential medium supplemented with 15 mM Hepes,
lx nonessential amino acids, 1 mM sodium pyruvate, 0.075% sodium
bicarbonate, penicillin, streptomycin, and 1 mM glutamine
SEA, staphylococcal enterotoxin A
sec, seconds
Strep, streptomycin 20 pg/ml final concentration
TGEa, transforming growth factor a
Tjj, T helper cell
Tg, T suppressor cell
VGF, vaccinia virus growth factor
vi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF MURINE INTERFERON GAMMA PRODUCTION
BY EPIDERMAL GROWTH FACTOR
By
Na'eem Adib Abdullah
December, 1988
Chairman: Howard M. Johnson
Major Department: Immunology and Medical Microbiology
It has recently been shown that the epidermal growth factor (EGF)
molecule is capable of positive regulation of immune function by inducing
the production of gamma interferon (IFNY), establishing a functional
relationship between nonhematopoietic growth factors and the immune
system. In order to study this relationship further EGF and EGF-related
growth factors, transforming growth factor a (TGFa), and vaccinia virus
growth factor (VGF), which all exert their effects on cells through the
EGF receptor on cells, were studied for their functional and physicochem
ical effects on IFNY production at the receptor level.
IFN yproduction requires T helper (Tg) cell function. Both purified
murine EGF and recombinant human EGF were capable of restoring competence
for IFNY production by murine spleen cell cultures depleted of Tjj cells.
In agreement with this observation, anti-EGF antibodies were capable of
specifically neutralizing the EGF effect. Interestingly, EGF receptors
are not constitutively expressed on normal or primary lymphocytes. We
observed that treatment of lymphocytes with T-cell mitogens induced the
vii

expression of the EGF receptor, analogous to the induction of the high
affinity receptor on lymphocytes for interleukin-2. Neither TGFa nor VGF
were capable of restoring competence for IFNy production by Tjj cell
depleted spleen cell cultures. Furthermore, neither TGFa nor VGF could
block the ability of EGF to restore competence for IFN yproduction,
suggesting that the induced EGF receptor on lymphocytes is different from
that which is expressed on nonhematopoietic cells such as mouse 3T3
fibroblasts.
Consistent with the functional data for a novel inducible EGF
receptor on lymphocytes, the receptor was also detected by binding
studies with ^-I-labeled EGF. The receptor has a dissociation constant
of about 5 nanomolar. The receptor is optimally expressed after 48 hr
treatment of lymphocytes by T-cell mitogen. TGFa did not compete with
l^I-EGF for the lymphocyte EGF receptor. Both TGFa and VGF induced
proliferation of mouse 3T3 cells. Furthermore, consistent with previous
studies, both mitogens competed with EGF in receptor binding studies on
3T3 cells. Thus, the failure of TGFa and VGF to functionally mimic the
EGF help in IFNyproduction, and the failure of TGFa to compete for
receptor on lymphocytes, is compatible with the hypothesis that lympho
cytes express a novel inducible EGF receptor that differs from the
classic receptor expressed on cells such as 3T3 fibroblasts.
viii

CHAPTER 1
INTRODUCTION
Interferon Gamma
The Interferon Gamma Molecule
In 1957 an antiviral substance was discovered by Isaacs and
Lindenraann (1). This substance, which they termed interferon (IFN),
plays an important role in host defense against viral infection. There
is a large body of evidence that IFNs, particularly IFNy, play a central
role in the regulation of a variety of immune functions.
The human and murine IFNs are classified into three broad groups
which are based on antigenic properties, analogous to the classification
of immunoglobulin isotypes (2). They are designated IFNa, IFNS, and
IFN Y. Induction of IFNa and IFNS in appropriate cell types is achieved
by the addition of virus or polyribonucleotides, while IFNy is induced in
T lymphocytes or natural killer (NK) cells by specific antigens or T cell
mftogens (Table 1-1) (3).
The genes for all of the known IFNs have been cloned. The cloning
data has revealed 23 IFNa genes, at least 15 of which code for full
length proteins (4). IFNa is apparently glycosylated. The IFNa genes,
none of which contain introns, are clustered near each other on the same
chromosome. IFNa induces rearrangement of the T cell antigen receptor a-
chain and matures T lymphocyte clones to cytotoxicity ini vitro (5,6).
There are at least 3 IFNS genes, one of which does not contain introns,
while the other two appear to contain 4 introns (7-12). The intron-
1

2
containing IFN 3 genes are called IFNf$2 (7). IFNfio has a number of
interesting properties in addition to its antiviral properties (8-13).
While IFNP2 was initially identified as an antiviral protein isolated
from fibroblasts, others have found that IFNf^ from T cells acted as a B-
cell differentiation factor (BSF-2) that can enhance antibody secretion
and induce B-cell proliferation (8,10). IFNB2 is also called inter
leukin-6 (IL-6) (13).
IFN y has been cloned from the human (14,15), mouse (16), and bovine
(17) genomes. Only one gene has been found in all 3 species and it
contains 3 introns. The precursor polypeptide is processed and glyco
sylated. Based on the cDNA structure of the human and mouse genes the
mature IFNY molecule is composed of 133 amino acids and has a mass of
1.7,000 daltons (14,16). The molecular weight of natural IFNy varies from
20,000 to 25,000 daltons by SDS-PAGE due to glycosylation of the mature
molecule (18). Gel filtration studies have assigned IFN Ya mass of
40,000 to 50,000 daltons suggesting a natural multimer formed by the
mature molecule (19).
Regulation of IFNY Production
The cellular requirements for IFNY production have been studied in
detail in both the human and mouse systems (20-22). IFNY production in
the C57BL/6 mouse spleen cell system is regulated by a dynamic inter
action between helper cells (Lyt l+,2-), suppressor cells (Lyt l+,2+),
and IFNy-producing cells (Lyt 1",2+) (Figure 1-1) (3). Helper cells aid
IFN Y production via production of interleukin-2 (IL-2), which acts in
concert with a T-cell mitogen, staphylococcal enterotoxin A (SEA), to
induce IFNY- Suppressor cells inhibit or block IFN Y production by

3
adsorption or sequestration of IL-2. The IL-2 helper signal for induc
cin of IFN Yis dissociated from its ability to stimulate T-cell pro
liferation (3).
The question arose as to whether nonhematopoietic growth factors
were also able to stimulate IFNY production. Epidermal growth factor
(EGF), platelet-derived growth factor (PDGF), and fibroblast growth
factor (FGF), were shown not to stimulate lymphocyte proliferation
(23,24). It was demonstrated that these growth factors induce the
production of IFNY in conjunction with a T-cell mitogen signal (23,24).
By comparison, neither rat growth hormone nor human growth hormone are
capable of inducing IFNY production.
Immunoregulatorv Functions of IFNY
Interest in the immunoregulatory functions of IFNs began with the
observation that murine IFNa and IFN(3 suppressed antibody production m
in vitro assays (25,26). Concurrent studies showed that IFN could also
regulate cellular immunity and delayed type hypersensivity (27,28). Once
it was established that IFN enhanced natural killer (NK) cytotoxic
activity, the immunology community began a more rigorous study of the
immunomodulatory effects of the IFNs. It became apparent that the scope
of the immunoregulatory effects of the IFNs went far beyond the initial
observations, as shown in Table 1-2 (25-50).
Some of the immunoregulatory properties of IFNY are described herein.
Class I MHC molecules are up-regulated upon the addition of IFNY (44,45).
This surface antigen is important for the efficient killing of virally
infected cells. Class II MHC antigens are also up-regulated by IFNY
(43). The class II molecules are required for the cellular interactions
important for immune function such as antigen presentation. The expres-

-sion of class II molecules on the surface of macrophages is modulated by
IFNY as shown by their induction upon the addition of recombinant IFNY
(43,51). Indeed there are other surface molecules which are also up
regulated by IFNY, including the IL-2 receptor and Fc receptors (48,52).
Another important function of IFNY is its activity as a macrophage
activating factor (MAF) which augments various functions of macrophages,
including increased secretion of proteinases, hydrolases, oxygen metabo
lites, and increased killing capacity for tumor or virally infected
cells. A lvmphokine MAF activity was identified when it was observed
that activated T lymphocytes or their soluble products could prime or
activate macrophages for increased tumor cell killing (53). After
activation, macrophages are triggered to kill tumor cells by the addition
of a small amount of lipopolysaccharide which acts as a second signal.
Studies using purified IFN Y, recombinant IFNY and IFN Yneutralizing
antibodies have shown that the MAF activity is predominantly, if not
exclusively, due to the IFNY molecule. This data does not preclude there
being other structurally distinct MAFs. For example, IFNct and IFN& are
also MAFs, though not as potent as IFNY (54). As mentioned previously, T
killer lymphocyte and natural killer (NK) cytotoxic activity against
tumor cells and virally infected cells is also enhanced by the addition
of IFNs, especially IFNY (3).
Finally, IFNYbas been shown to modulate the differentiation of
myeloid and B lymphocyte cells (50,55). In the latter case the
differentiation signal provided by recombinant IFNY, was shown to be
sufficient to induce the secretion of antibodies (50).

5
Thus, IFNy is an immunoregulatory molecule which can modulate a wide
range of physiological states in a variety of cell types; its biological
functions augment both cellular and humoral defense systems as well as
identify potential targets for immune responses. An understanding of the
biological signals which regulate the production of IFNy will help in
understanding the initiation of the complex immune response. The work
presented here addresses the regulation of production of IFNy by EGF with
particular emphasis on receptor recognition.
Epidermal Growth Factor
The EGF Molecule
EGF is mitogenic for a wide variety of cell types. Studies on EGF
and its receptor have helped to increase our understanding of cell
growth, ligand-receptor interactions, receptor activation, and oncogenes
(56,57).
EGF was discovered in 1959 when it was observed that murine sub
maxillary gland extracts, when injected into newborn mice, induced early
eyelid opening and incisor eruption (56,58). Purification of the respon
sible substance showed that the active factor was EGF and not nerve
growth factor which is also present in extracts of murine submaxillary
glands (56). The purified EGF was also shown to stimulate proliferation
of epidermal cells and keratinocytes (59,60).
Mature murine submaxillary gland EGF is composed of 53 amino acids
with a molecular mass of 6,000 daltons (61). It has three disulfide
bridges which are required for biological activity. EGF is not glyco
sylated. There is 70% homology between mouse and human EGF and the two
molecules behave identically in cell and biochemical assays (56,57,62).

6
The EGF gene encodes a large precursor molecule containing EGF and 7
related peptides (63,64). The mature EGF molecule is contained in the
carboxyl terminal region of the precursor molecule and is obtained by
processing of the polyprotein. The functions of the other EGF-related
peptides in the polyprotein are currently unknown.
As indicated above, the submaxillary gland is a major source of EGF
in the mouse, whereas platelets are a major source of EGF in humans
(65,66). The submandibular and Brunners glands are also thought to be
sources of EGF in humans (67). Studies are in progress to further
determine the roles of various hematopoietic cells as sources of EGF and
related growth factors (68,69).
Functional Roles of EGF
Purified EGF has a wide range of biological effects. These include
(1) augmentation of ectodermal and mesodermal cell (e.g. fibroblast)
proliferation (56); (2) regulation of spermatogenesis (70,71); (3)
inhibition of gastric acid secretion (72); and (4) regulation of immune
functions such as IFNY induction (22,23).
With regard to the first point, the proliferative effect of EGF on
ectodermal and mesodermal cells types in vitro suggests that it may play
a role in wound healing. In controlled experiments EGF does indeed aid
in the wound healing process (73). With regard to the second point, EGF
apparently plays a major role in spermatogenesis. Male mice which have
been treated so as to not produce EGF (by submaxillary gland removal)
have low sperm and spermatid counts, while the addition of exogenous EGF
to such mice reverses this condition. Humans also have an EGF-like
molecule in their seminal plasma. Thus, EGF is probably a differenti-

7
ation signal for spermatogenesis in mammals. The third point, addressing
EGF's ability to inhibit gastric acid secretion, suggests that EGF can
inhibit gastric acid secretion without stimulating cell growth. Thus,
EGF may exert its effects in the alimentary canal not just as a growth
factor but also as a regulator of gastric acid secretion.
Finally, EGF has recently been shown to modulate the production of
the immunoregulatorv molecule IFNY. The observation is unique because
nonhematopoietic growth factors were heretofore thought not to play a
role in the immune system. Also, the literature suggests that hemato
poietic cells do not constituively express EGF receptors (57). Thus,
the observation implies that under certain conditions hematopoietic T
cells do express EGF receptors which, when bound by the EGF ligand,
initiate the production of an important biological response modifier,
IFNY.
EGF Related Peptides Transforming Growth Factor a and Vaccinia Virus
Growth Factor
Transforming growth factor a (TGFa) and vaccinia virus growth factor
(VGF) are structural and functional relatives of EGF. Both molecules, or
their relevant functional sequences, have (1) approximately 30% homology
with the EGF molecule (74-76); (2) have the same number of cysteines and
disulfide linkage alignment (77); (3) compete with EGF for its receptor
and use the EGF receptor solely for their known functions (69,75,76); and
(4) are mitogenic for the same tissues as EGF (57). The structural and
functional similarities imply that the three growth factors evolved from
a common ancestral gene (78,79).
TGFa was initially found in the supernatants of transformed cells
and was thought to be related to oncogenesis (80). But it is now known

8
to be a growth factor encoded in the genome of several species. The TGFa
gene encodes a messenger RNA (mRNA) that synthesizes a precursor poly
peptide, but unlike EGF there is no evidence of TGFa related peptides in
the precursor molecule. The mature TGFa molecule is a 6,000 dalton
protein of 50 amino acids (7A,75) and like EGF it is not glycosylated.
Biological properties that TGFa has demonstrated are mitogenic effects on
fibroblasts (57,69), and wound healing by inducing keratinocyte prolifer
ation and migration (81). TGFa is synthesized by both macrophages and
keratinocytes and thus may contribute directly to wound healing in vivo
as both cells are found in such lesions (68,82,83).
VGF, like EGF and TGFa, is synthesized as a precursor polypeptide.
The mature VGF molecule has 77 amino acids, is glycosylated, and has a
molecular mass of 23,000 daltons as determined by SDS polyacrylamide gel
electrophoresis (76). The structural relationship of EGF, TGFa, and VGF
is shown in Figure 1-2.
VGF has several EGF-like and other biological activities which
include: (1) mitogenic activity for fibroblast cells in vitro (57); (2)
initiation of mitogenesis and migratory activities for keratinocytes to
aid in wound healing (81); (3) augmentation of production of viral
progeny from infected cells (84). This latter property suggests that VGF
aids the lifecycle of the vaccinia virus through its cellular prolifera
tive properties.
Again, it should be emphasized that all of the known effects of TGFa
and VGF are mediated through the EGF receptor.
The EGF Receptor
The EGF receptor, also called c-erb B-l (57,85), has been found on
almost all tissues but has not been shown to be constitutiveiy produced

9
in hematopoietic cells. Studies have not been carried out to determine
if the receptor is inducible in such cells as primary lymphocytes in a
manner analogous to the receptor for IL-2, where the high affinity IL-2
receptor is not constitutively expressed but is induced by lymphocyte
mitogens (86). The EGF receptor is a 170,000 dalton single chain
glycoprotein with intrinsic tyrosine kinase activity (87). The mature
receptor is composed of three major domains, which are: (1) a large
glycosylated extracellular domain, (2) a transmembrane hydrophilic region
of 23 amino acids, and (3) a cytoplasmic region containing the tyrosine
kinase domain (57) that is composed of residues that are characteristic
of the tyrosine kinase family (88). Binding of EGF to its receptor
results in activation of the protein tyrosine kinase and thus triggers
subsequent intracellular events (57,89). The binding site of EGF on its
receptor is between residues 294 to 543 (90). Signal transduction is
mediated through the autophosphorylation of tyrosine residue 1173 (91).
Binding experiments with radiolabeled EGF to its receptor demon
strate a stoichiometric ligand-receptor interaction of one-to-one (92).
Scatchard analysis of EGF binding to intact cells, with high receptor
numbers, suggests the presence of different receptor classes with
distinct affinities toward EGF. High affinity receptors comprise 5 to
10% of the total, while the remaining receptors are of low affinity (93).
In EGF receptor negative 3T3 cells the introduction of wild type EGF
receptor showed both high and low affinity binding (94). Thus, the basis
for the same EGF receptor showing different affinities is not known.
Cells which are treated with the tumor promoter phorbol myristate
acetate (PMA) or platelet-derived growth factor (PDGF) abolish the high-

10
affinity receptor state (93,95-99). This "receptor transmodulation"
process also reduces the tyrosine kinase activity of the EGF receptor.
Receptor transmodu'lators bind to their own distinct receptors (100) and
activate protein kinase C (and/or other kinases) which phosphorylates EGF
receptor threonine (Thr) 654. Phosphorylation of Thr 654 on the EGF
receptor by PMA has been shown to block the mitogenic effects of EGF, but
this phosphorylation event occurs before phosphorylation by the tyrosine
kinase phosphorylase (101).
The EGF receptor has a half-life of greater than 5 hr (89). After
ligand binding the half-life decreases to approximately 1 hr. Receptor
degradation is mediated by coated pit internalization and subsequent
lysosomal enzyme degradation (56). EGF receptor mutation studies have
shown that the kinase activity is also essential for degradation of the
wild type receptor (89,100).
The EGF receptor molecule has several relatives. The c-erb B-2 gene
product is a 185,000 dalton glycosylated protein that shares about 50%
homology with the EGF receptor (102). It has an extensive extracellular
domain like its c-erb B-l counterpart yet it does not bind EGF or any
known ligand. C-erb B-2 transcripts have been detected in placenta,
kidney, embryonic tumors, stomach adenocarcinomas, and neuroblastomas.
Greater than 80% of the tyrosine kinase domain in c-erb B-2 is homologous
with its EGF receptor counterpart. The c-erb B-2 receptor is phos-
phorylated via interaction of EGF with the c-erb B-l receptor and is
believed to be a substrate for the c-erb B-l phosphorylating activity
(103,104). Four independent cell lines have the identical mutation from
valine 664 to glutamic acid in c-erb B-2 in the transmembrane region,

11
which subsequently converts it to the oncogenic form called neu
(105,106). The neu oncogene product of c-erb B-2 differs from its wild
type counterpart by this single point mutation which drastically alters
the function of the gene product. Another related oncogene is the v-erb
B gene product from the avian erythroblastosis virus (107). It is 68,000
daltons and has approximately 95% homology to the EGF receptor within a
stretch of 400 cytoplasmic domain residues (57). The amino terminal of
this molecule is truncated and thus does not bind EGF and there is also a
small truncation at the carboxyl terminus. The v-erb B oncogene has
autophosphorylation activity (108,109).
Thus, the interaction of EGF with its receptor initiates a complex
series of events which are responsible for EGE induced physiological cell
functions.
Specific Aims
The objective of the work outlined in this dissertation is to
characterize the EGF helper signal for induction of IFNy in a C57B1/6
murine splenic T cell system. We will focus on the functional and
receptor binding properties of EGF on mouse splenic lymphocytes as
compared to the interactions of EGF with its classic receptor on murine
3T3 fibroblast cells. We propose to achieve the objective through the
following specific aims: (1) definitively determine if EGF can provide
the helper signal for IFNy induction by using both EGF purified to
homogeneity and recombinant EGF in IFNy production studies; (2) determine
if the EGF related growth factors TGFa and VGF have the ability to
provide the helper signal for IFNy production and/or modulate the EGF
helper signal through competition for the receptor. Such studies should

12
provide insight into the relationship of the EGF receptor on lymphocytes
to the well characterized EGF receptor on 3T3 fibroblasts to which all
three mitogens bind and induce cellular proliferation; (3) determine if
the EGF receptor on splenic lymphocytes is constitutively produced or is
induced upon stimulation with SEA, and determine the binding character
istics of EGF with this receptor; (A) determine if TGFa and/or VGF can
compete with EGF for the EGF receptor on splenic lymphocytes.

13
Figure 1-1. IFNY production is regulated by the dynamic interaction
between helper (T^j), suppressor (T3), and IFNy producer cells. Helper
cells provide IL-2. Suppressor ceils absorb IL-2. Arrows: ( ),
positive signal; ( ), negative signal (3).

14
4 O
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70
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VGF I D G Y
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mEGF LOS Y
hEGF L D K _Y.
Q T R D
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90
V D Y Q R
L A
R W W E L R
l|k W W E L R
Figure 1-2. Alignment of the VGF, rTGFa (rat), mEGF (mouse), and hEGF
(human). The sequences of the mature peptide growth factors are shown in
their entirety, numbered from the N-terminus of the precursor VGF.
Residues conserved in all four sequences are boxed. The VGF sequence
represents the recombinant subcloned VGF used in the present studies and
was provided by Oncogen Inc., Seattle, WA. Adapted from Brown et al.
Nature 313:491 (1985) Reprinted from Nature, Vol. 313, No. 6002, pp.
491-492. Copyright (c) 1985 by Macmillan Journals Limited (77).

15
Table 1-1
Classification of I.FN's
Former
Interferon Cellular Source
Inducer
Nomenclature
IFN-cx
Lymphocytes (B, null,
Viruses, polyribonucleo-
Leukocyte,
and T), macrophage?
tides, tumors, chemicals
Type I
IFN-3
Fibroblast, epithelial,
Viruses, polyribonucleo-
Fibroblast,
macrophage
tides, chemicals
Type 1
IFN-Y
T lymphocyte
Antigens, T cell mitogens
Immune,
Type II
Reprinted from Lymphokines 1J^:33, 1985. Copyright (c) 1985 by Academic
Press, Inc. (3)

Table 1-2
Some Immunoregulatory Effects of IFN
Effects
References
Suppression and enhancement of antibody production
25,26,29-31
Suppression of antigen- and mitogen-induced
lymphocyte proliferation
27,28,32
Enhancement of specific cytotoxicity of T lymphocytes
33
Enhancement of NK cell cytotoxicity
34-37
Enhancement of antibody-dependent cell-mediated
cytotoxicity
38
Activation of macrophages for enhanced tumor ceil
killing
39-41
Modulation of expression of products of the major
histocompatibility complex on the cell membrane
42-45
Modulation of expression of Fc receptors on the
cell membrane
46-48
Modulation of expression of interleukin 2 receptors
on the cell membrane
49
Maturation of B cells for immunoglobulin production
and secretion
50
Reprinted from Lymphokines 11:33, 1985. Copyright (c) 1985 by Academic
Press, Inc. (3)

CHAPTER 2
MATERIALS AND METHODS
Mice
C57B1/6 female mice, 8 to 12 week old, are obtained from the Jackson
Laboratory, Bar Harbor, ME.
Reagents
The T cell mitogen SEA is purchased from Toxin Technology, Madison,
WI. Ultrapure mouse submaxillary gland EGF is obtained from Toyobo Co.,
Ltd., New York, NY. Recombinant human EGF is obtained from Scott
Laboratories, Fiskeville, RI. Highly purified synthetic rat TGFa is
obtained from Peninsula Laboratories, Inc., Belmont, CA. Recombinant VGF
is a gift from Dr. Gregory Bruce of Oncogen, Inc., Seattle, WA.
Recombinant c-sis PDGF (PDGF B) is from AMGen Biologicals, Thousand Oaks,
CA. -'-^-Iodide as a sodium salt (15 mCi/pg) and tritiated thymidine (21
mCi./mg) are obtained from Amersham Corp., Arlington Heights, IL.
Monoclonal anti-Lyt 1.2 antibody and anti-Lyt 2.2 antibody are obtained
from New England Nuclear, Boston, MA. The source of complement is serum
from New Zealand white rabbits. Endotoxin-free fetal bovine serum (FBS)
is obtained from HyClone Laboratories, Logan, UT. Trifluoroacetic acid
and radioimmunoassay grade bovine serum albumin (BSA) are obtained from
Sigma, St. Louis, MO. Dibutyl phthalate (gold label) and dioctyl
phthalate are obtained from Aldrich Chemical Co., Milwaukee, WI. Rabbit
anti-mouse EGF antibody (IgG fraction) is obtained from Collaborative
17

18
Research Inc., Bedford, MA. HPLC grade acetonitrile is obtained from
Fisher Scientific, Orlando, FL.
IFN-Y production
Spleen cells (1 ml at 1.25 x 10^ cell/ml), 0.1 ml of a 10-^ dilution
of monoclonal anti-Lyt 1.2 antibody and rabbit complement (0.1 ml of
serum) are mixed and incubated at 37C for 1.5 hr in RPMI 1640 containing
10% FBS. The helper cell-depleted cultures are then washed twice,
suspended in the RPMI 1640 media containing 10% FBS, and cultured in
duplicate at 1.25 x 10^ viable cells/ml. Whole spleen cell- and helper
cell-depleted cultures are stimulated with 0.5 pg/ml of SEA for 1 to 3
days in the presence of growth factors, and the supernatants are assayed
for IFNy activity on mouse L cells by using vesicular stomatitis virus as
described (110). The IFN produced is identified as IFNy by
neutralization with specific antisera.
Balb/c 3T3 Fibroblast Mitogenic Assay
The 3T3 growth factor mitogenic assay is performed using a
modification of Kobavashi et al. (111). The 3T3 fibroblasts are grown to
subconfluent monolayers in supplemented Dulbecco's minimal essential
medium (SDMEM) containing 10% FBS. Note, 3T3 fibroblasts are not used
beyond passage number 40. The cells are harvested, washed and seeded in
96 well plates (Falcon) at 5 x 10^ cells/well in 0.2 ml. All peripheral
wells receive 0.2 ml of medium only to keep the humidity of the target
wells constant. After 24 hrs in 5% CO2, 37C the spent medium is
replaced with 0.1 ml of SDMEM containing either 10% FBS or 0.2% FBS with
or without dilutions of growth factors in duplicate. After 22 hrs at 5%
C0'2, 37C, 0.1 pCi of -%-thymidine in 10 ,ul is added to each well. The
samples are incubated another 2 hrs at 37C, 5% CO2- After washing twice

19
with phosphate buffered saline (PBS), cells are solublized by treatment
with 0.1 M NaOH (0.1 ml/well). The contents of the wells are then
harvested and counted on a LKB beta liquid scintillation counter
(Gaithersburg, MD).
Anti-EGF Antibody functional Studies
Rabbit anti-mouse EGF antibodies are used to block EGF functional
interactions with EGF receptors on Balb/c 3T3 cells and on C57B1/6 mouse
spleen cells. Mouse EGF and anti-EGF are mixed and incubated for 1 hr at
37C prior to addition to either 3T3 cells or spleen cells. 3T3
proliferation and IFNy production assays are carried out as previously
described. Recombinant human c-sis PDGF is also incubated with anti
mouse EGF as described above and is added to spleen cell cultures.
Radioiodination of EGF
EGF is labeled with ^-^1 using a modification of Das et al. (112).
Briefly, 2 Mg in 10 pi of EGF (200 pg/ml in 0.4 M KP04, pH 7.5) is added
to a 1.5 ml Eppendorf test tube. Five pi of Na ^--"Iodide (500 pCi) and
10 pi of freshly dissolved chloramine-T (6.25 mg/ml) are added. After 1
min the reaction is quenched by the addition of 10 pi of Na
metabisulfide (12.5 mg/ml) and 10 pi of KI (80 mg/ml). Then 10 pi of BSA
(Sigma) (20 mg/ml) is added and the sample is run over a 7 ml Sephadex G-
10 column (Pharmacia) equilibrated with 50 mM KP04 (pH 7.5) containing
0.15 M NaCl and 2 mg BSA/ml. Fractions are counted and those with the
highest counts are pooled. The specific activity of the pooled sample is
generally from 100-125 pCi/pg. ^^I-EGF is aliquoted and stored at -70C
for no more than 3 weeks prior to use.

20
Balb/c 3T3 Fibroblast Growth Factor Binding Assays
The 3T3 growth factor binding assay is performed using a
modification of Kobayashi et al. (ill). 3T3 fibroblasts are grown as
above and seeded at 6 x 10^ cells/weil in 0.2 ml volume. All the
peripheral wells are treated as above. After 24 hrs at 37C, 5% CO2
confluent monolayers are washed 3 times with Earle's balanced salt
solution (EBSS) lacking calcium (57) containing Pen/Strep and 20 mM NaNg,
2 mg BSA/ml and aspirated to dryness. Then 25 pi of EBSS or dilutions of
growth factors are added to wells in triplicate. After 15 min, 25 pi of
l^I-EGE is added. After 1 hr at room temperature the wells are washed 3
times with EBSS and NaOH (50 pi of 0.1 M) is added to each well to
solubilize the cells. The liquid is absorbed by cotton-tipped
applicators and counted on an LKB gamma counter.
Murine Spleen Lymphocyte Binding Assays
Spleen cells are resuspended at 5 x 10^ cells/ml in SDMEM containing
10% FBS in the presence or absence of 0.5 pg SEA/ml. The cells are
seeded at 25 ml per 150 cm^ Corning flask and incubated at 37C, 5% CO2
for 48 hr. Nonadherent cells are then collected and centrifuged at 1,000
rpm for 10 min. Red blood cells are lysed by the addition of cold
distilled water (1.5 ml per 3 spleens) to the completely dispersed
pellet. After 10 seconds, cold EBSS (5.5 ml per 3 spleens) lacking
calcium and containing 0.15 M NaCl, 15% FBS, 20 mM NaNg, and 2 mg/ml RIA-
BSA is added to the spleen cells. After 10 minutes on ice the cells are
placed into a 15 ml Corning test tube and centrifuged at 133 X g for 40
sec (adding 30 sec for each additional 5 ml). The supernatant is
aspirated and the cells are washed twice in cold EBSS containing 20 mM

21
NaNg and 2 mg./ml RIA-BSA (7 ml) exactly as above. The cells are
resuspended in 0.1 ml of the above buffer and the debris is allowed to
settle for 2 min. The cells are counted and resuspended in the above
buffer at a final concentration of 4 x 10^ cells/ml final concentration.
Cells are incubated on ice or at room temperature for 15 min in the
presence of cold growth factors or buffer prior to the addition of I-
EGF. The reaction volume is 40 pl/tube and all experiments are performed
in triplicate. Binding is carried out on ice or at room temperature for
1 hr at which time samples are carefully layered into 0.4 ml
polypropylene tubes over a phthalate oil mixture (1:1.27 ratio of dioctyl
phthalate to dibutyl phthalate, v/v). Samples are centrifuged at 12,000
rpm for 30 sec to separate free ^-*I-EGF from bound ^~I~EGF. Test tubes
are then cut and cell pellets are counted on an LKB gamma counter.
Reverse Phase High Performance Liquid Chromatography of Radioiodinated
EGF
The HPLC apparatus uses a Perkin-Elmer Series 400 pump (Norwalk,
CT), a Rheodvne model 7125 injector (Cotati, CA) with a 50 pi
loop, a 25 cm x 4.6 mm Pecosil C-^g 10 pm particle size reversed phase
cartridge column and a Pellicular C^g packed guard column (Perkin-Elmer).
Fractions are collected on a LKB 2111 Multirac fraction collector.
Murine and human EGF are each radioiodinated as described and 10^ cpm in
0.8 ml are loaded onto the column. Elution is accomplished by a linear
gradient of 10% acetonitrile and 0.1% trifluoroacetic acid at pH 3.1 to
100% acetonitrile over 60 min (1 min per fraction). Samples are
collected and counted on a gamma counter. A salt peak of free LZ-JI
eluted early in each run and is not shown in the figures.

CHAPTER 3
RESULTS
It was previously shown that electrophoretically pure mouse sub
maxillary gland EGF (from Collaborative Research) could replace the IL-2
requirement for IEN Yproduction in the mouse (C57B1/6) spleen cell system
(23,24). To confirm the above, another source of mouse submaxillary
gland EGF (Toyobo) that was purified to homogeneity was tested for its
ability to replace the IL-2 requirement, and was observed to provide
essentially maximal help for IFNy production at 1 nM (Table 3-1).
Purified recombinant human EGF (Scott Labs) also provided the helper
signal at 1 nM as shown in Table 3-1. Thus, both EGF preparations have
equivalent potency for inducing IFNy. Moreover, specific anti-murine EGF
antibodies (Collaborative Research) could completely block EGF-induced
IFNy production while this same antibody preparation had no effect on the
recombinant c-sis PDGF (AMGen) helper signal for IFNY production (Table
3-2). Purified murine submaxillary gland EGF and recombinant human EGF
were radioiodinated and passed over a reverse phase high performance
liquid chromatography column in order to confirm their indicated purity.
In both cases these labeled EGFs eluted as a single peak as indicated in
Figures 3-1 and 3-2. This provides further evidence that the helper
signal provided by EGF for IFNy production is due to the EGF molecule and
not to a contaminant in these preparations.
22

23
As indicated, the growth factors TGFa and VGF are related to EGF
(Figure 1-2). They are in the family of EGF-like peptides as determined
by structural similarity, the placement of cysteine residues within the
molecules, and, most importantly, they exert their mitogenic effects on
fibroblasts via the EGF receptor (57,74-77). Thus, synthetic rat TGFa
(Peninsula Labs) and recombinant VGF (Oncogen) were examined for their
ability to provide the helper signal for IFNYproduction. Unlike EGF,
neither TGFa nor VGF could restore competence for IFNy production by
mouse C57B1/6 spleen cell cultures that were depleted of helper cell
function (Figure 3-3) (113). Both growth factors were as competent as
EGF in stimulating proliferation of 3T3 fibroblasts (Figure 3-4), so
their inability to provide the helper signal for IFNy production was not
due to factors such as lack of biological activity. Rather, it is
possible that the EGF receptor on lymphocytes is different from that on
3T3 cells and thus novel, or that TGFa and VGF can bind to the EGF
receptor on lymphocytes, but cannot trigger the signal for IFNy
induction. If the former were true, then TGFa and VGF should not
functionally compete with EGF for the receptor and thus block its helper
signal, whereas if the latter were true, then they should block the EGF
helper signal for IFNy production. As shown in Table 3-3, neither TGFa
nor VGF in molar excess blocked the helper signal of EGF for production
of IFNy by lymphocytes (113). Additionally, neither growth factor
blocked IFNy production by spleen cells that were not depleted of helper
cell function (Table 3-4). Thus, the functional data suggest that EGF
provides its helper signal for IFNy production by interaction with a
novel receptor on lymphocytes.

24
Hematopoietic cells have been reported to lack EGF receptors (57),
thus EGF receptor binding studies were performed with untreated splenic
lymphocytes cultured for 48 hr and splenic lymphocytes that were stimu
lated for 48 hr with the T-cell mitogen SEA. Exogenous SEA is required
for several aspects of IFNY production (3). First, SEA acts as a T cell
mitogen by inducing IL-2 production by Ty- cells (3). Second, SEA is a
required second signal, along with IL-2, to initiate T cell production of
IFNY.
As shown in Table 3-5, specific binding of -"^I-EGF (125 pCi/ug
protein) was observed only in cultures that were stimulated with SEA for
48 hr (113). Specific binding was generally 25 to 50 percent of total
binding. The data indicate that the EGF receptor is induced on
lymphocytes and not constitutively expressed as in 3T3 fibroblasts.
Similar data have been obtained in at least 3 separate experiments. A
saturation binding curve for ^-I-EGF on splenic lymphocytes is
presented in Figure 3-5, and indicates a of 5 to 10 nM. Since the
cultures were depleted of adherent cells, it is unlikely that the binding
observed was due to macrophages, but it cannot be ruled out that small
amounts of contaminating macrophages may play some role in EGF binding
to lymphocytes. The question has arisen that the novel EGF receptor
described above may actually be the IL-2 receptor on lymphocytes, since
the high affinity IL-2 receptor is also induced by T-cell mitogens. It
has previously been shown, however, that EGF did not compete with IL-2
for the IL-2 receptor on lymphocytes (86). Therefore, the novel EGF
receptor on lymphocytes is different from the IL-2 receptor on
lymphocytes.

25
EGF, TGFa, and VGF compete similarly for the EGF receptor on 3T3
cells (69,75,76). We confirmed this in competitive binding experiments
with -'-^-I-EGF and the EGF, TGFa, and VGF that were used in the functional
studies above (Figure 3-6). PDGF does not bind to the EGF receptor, but
does slightly down regulate the expression of high affinity receptors
(97-99). Thus, ^^^I-EGF binding to 3T3 cells was reduced by PDGF
treatment, but not nearly as much as by the ligands that compete for the
receptor. Thus, the data in Figure 3-6 are consistent with and confirm
previous observations on the properties of the EGF receptor on 3T3
fibroblasts.
As indicated, the functional data presented suggest that EGF exerts
its effects on lymphocytes by binding to a novel receptor, since TGFa and
VGF could not provide the helper signal for IFNy production, and could
not functionally block the help of EGF. The binding competition data of
Figure 3-7, where cold submaxillary gland EGF blocked ^^I-EGF binding to
lymphocytes, but TGFa at the same concentrations was without effect is
further evidence that the EGF receptor on lymphocytes is novel. We
currently do not know why relatively high concentrations of cold EGF are
required in receptor competition experiments with lymphocytes, but the
relatively low specific binding to total binding may play a role. Also,
it is possible that the binding studies using submaxillary gland -^JI-EGF
could involve a contaminant in the EGF preparation, but this is unlikely
since purified recombinant human EGF inhibited submaxillary gland iiJI-
EGF binding as effectively as did cold submaxillary gland EGF
(Table 3-6). Similar competitive binding patterns have been observed in
other systems such as the adrenergic receptors (114).

26
The above data raise questions about functional sites on the EGF,
TGFa, and VGF molecules that are involved in receptor interactions. It
has been suggested that the third disulfide loop of these growth factors
comprises the binding site for the EGF receptor on such cells as 3T3
fibroblasts (115). The data presented here raise questions about a
common binding site for these 3 growth factors with respect to
interactions with lymphocytes. In order to gain further insight into the
structural relationship of these molecules we generated composite surface
profile plots which are based on hydrophilicity, accessibility, and
flexibility of secondary structure (116,117). Mouse and human EGF showed
very similar composite surface profiles (Figure 3-8a and 3-8b). Their
profiles were different from those of TGFa and VGF (Figure 3-8c and 3-
8d). The composite surface profile plots suggest structural differences
between EGF and TGFa and VGF. A precise delineation of these differences
could possibly provide insight into EGF interaction with lymphocytes in
the absence of such an interaction by TGFa and VGF.
We feel that the data presented are quite interesting and allow
us to draw the following conclusions: (1) EGF provides a helper signal
for the induction of IFNy, (2) EGF provides its signal by interaction
with a novel receptor on lymphocytes based on functional and receptor
competition data with TGFa and VGF, (3) the novel EGF receptor on
lymphocytes is not expressed constitutively but is induced by a T cell
mitogen.
Related to our discovery of a novel EGF receptor on lymphocytes is a
recent finding of a novel PDGF receptor on human fibroblast and baby
hamster kidney cells (118-120).

27
Figure 3-1. HPLC profile of murine EGF. Murine submaxillarv gland EGF
(Toyobo) was radioiodinated and passed over a reverse phase HPLC
column. Fractions are eluted and counted in a gamma counter.

40 i
28
I
I I
20 40
Figure 3-2. HPLC profile of recombinant human EGF.
EGF (Scott Labs) was radioiodinated and passed over
HPLC column. Fractions are eluted and counted in a
60
Recombinant human
a C^g reverse phase
gamma counter.

iriiy (ii/iiin
29
GROWTH FAC i OR (fir!)
Figure 3-3. Ability of growth factors to restore competence for IFN y
production by Lyt L-, 2"*" spleen cells. EGF iO)< TGFa (A), and VGF (Q)
were incubated with Lyt 1-depleted spleen cells in the presence of the T-
cell mitogen SEA. IFNYtiters are from day 3 of culture. Reprinted from
Progress in Leukocyte Biology 8:159, 1988. Copyright (c) by Alan R.
Liss, Inc. (113).

30
0-
!
0.001
1 i
0,01 0,1
GROWTH FACTOR (nil)
~ I
10 30
Figure 3-4. Relative mitogenic activity of growth factors on Balb/c 3T3
cells. Cells were incubated for 24 hr in SDMEM containing 10% FBS,
washed extensively, and growth factors were added at the indicated
concentrations. After 24 hr at 37C in the presence of 5% CC>2, JH-
thymidine was added for 2 hr. Cells were then harvested and counted on a
beta scintillation counter. Symbols: EGF (A); TGFa (Q)? VGF ().

31
Figure 3-5. Binding of 125I-EGF to spleen cells. Spleen cells were
incubated with 3.3 pM EGF for 15 min prior to a 1 hr incubation with
l25I-EGF at the indicated concentrations. Cells were then harvested and
counted on a gamma counter. Data are plotted as bound i2 I-EGF vs. free
125I-EGF.

-EOF (CPM)
32
:,800
2,400-

o-
i
0
ih
[
0.1
I I
1 10
GROWTH FACTOR CnM)
Figure 3-6. Competitive binding of ^^I-EGF to Baib/c 3T3 cells in the
presence of growth factors. Confluent monolayers were incubated for 15
min with cold EGF (.), TGFa {Q), VGF (A), PDGF B (Q), or buffer (A).
125i-EGF was then added at a final concentration of 5 nM. After 1 hr
incubation at room temperature, cells were harvested and counted on a
gamma counter.

33
Figure 3~7. Competitive binding of ^-I-EGF to spleen lymphocytes in the
presence of growth factors. SEA-stimulated spleen lymphocytes were
harvested as previously described and incubated for 15 min with EGF (O)
TGFa (A), or buffer (CD). -EGF was then added at a final concen
tration of 5 nM. After 1 hr incubation at room temperature, cells were
harvested and counted on a gamma counter.

100
Figure 3-3. Surface profile of EGF-related proteins. Composite surface profiles of mouse EGF (A),
human EGF (B), rat TGFot (C), and VGF (D) were developed as described by using a computer program
i'118) This program takes into account HPLC mobility, accessibility, and segmental mo i lty l
values) of amino acids in model proteins and peptides. Residues with high composite values are most
likely to reside on the surface of a protein molecule. The abscissa equals the surface value. e
ordinate equals the residue number.

Table 3-1
Relative abilities of mouse EGF and recombinant human EGF
to provide the helper signal for IFNy production
by Lyt 2+ spleen cells
GFa
(nM)
IFNy(U/ml
moEGF
+ S.D. )
huEGF
0
<3
<3
0.1
<3
<3
1
65 7
55 + 7
10
60 .14
85 21
100
95 7
200 141
aSamples were from day 3 of culture. Whole spleen cells
had 95 35 IFN Yunits/ml.

36
Table 3-2
Ability of anti-EGF antibodies to block EGF
helper signal for IFNy production
by Lyt 2+ spleen cells
Anti-EGF Antibodies3
(Mg/ml)
IFN y(U/ml + S.D. )
EGF 5nM PDGF 5nM
0
120 28
105 21
0.5
80 14
220 28
5
<3
85 21
50
<3
135 21
aSampies were from day 3 of culture. Whole spleen cells had 80 28
IFNYunits. Anti-Lyt 1.2 + C control cells had <3 IFNyunits.

37
Table 3-3
Failure of TGFa and VGF to block helper signal for
FNY production by mouse spleen cells
SEA-stimulated cultures3
IFNY (U/ml SD)
Ly t 1
> 2+
cells
<7
Lyt 1"
, 2+
cells +
EGF
270
42
Lyt 1"
, 2+
cells +
EGF
+ TGFa
290
14
Lyt 1-
, 2+
cells +
EGF
+ VGF
295
7
Whole
spleen cells
215
21
aSamples are from day 3 of culture. Concentration of
factors: EGF, 5 nM; TGFa, 14 nm; VGF, 17 nM.
Reprinted from Progress in Leukocyte Biology 8:159, 1988.
Copyright (c) 1988 by Alan R. Liss, Inc. (113)

38
Table 3-4
Failure of TGFa and VGF to block IFN Y production
in spleen cells
SEA-stimulated cultures3
IFN Y (U/ml SD)
TGFa
275 35
VGF
350 71
Control
275 106
aSamples are from day 3 of culture. Concentration of
factors: TGFa, 14 nM; VGF, 17 nM.

39
Table 3-5
Induction of
EGE receptor
by SEA on mouse spleen
lymphocytes3
SEA
Cold
CPM
p-value
treatment,
EGF,
0.5 Mg/ml
3 i_iM
-
-
11.15 256
-
+
1009 49
N.S.
+
-
1539 64
+
+
1158 54
<0.005
aSpleen cells were incubated with SEA for 48 hr, at which
time cultures were depleted of macrophages and red blood
cells. CPM (counts per minute) are from lymphocyte bound
^^I-EGF. Reprinted from Progress in Leukocyte Biology
8:159, 1988. Copyright (c) by Alan R. Liss, Inc. (113).

AO
Table 3-6
Ability of recombinant human EGF to compete with
murine purified submaxillary lz,Jl-EGF for the
EGF spleen cell receptor3
rHuEGF
(nM)
MuEGF
(nM)
CPM
+
SD
-
-
1488
+
269
10
-
1424
+
197
100
-
1463
+
235
330
-
1189
+
183
1000
-
1184
+
97
3300
-
1100
+
108
-
3300
1152
+
78
aSpleen cells were stimulated with SEA for 48 hr
and washed extensively prior to binding with
-^-I-EGF at 5 nM final concentration.

CHAPTER 4
DISCUSSION
EGF is a molecule with a wide variety of biological functions.
These include: (l) the proliferation of ectodermal and mesodermal cells
(56) and thus wound healing (73); (2) regulation of spermatogenesis
(70,71); (3) inhibition of gastric acid secretion (72); and (4)
regulation of IFNy production (22,23). The first three functions are
believed to be mediated through the well characterized EGF receptor
(57,100).
The EGF receptor is constitutively found on almost every tissue
studied except for the hematopoietic cell types (57). It has been cloned
and sequenced (87). It is a 170,000 dalton glycoprotein which has been
extensively characterized in its structure and its functions. The EGF
receptor has three major domains: (1) an extracellular domain which is
responsible for the signal transduction after ligand binding; (2) a
transmembrane region; and (3) the intracellular region which contains the
tyrosine kinase domain responsible for the signal transduction after
ligand binding onto the receptor (57,100). Signal transduction is
mediated through autophosphoralation of the EGF receptor at tyrosine
1173.
The EGF-related peptides TGFa and VGF are structurally related to
EGF and compete with this ligand for its receptor (57,74-76). They also
share known biological functions that are mediated through the EGF
41

42
receptor. These include mitogenic effects on fibroblasts (57,69,76) and
wound healing (81). VGF can also augment the production of viral progeny
from infected cells (84). As indicated in the Results EGF, TGFa, and VGF
molecules are structurally and functionally related to each other,
suggesting evolution from a common gene (78,79). The third disulfide
loop, a region that is most common to all three ligands in sequence and
structure, is believed to be the ligand binding region which is
recognized by the classic EGF receptor (115). In the data presented here
we have confirmed that TGFa and VGF, like EGF, can stimulate the
proliferation of 3T3 fibroblasts as well as compete for binding to the
EGF receptor on these cells using ^JI-labeled EGF. The question arises
as to whether purified and recombinant EGF from other sources and whether
TGFa and VGF can similarly provide the helper signal for induction of
IFN y .
In the course of studies on the regulation of murine IFNY production
by various growth factors it was found that EGF was capable of inducing
IFNY production in spleen cell cultures depleted of Tg cells (23,24).
The mouse submaxillary gland EGF preparation used in these studies (from
Collaborative Research) was electrophoretically pure. This preparation
was found, however, to restore the production of IFNYat 17 nM. This
concentration is higher than that required for the proliferative
functions of EGF for fibroblasts. Thus, the question was raised that a
possible contaminant of the EGF preparation was responsible for the
helper signal for IFNY production. To address this question and to
further extend our knowledge of the effects of EGF in the production of
IFNY, we obtained EGF from several other sources. We have shown here

43
that purified mouse submaxillary gland EGF (Toyobo) and recombinant human
EGF (Scott Labs) were both capable of inducing the production of IFNy at
1 nM. Thus, unlike the relatively high EGF concentrations required to
induce IFNY in the previous study, the IFNY inducing concentrations of
these EGF preparations approximated those concentrations required to
detect mitogenic effects of EGF on fibroblasts. The initial observation
that EGF can provide the helper signal for IFNY production, then,
apparently involved the use of EGF that was not fully active. The EGF
preparations that we used in these studies were radioiodinated, eluted
from a reverse phase column and only one peak was found in each sample,
indicating only one major species in each sample. These data excluded
the possibility of there being a contaminant in these preparations that
was responsible for the induction of IFNy* Thus, the data presented here
show EGF to provide the help for IFNy production at concentrations
similar to those for its other biological effects. Also anti-EGF
antibodies inhibited EGF induction of IFNY in a specific manner.
The above data demonstrate that EGF can regulate lymphokine
production. As indicated earlier, lymphocytes do not constitutively
express an EGF receptor. Although EGF receptors are generally thought
not to be expressed on hematopoietic cells there is one preliminary
report of induction of an EGF receptor on B lymphoblastoid cells (121).
The B lymphoblastoid lines RPMI 1788 and RPMI 8392 were both shown to
bind -*-25i_egf after treatment with the B cell mitogen staphylococcal
B cell mitogen (STM). RPMI 1788 cells expressed between 500 to 1,000 EGF
receptors while RPMI 8392 expressed between 2,000 to 4,000 EGF receptors.
Normal lymphocytes from blood or spleen could also be induced to express

44
EGF receptors after treatment with B cell mitogens. Induction of
receptor was not observed when the cells were treated with the T-cell
mitogen Concanavalin A. Other T-cell mitogens were not tested. This EGF
receptor was not further characterized. Interestingly, in our studies we
found an EGF receptor on splenic lymphocytes that required induction by
the T-cell mitogen SEA. The splenic lymphocyte EGF receptor was
detectable after a 48 hr incubation in the presence of SEA.
Approximately 25% of total binding was specific, which would suggest the
presence of low receptor numbers. The splenic lymphocyte EGF receptor
has an apparent dissociation constant of 7 nM.
The IL-2 receptor is another example of an inducible lymphocyte
receptor (86). This receptor is induced by a T-cell mitogen and takes
several days before it can be detected on the surface of lymphocytes. The
induced IL-2 receptor does not recognize the EGF molecule as a
competitive ligand in the presence of labeled IL-2. Therefore, the
induced EGF receptor is different from the IL-2 receptor on lymphocytes.
Since we had shown that the EGF molecule itself, and not a
contaminant, regulated the production of the IFNY lymphokine, we then
determined if the EGF-related growth factors TGFa (synthetic) and VGF
(recombinant) also induced IFNy production. We were surprised to find
that, unlike EGF, neither TGFa nor VGF were able to induce the production
of IFNY(Figure 3-3). Moreover, neither peptide functionally blocked the
ability of EGF to induce IFNY; nor were these peptides able to suppress
the production of IFNY in whole spleen cells. Thus while TGFa and VGF
were biologically active in induction of 3T3 fibroblast proliferation
(Figure 3-4), unlike EGF. they were not capable of inducing IFNy in

lymphocytes. Finally, in competitive binding studies we demonstrated
that TGFa did not compete with ^-*I-EGF for the induced lymphocyte EGF
receptor while it did compete for receptor on 3T3 cells. We conclude
that there is a novel EGF receptor on lymphocytes which, unlike the
classic EGF receptor, does not functionally or physicochemically (in
binding studies) recognize the EGF-related peptides TGFa and VGF.
The induction of IFNy by EGF is a newly described function for this
nonhematopoietic growth factor. Its effect is mediated through a novel
EGF receptor. This EGF receptor differs from the classic EGF receptor in
that it is inducible by a T-cell mitogen, and fails to recognize the EGF-
related growth factors TGFa and VGF.

APPENDIX
PRELIMINARY DATA
Previous studies have shown that rabbit polyclonal antibodies to the
classic EGE receptor blocks EGP induced proliferation of mouse 3T3
fibroblasts (122). We compared the effects of these antibodies on both
EGF induced 3T3 proliferation and EGF helper signal for IFNy production.
Table A-l shows partial suppression of %-thymidine incorporation into
3T3 cells. Normal rabbit serum was not inhibitory and at higher
concentrations had an enhancing effect. These data are similar to that
of Adamson and colleagues (122). Anti-EGF receptor antibodies had no
effect on EGF helper signal for IFNy production (Table A-2). The anti-
EGF receptor antibody data supports our previous conclusion that the
lymphocyte EGF receptor is different iron the classic EGF receptor.
In order to determine the size of the lymphocyte EGF receptor, we
labeled EGF with and used this as a probe. We. confirmed the size of
the classic EGF receptor by cross-linking the -^^I-EGF onto Balb/c 3T3
fibroblasts in the absence and presence of cold EGF. The preparations
were prepared and run on an 8% SDS-PAGE system. After autoradiographic
exposure the dominant 170,000 dalton EGF receptor was seen as expected
(Figure A-l). The minor bands at 120,000 and 78,000 dalton bands are
known EGF receptor breakdown products (58). As expected there was no
radiolabeled signal in the preparation containing cold EGF. The same
46

procedure using radiolabeled EGF was carried out with 48 hr 5EA-
stimulated lymphocytes. The detected bands migrated at 130,000 daltons,
100,000 daltons, and 50,000 daltons upon autoradiographic exposure
(Figure A-2). The 100,000 dalton and 50,000 dalton bands could represent
degradation products of the 130,000 dalton band or the 3 bands could
represent a complex EGF receptor on lymphocytes. Further studies need to
be conducted to better delineate the nature of the induced lymphocyte EGF
receptor.

48
1 2
Figure A-L. SDS-PAGE in 8% acrylamide/bis with L^^I-EGF cross-linked
onto 3T3 fibroblasts in the absence (lane 1) and presence (lane 2) of 1
mM cold EGF. Confluent 3T3 cell monolayers were incubated in the
absence and presence of 1 uM cold EGF for 15 min prior to the addition of
5 nM 1-251-egf. After 1 hr at 0C, cells were washed and cell-bound EGF
was covalently cross-linked to the cellular receptor by the addition of
Bis (suifosuccinimidyl) suberate (BSJK After I hr at 0C, the BSJ was
removed. Cells were suspended in SDS-PAGE sample buffer. Samples were
electrophoresed under reducing conditions on an 8% separating gel.
Molecular weight markers were also run on the gel.

49
1
2
Figure A-2. SDS-PAGE in 8% acrylamide/bis with L^^1-EGF cross-linked
onto 48 hr SEA stimulated splenic lymphocytes in the presence (lane 1)
and absence (lane 2) of 3.2 pM cold EGF. Samples are incubated for 15
min at 0C in the presence or absence of 3.2 pM final concentration cold
EGF prior to the addition of 5 nM ^*-^I-EGF. 'Ceils are washed without BSA
and cell-bound EGF is covalently cross-linked to the cellular receptor by
the addition of freshly prepared BS^. After 1 hr at 0C the BS^ was
removed and the spun sample pellets treated with SDS-PAGE sample buffer
and sonicated. Samples were centrifuged to remove debris and superna
tants were boiled prior to being electrophoresed. Samples were electro-
phoresed on an 8% separating gel as in the 3T3 EGF receptor SDS-PAGE
protocol. The gel was autoradiographed at -70C for 6 weeks with
intensifying screens prior to film development.

50
Table A~1
Ability of anti-EGF receptor antibodies to block EGF-induced
thymidine incorporation by 3T3 fibroblasts3
Immunoglobulin Percent of control
protein (ug/ml)^ ^H-thymidine incorporation
Anti-EGF Normal
receptor antibody rabbit serum
0
100
100
30
64
98
LOO
64
126
300
45
221
1000
42
263
aBalb/c 3T3 fibroblasts were incubated in duplicate with either rabbit
anti-EGF receptor antibodies or control normal rabbit serum for 1 hr
prior to the addition of EGF (2 nM). After 24 h at 37C in the presence
of 5% CO'), %-thymidine was added for 2 hr. Cells were then harvested
and counted on a beta scintillation counter.
^Rabbit polyclonal anti-EGF receptor antibody was provided bv Dr. E.
Adamson (122). The antibody and normal rabbit serum were ammonium
sulfate precipitated, dialysed in PBS, aliquoted, and frozen.

51
Table A-2
Effect of anti-EGF receptor antibodies on IFNy production by
Mitomycin C-treated spleen cells in the presence of EGF
EGF
5nM
Anti-EGF receptor
antibodies (pg/ml)
NRS
(Mg/ml)
IFNy3
(U/ml SD)
-
-
-
<3
-
-
35 7
t*
30
-
25 7
+
100
-
28 4
+
300
-
33 4
+
1,000
-
28 4
+
-
30
45 21
f
-
100
65 21
+
-
300
50 28
+
-
1,000
28 4
a£FNY titers are from Day 3 of culture. Untreated spleen cells
stimulated with SEA produced 85 21 units IFNY/ml on Day 3 of culture.
Normal rabbit serum (NRS) was used as a control.

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BIOGRAPHICAL SKETCH
On September 15, 1956, in Lorain, Ohio, Keith Anderson was born to
Dr. Marvin Moore Anderson and Mrs. Dorothy-Jean Barbee Anderson. He has
4 brothers who are Mansur Abdullah, Clarence, Christopher and Eric
Anderson. As a child and young adult he had a strong inclination toward
natural sciences. His parents encouraged such activities. In 1971 he
joined the Nation of Islam. In 1975 he graduated from Admiral King High
School and became a Muslim. That year he enrolled in the Biology
Department of Bradley University in Peoria, Illinois. He graduated with
a Bachelor of Science degree from Bradley University in 1979.
In 1979 he enrolled in the Department of Immunology and Medical
Microbiology, College of Medicine at the University of Florida,
Gainesville, Florida. In 1981 he began research in the laboratory of Dr
George E. Gifford. In July 1984 Keith Anderson legally changed his name
to Naeem Adib Abdullah. He joined the laboratory of Dr. Howard M.
Johnson in 1986 to work on the regulation of interferon gamma production
in the murine system. He received his Doctorate of Philosophy degree in
December 1988.
63

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Howard M. Johnson,
Graduate Research Professor of
Immunology and Medical Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Ac QC /
/L b",/ ..1
Donna H. Duckworth
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
LindseyHutt-Fletcher
Associate Professor of Pathology and
Laboratory Medicine
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Arthur K. Kimura
Associate Professor of Pathology and
Laboratory Medicine

This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December, 1988
Dean, College of Medicine
Dean, Graduate School



and tracking materials, reagents and scientific correspondences required
for my experiments as well as helping to set up actual experiments.
I wish to thank my parents, Marvin Moore Anderson, M.D., and
Dorothy-Jean Barbee Anderson and brothers Mansur Abdullah, Clarence H.
Anderson, Christopher A. Anderson, Eric Anderson, M.D., and his wife
Ellen, who gave me tremendous love, support and encouragement throughout
my graduate studies. A special thanks to Juanita Maria Maxwell whose
encouragement and companionship were appreciated. They will never know
how much their support meant to me.
I must thank my many other relatives, friends, colleagues, and
associates who are too numerous to mention but whose support and
encouragement were deeply appreciated and spurred me on to obtain the
Doctorate of Philosophy. Finally, thank you Jennifer (Maynard) for
preparing and retyping this manuscript through its numerous revisions and
impending deadlines.
iii


57
59. Cohen, S. The stimulation of epidermal proliferation by a specific
protein (EGF). Develop. Biol. 1_2:394, 1965.
60. Cohen, S. and Elliott, G.A. The stimulation of epidermal
kerafinization by a protein isolated from the submaxillary gland of
the mouse. J. Invest. Dermatol. 40:1, 1963.
61. Savage, Jr., C.R., Inagami, T. and Cohen, S. The primary structure
of epidermal growth factor. J. Biol. Chem. 247:7612, 1972.
62. Gregory, H. and Preston, B.M. The primary structure of human
urogastrone. Int. J. Peptide Protein Res. 9:107, 1977.
63. Gray, A., Dull, T.J. and Ullrich, A. Nucleotide sequence of
epidermal growth factor cDNA predicts a 128,000-molecular weight
protein precursor. Nature 303:722, 1983.
64. Scott, J., Urdea, M., Quiroga, M., Sanchez-Pescador, R., Fong, N.,
Selby, M. Rutter, W.J.., Bell, G.I. Structure of a mouse
submaxillary messenger RNA encoding epidermal growth factor and
seven related proteins. Science 221:236, 1983.
65. Bowen-Pope, D.F. and Ross, R. Is epidermal growth factor present
in human blood? Interference with the radioreceptor assay for
epidermal growth factor. Biochem. Biophys. Res. Comm. 114:1036,
1983.
66. Oka, Y. and Orth, D.N. Human plasma epidermal growth factor/p-
urogastrone is associated with blood platelets. J. Clin. Invest.
72:249, 1983.
67. Elder, J.B., Williams, G., Lacey, E. and Gregory, H. Cellular
Localisation of human urogastrone/epidermal growth factor. Nature
271:466, 1978.
68. Madtes, D.K., Raines, E.W., Sakariassen, K.S., Assoian, R.K.,
Sporn, M.B., Bell, G.I. and Ross, R. Induction of transforming
growth factor-a in activated human alveolar macrophages. Cell
53:285, 1988.
69. Derynck, R. Transforming growth factor a. Cell 54:593, 1988.
70. Tsutsumi, 0., Kurachi, H. and Oka, T. A physiological role of
epidermal growth factor in male reproductive function. Science
233:975, 1986.
71. Elson, S.D., Browne, C.A. and Thorburn, G.D. Identification of
epidermal growth factor-like activity in human male reproductive
tissues and fluids. J. Clin. Endocrin. Metabol. 58:589, 1984.


rpm, revolutions per minute
SDMEM, Dulbecco's minimal essential medium supplemented with 15 mM Hepes,
lx nonessential amino acids, 1 mM sodium pyruvate, 0.075% sodium
bicarbonate, penicillin, streptomycin, and 1 mM glutamine
SEA, staphylococcal enterotoxin A
sec, seconds
Strep, streptomycin 20 pg/ml final concentration
TGEa, transforming growth factor a
Tjj, T helper cell
Tg, T suppressor cell
VGF, vaccinia virus growth factor
vi


62
118. Heldin, C.-H., Backstrom, G. Ostman, A., Hammacher, A.,
Ronnstrand, L., Rubin, K., Nister, M., and Westermark, B. Binding
of different dimeric forms of PDGF to human fibroblasts: Evidence
for two separate receptor types. The EMBO J. 2:1!387, 1988.
119. Nister, M., Hammacher, A., Mellstrom, K., Siegbahn, A.,
Ronnstrand, L., Westermark, B., and Heldin, C.-H. A glioma-derived
PDGF A chain homodimer has different functional activities from a
PDGF AB heterodimer purified form human platelets. Cell 52:791,
1988.
120. Gronwald, R.G.K., Grant, F.J., Haldeman, B.A., Hart, C.E.,
O'Hara, P.J., Hagen, F.S., Ross, R., Bowen-Pope, D.F., and Murray,
M.J. Cloning and expression of a cDNA coding for the human
platelet-derived growth factor receptor: Evidence for more than
one receptor class. Proc. Natl. Acad. Sci. USA 85:3435, 1988.
121. Niebergs, A., Das, M., Bishayee, S,, and Cohen, S. Binding of
epidermal growth factor by lymphoid cells. Fed. Proc. Fed. Am.
Soc.. Exp. Biol. 42:410, Abstract #691, 1983.
122. Weller, A., Meek, J., and Adamson, E., D. Preparation and
properties of monoclonal and polyclonal antibodies to mouse
epidermal growth factor (EGE) receptors: Evidence for cryptic EGF
receptors in embryonal carcinoma cells. Development 100:351, 1987.


CHAPTER 1
INTRODUCTION
Interferon Gamma
The Interferon Gamma Molecule
In 1957 an antiviral substance was discovered by Isaacs and
Lindenraann (1). This substance, which they termed interferon (IFN),
plays an important role in host defense against viral infection. There
is a large body of evidence that IFNs, particularly IFNy, play a central
role in the regulation of a variety of immune functions.
The human and murine IFNs are classified into three broad groups
which are based on antigenic properties, analogous to the classification
of immunoglobulin isotypes (2). They are designated IFNa, IFNS, and
IFN Y. Induction of IFNa and IFNS in appropriate cell types is achieved
by the addition of virus or polyribonucleotides, while IFNy is induced in
T lymphocytes or natural killer (NK) cells by specific antigens or T cell
mftogens (Table 1-1) (3).
The genes for all of the known IFNs have been cloned. The cloning
data has revealed 23 IFNa genes, at least 15 of which code for full
length proteins (4). IFNa is apparently glycosylated. The IFNa genes,
none of which contain introns, are clustered near each other on the same
chromosome. IFNa induces rearrangement of the T cell antigen receptor a-
chain and matures T lymphocyte clones to cytotoxicity ini vitro (5,6).
There are at least 3 IFNS genes, one of which does not contain introns,
while the other two appear to contain 4 introns (7-12). The intron-
1


18
Research Inc., Bedford, MA. HPLC grade acetonitrile is obtained from
Fisher Scientific, Orlando, FL.
IFN-Y production
Spleen cells (1 ml at 1.25 x 10^ cell/ml), 0.1 ml of a 10-^ dilution
of monoclonal anti-Lyt 1.2 antibody and rabbit complement (0.1 ml of
serum) are mixed and incubated at 37C for 1.5 hr in RPMI 1640 containing
10% FBS. The helper cell-depleted cultures are then washed twice,
suspended in the RPMI 1640 media containing 10% FBS, and cultured in
duplicate at 1.25 x 10^ viable cells/ml. Whole spleen cell- and helper
cell-depleted cultures are stimulated with 0.5 pg/ml of SEA for 1 to 3
days in the presence of growth factors, and the supernatants are assayed
for IFNy activity on mouse L cells by using vesicular stomatitis virus as
described (110). The IFN produced is identified as IFNy by
neutralization with specific antisera.
Balb/c 3T3 Fibroblast Mitogenic Assay
The 3T3 growth factor mitogenic assay is performed using a
modification of Kobavashi et al. (111). The 3T3 fibroblasts are grown to
subconfluent monolayers in supplemented Dulbecco's minimal essential
medium (SDMEM) containing 10% FBS. Note, 3T3 fibroblasts are not used
beyond passage number 40. The cells are harvested, washed and seeded in
96 well plates (Falcon) at 5 x 10^ cells/well in 0.2 ml. All peripheral
wells receive 0.2 ml of medium only to keep the humidity of the target
wells constant. After 24 hrs in 5% CO2, 37C the spent medium is
replaced with 0.1 ml of SDMEM containing either 10% FBS or 0.2% FBS with
or without dilutions of growth factors in duplicate. After 22 hrs at 5%
C0'2, 37C, 0.1 pCi of -%-thymidine in 10 ,ul is added to each well. The
samples are incubated another 2 hrs at 37C, 5% CO2- After washing twice


-sion of class II molecules on the surface of macrophages is modulated by
IFNY as shown by their induction upon the addition of recombinant IFNY
(43,51). Indeed there are other surface molecules which are also up
regulated by IFNY, including the IL-2 receptor and Fc receptors (48,52).
Another important function of IFNY is its activity as a macrophage
activating factor (MAF) which augments various functions of macrophages,
including increased secretion of proteinases, hydrolases, oxygen metabo
lites, and increased killing capacity for tumor or virally infected
cells. A lvmphokine MAF activity was identified when it was observed
that activated T lymphocytes or their soluble products could prime or
activate macrophages for increased tumor cell killing (53). After
activation, macrophages are triggered to kill tumor cells by the addition
of a small amount of lipopolysaccharide which acts as a second signal.
Studies using purified IFN Y, recombinant IFNY and IFN Yneutralizing
antibodies have shown that the MAF activity is predominantly, if not
exclusively, due to the IFNY molecule. This data does not preclude there
being other structurally distinct MAFs. For example, IFNct and IFN& are
also MAFs, though not as potent as IFNY (54). As mentioned previously, T
killer lymphocyte and natural killer (NK) cytotoxic activity against
tumor cells and virally infected cells is also enhanced by the addition
of IFNs, especially IFNY (3).
Finally, IFNYbas been shown to modulate the differentiation of
myeloid and B lymphocyte cells (50,55). In the latter case the
differentiation signal provided by recombinant IFNY, was shown to be
sufficient to induce the secretion of antibodies (50).


51
Table A-2
Effect of anti-EGF receptor antibodies on IFNy production by
Mitomycin C-treated spleen cells in the presence of EGF
EGF
5nM
Anti-EGF receptor
antibodies (pg/ml)
NRS
(Mg/ml)
IFNy3
(U/ml SD)
-
-
-
<3
-
-
35 7
t*
30
-
25 7
+
100
-
28 4
+
300
-
33 4
+
1,000
-
28 4
+
-
30
45 21
f
-
100
65 21
+
-
300
50 28
+
-
1,000
28 4
a£FNY titers are from Day 3 of culture. Untreated spleen cells
stimulated with SEA produced 85 21 units IFNY/ml on Day 3 of culture.
Normal rabbit serum (NRS) was used as a control.


55
34. Djeu, J.Y., Heinbaugh, J.A., Holden, H.T. and Herberman, R.B.
Augmentation of mouse natural killer cell activity by interferon
and interferon inducers. J. Immunol. 122:175, 1979.
35. Gidlund, M._, Orn, A., Wigzell, H., Senik, A. and Gresser, I.
Enhanced NK cell activity in mice injected with interferon and
interferon inducers. Nature 273:759, 1978.
36. Trinchieri, G., Santoli, D., Dee, R.R. and Knowles, B.B. Anti
viral activity induced by culturing lymphocytes with tumor-derived
or virus-transformed cells. J. Exp. Med. 147:1299, 1978.
37. Weigent, D.A., Stanton, G.J. and Johnson, H.M. Interleukin 2
enhances natural killer cell activity through induction of gamma
interferon. Infect. Immun. 41:992, 1983.
38. Herberman, R.R., Ortaldo, J.R. and Bonnard, G.D. Augmentation by
interferon of human natural and antibody-dependent cell-mediated
cytotoxicity. Nature 277:221, 1979.
39. Chirigos, M.A. Antitumor action of interferon: Animal systems.
Texas Repts. Biol. Med. 41:610, 1982.
40. Kleinschmidt, VJ.J. and Schultz, R.M. Similarities of murine gamma
interferon and the lymphokine that renders macrophages cytotoxic.
J. Interferon Res. 2:291, 1982.
41.Pace, J.L., Russell, S.W., Torres, B.A., Johnson, H.M. and Gray,
P.W. Recombinant mouse y interferon induces the priming step in
macrophage activation for tumor cell killing. J. Immunol.
130:2011, 1983.
42. Fellous, M., Kamoun, M., Gressor, I. and Bono, R. Enhanced
expression of HLA antigens and -microglobulin on interferon-
treated human lymphoid cells. Eur. J. Immunol. 9:446, 1979.
43. Steeg, P.S., Moore, R.N., Johnson, H.M. and Oppenheim, J.J.
-Regulation of murine macrophage la antigen expression by a
lymphokine with immune interferon activity. J. Exper. Med.
156:1780, 1982.
44.Vignaux, F. and Gresser, I. Differential effects of interferon on
the expression of H-2K, H-2D, and la antigens on mouse lymphocytes.
J. Immunol. 118:721, 1977.
45.VJallach, D., Fellous, M. and Revel, M. Preferential effect of y
interferon on the synthesis of HLA antigens and their mRNAs in
human cells. Nature 299:833, 1982.
46.Fridman, W.H., Gresser, I., Bandu, M.T., Aguet, M. and Neauport-
Sautes, C. Interferon enhances the expression of Fc receptors.
J. Immunol. 124:2436, 1980.


53
10. Hirano, T. Yasukawa, K., Harada, H., Taga, T. Watanabe, Y.,
Matsuda, T., Kashiwamura, S-I., Nakajima, K., Koyaraa, K., Iwamatsu,
A., Tsunasawa, S., Sakiyama, F., Matsui, H., Takahara, Y.,
Taniguchi, T. and Kishimoto, T. Complementary DNA for a novel
human interleukin (BSF-2) that induces B lymphocytes to produce
immunoglobulin. Nature 324:73, 1986.
11. May, L.T., Helfgott, D.C. and Sehgal, P.B. Anti-f3-interferon
antibodies inhibit the increased expression of HLA-B7 rnRNA in tumor
necrosis factor-treated human fibroblasts: Structural studies of
the $2 interferon involved. Proc. Natl. Acad. Sci. USA 83:8957,
1986.
12. Zilberstein, A., Ruggieri, R., Korn, J.H. and Revel, M. Structure
and expression of cDNA and genes for human interferon-beta-2, a
distinct species inducible by growth-stimulator cytokines. 'The
EMBO J. 5:2529, 1986.
13. Poupart, P., Vandenabeele, P., Cayphas, S., Van Snick, J.,
Haegeman, G., Kruys, V., Fiers, W. and Content, J. B cell growth
modulating and differentiating activity of recombinant human 26-kd
protein (BSF-2, HuIFN-fJ2. HPGF). The EMBO J. 6:1219, 1987.
14. Gray, P.W. and Goeddel, D.V. Structure of the human immune
interferon gene. Nature 298:859, 1982.
15. Devos, R., Cheroutre, H., Taya, Y"., Degrave, W. Van Heuverswyn, H.
and Fiers, W. Molecular cloning of human immune interferon cDNA
and its expression in eukaryotic cells. Nucleic Acids Res.
10:2487, 1982.
16. Gray, P.W. and Goeddel, D.V. Cloning and expression of murine
immune interferon cDNA. Proc. Natl Acad. Sci. USA 80:5842, 1983.
17.Cerretti, D.P., McKereghan, K., Larsen, A., Cosman, D., Gillis, S.
and Baker, P.E. Cloning, sequence, and expression of bovine
interferon-Y. J. Immunol. 136:4561, 1986.
18.Yip, Y.K., Barrowclough, B.S., Urban, C. and Vilcek, J. Molecular
weight of human gamma interferon is similar to that of other human
interferons. Science 215:411, 1982.
19. Langford, M.P., Georgiades, J.A., Stanton, G.J., Dianzani, F. and
Johnson, H.M. Large-scale production and physicochemical
characterization of human immune interferon. Infect. Immun. 26:36,
1979.
20. Torres, B.A., Farrar, W.L. and Johnson, H.M. Interleukin 2
regulates immune interferon (IFNY) production by normal and
suppressor cell cultures. J. Immunol. 128:2217, 1982.


50
Table A~1
Ability of anti-EGF receptor antibodies to block EGF-induced
thymidine incorporation by 3T3 fibroblasts3
Immunoglobulin Percent of control
protein (ug/ml)^ ^H-thymidine incorporation
Anti-EGF Normal
receptor antibody rabbit serum
0
100
100
30
64
98
LOO
64
126
300
45
221
1000
42
263
aBalb/c 3T3 fibroblasts were incubated in duplicate with either rabbit
anti-EGF receptor antibodies or control normal rabbit serum for 1 hr
prior to the addition of EGF (2 nM). After 24 h at 37C in the presence
of 5% CO'), %-thymidine was added for 2 hr. Cells were then harvested
and counted on a beta scintillation counter.
^Rabbit polyclonal anti-EGF receptor antibody was provided bv Dr. E.
Adamson (122). The antibody and normal rabbit serum were ammonium
sulfate precipitated, dialysed in PBS, aliquoted, and frozen.


31
Figure 3-5. Binding of 125I-EGF to spleen cells. Spleen cells were
incubated with 3.3 pM EGF for 15 min prior to a 1 hr incubation with
l25I-EGF at the indicated concentrations. Cells were then harvested and
counted on a gamma counter. Data are plotted as bound i2 I-EGF vs. free
125I-EGF.


40 i
28
I
I I
20 40
Figure 3-2. HPLC profile of recombinant human EGF.
EGF (Scott Labs) was radioiodinated and passed over
HPLC column. Fractions are eluted and counted in a
60
Recombinant human
a C^g reverse phase
gamma counter.


26
The above data raise questions about functional sites on the EGF,
TGFa, and VGF molecules that are involved in receptor interactions. It
has been suggested that the third disulfide loop of these growth factors
comprises the binding site for the EGF receptor on such cells as 3T3
fibroblasts (115). The data presented here raise questions about a
common binding site for these 3 growth factors with respect to
interactions with lymphocytes. In order to gain further insight into the
structural relationship of these molecules we generated composite surface
profile plots which are based on hydrophilicity, accessibility, and
flexibility of secondary structure (116,117). Mouse and human EGF showed
very similar composite surface profiles (Figure 3-8a and 3-8b). Their
profiles were different from those of TGFa and VGF (Figure 3-8c and 3-
8d). The composite surface profile plots suggest structural differences
between EGF and TGFa and VGF. A precise delineation of these differences
could possibly provide insight into EGF interaction with lymphocytes in
the absence of such an interaction by TGFa and VGF.
We feel that the data presented are quite interesting and allow
us to draw the following conclusions: (1) EGF provides a helper signal
for the induction of IFNy, (2) EGF provides its signal by interaction
with a novel receptor on lymphocytes based on functional and receptor
competition data with TGFa and VGF, (3) the novel EGF receptor on
lymphocytes is not expressed constitutively but is induced by a T cell
mitogen.
Related to our discovery of a novel EGF receptor on lymphocytes is a
recent finding of a novel PDGF receptor on human fibroblast and baby
hamster kidney cells (118-120).


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABBREVIATIONS v
ABSTRACT vii
CHAPTERS
1 INTRODUCTION 1
Interferon Gamma 1
The Interferon Gamma Molecule 1
Regulation of IFNy Production 2
Immunoregulatory Functions of IFN Y. 3
Epidermal Growth Factor. 5
The EGF Molecule 5
Functional Roles of EGF 6
EGF Related Peptides TGFa and VGF 7
The EGF Receptor 8
Specific Aims 11
2 MATERIALS AND METHODS 17
Mice 17
Reagents 17
IFN Y Production 18
Balb/c 3T3 Fibroblast Mitogenic Assay 18
Anti-EGF Antibody Functional Studies 19
Radioiodination of EGF 19
Balb/c 3T3 Fibroblast Growth Factor Binding Assays 20
Murine Spleen Lymphocyte Binding Assays 20
Reverse Phase High Performance Liquid Chromatography
of Radioiodinated EGF 21
3 RESULTS 22
A DISCUSSION 41
APPENDIX 46
REFERENCES 52
BIOGRAPHICAL SKETCH. 63
iv


49
1
2
Figure A-2. SDS-PAGE in 8% acrylamide/bis with L^^1-EGF cross-linked
onto 48 hr SEA stimulated splenic lymphocytes in the presence (lane 1)
and absence (lane 2) of 3.2 pM cold EGF. Samples are incubated for 15
min at 0C in the presence or absence of 3.2 pM final concentration cold
EGF prior to the addition of 5 nM ^*-^I-EGF. 'Ceils are washed without BSA
and cell-bound EGF is covalently cross-linked to the cellular receptor by
the addition of freshly prepared BS^. After 1 hr at 0C the BS^ was
removed and the spun sample pellets treated with SDS-PAGE sample buffer
and sonicated. Samples were centrifuged to remove debris and superna
tants were boiled prior to being electrophoresed. Samples were electro-
phoresed on an 8% separating gel as in the 3T3 EGF receptor SDS-PAGE
protocol. The gel was autoradiographed at -70C for 6 weeks with
intensifying screens prior to film development.


33
Figure 3~7. Competitive binding of ^-I-EGF to spleen lymphocytes in the
presence of growth factors. SEA-stimulated spleen lymphocytes were
harvested as previously described and incubated for 15 min with EGF (O)
TGFa (A), or buffer (CD). -EGF was then added at a final concen
tration of 5 nM. After 1 hr incubation at room temperature, cells were
harvested and counted on a gamma counter.


CHAPTER 3
RESULTS
It was previously shown that electrophoretically pure mouse sub
maxillary gland EGF (from Collaborative Research) could replace the IL-2
requirement for IEN Yproduction in the mouse (C57B1/6) spleen cell system
(23,24). To confirm the above, another source of mouse submaxillary
gland EGF (Toyobo) that was purified to homogeneity was tested for its
ability to replace the IL-2 requirement, and was observed to provide
essentially maximal help for IFNy production at 1 nM (Table 3-1).
Purified recombinant human EGF (Scott Labs) also provided the helper
signal at 1 nM as shown in Table 3-1. Thus, both EGF preparations have
equivalent potency for inducing IFNy. Moreover, specific anti-murine EGF
antibodies (Collaborative Research) could completely block EGF-induced
IFNy production while this same antibody preparation had no effect on the
recombinant c-sis PDGF (AMGen) helper signal for IFNY production (Table
3-2). Purified murine submaxillary gland EGF and recombinant human EGF
were radioiodinated and passed over a reverse phase high performance
liquid chromatography column in order to confirm their indicated purity.
In both cases these labeled EGFs eluted as a single peak as indicated in
Figures 3-1 and 3-2. This provides further evidence that the helper
signal provided by EGF for IFNy production is due to the EGF molecule and
not to a contaminant in these preparations.
22


56
47. Itoh, K., Inoue, M., Kataoka, S. and Kumagai, K. Differential
effect of interferon expression of IgG- and IgM-Fcy receptors on
human lymphocytes. J. Immunol. 124:2589, 1980.
48. Vogel, S.N., Weedon, L.L., Moore, R.N. and Rosenstreich, D.L.
Correction of defective macrophage differentiation in C3H/HeJ mice
by an interferon-like molecule. J. Immunol. 128:380, 1982.
49. Johnson, H.M. and Farrar, W.L. The role of a gamma interferon-like
iymphokine in the activation of T cells for expression of
interleukin 2 receptors. Cell. Immunol. 7_5:154, 1983.
50. Sidman, C.L., Marshall, J.D., Shultz, L.D., Gray P.W. and Johnson,
H.M. Y -interferon is one of several direct B cell-maturing
lymphokines. Nature. 309: 801, 1984.
51. Sztein, M., Steeg, P.S., Johnson, H.M. and Oppenheim, J.J.
Regulation of human peripheral blood monocyte DR antigen expression
m vitro by lymphokines and recombinant interferons. J. Clin.
Invest. 7_3:556, 1984.
52. Johnson, H.M. and Farrar, W.L. The role of a gamma interferon-like
Iymphokine in the activation of T cells for expression of
interleukin 2 receptors. Cell. Immunol. 75:154, 1983.
53. Pace, J.L., Russell, S.W., Schreiber, R.D., Altman, A. and Katz,
D.H. Macrophage activation: Priming activity from a T-cell
hybridoma is attributable to interferon-y. Proc. Natl. Acad. Sci.
USA 80:3782, 1983.
54. Pace, J.L., Russell, S.W., LeBlanc, P.A. and Murasko, D.M.
Comparative effects of various classes of mouse interferons on
macrophage activation for tumor cell killing. J. Immunol. 134:977,
1985.
55. Perussia, B., Dayton, E.T., Fanning, V., Thiagarajan, P., Hoxie, J.
and Trinchieri, G. Immune interferon and leukocyte-conditioned
medium induce normal and leukemic myeloid cells to differentiate
along the monocytic pathway. J. Exp. Med. 158:2058, 1983.
56. Carpenter, G. and Cohen, S. Epidermal growth factor. Ann. Rev.
Biochem. 48:193, 1979.
57. Carpenter, G. Receptors for epidermal growth factor and other
polypeptide mitogens. Ann Rev. Biochem. 56:881, 1987.
58. Cohen, S. Isolation of a mouse submaxillary gland protein
accelerating incisor eruption and eyelid opening in the new-born
animal. J. Biol. Chem. 237:1555, 1962.


7
ation signal for spermatogenesis in mammals. The third point, addressing
EGF's ability to inhibit gastric acid secretion, suggests that EGF can
inhibit gastric acid secretion without stimulating cell growth. Thus,
EGF may exert its effects in the alimentary canal not just as a growth
factor but also as a regulator of gastric acid secretion.
Finally, EGF has recently been shown to modulate the production of
the immunoregulatorv molecule IFNY. The observation is unique because
nonhematopoietic growth factors were heretofore thought not to play a
role in the immune system. Also, the literature suggests that hemato
poietic cells do not constituively express EGF receptors (57). Thus,
the observation implies that under certain conditions hematopoietic T
cells do express EGF receptors which, when bound by the EGF ligand,
initiate the production of an important biological response modifier,
IFNY.
EGF Related Peptides Transforming Growth Factor a and Vaccinia Virus
Growth Factor
Transforming growth factor a (TGFa) and vaccinia virus growth factor
(VGF) are structural and functional relatives of EGF. Both molecules, or
their relevant functional sequences, have (1) approximately 30% homology
with the EGF molecule (74-76); (2) have the same number of cysteines and
disulfide linkage alignment (77); (3) compete with EGF for its receptor
and use the EGF receptor solely for their known functions (69,75,76); and
(4) are mitogenic for the same tissues as EGF (57). The structural and
functional similarities imply that the three growth factors evolved from
a common ancestral gene (78,79).
TGFa was initially found in the supernatants of transformed cells
and was thought to be related to oncogenesis (80). But it is now known


CHAPTER 4
DISCUSSION
EGF is a molecule with a wide variety of biological functions.
These include: (l) the proliferation of ectodermal and mesodermal cells
(56) and thus wound healing (73); (2) regulation of spermatogenesis
(70,71); (3) inhibition of gastric acid secretion (72); and (4)
regulation of IFNy production (22,23). The first three functions are
believed to be mediated through the well characterized EGF receptor
(57,100).
The EGF receptor is constitutively found on almost every tissue
studied except for the hematopoietic cell types (57). It has been cloned
and sequenced (87). It is a 170,000 dalton glycoprotein which has been
extensively characterized in its structure and its functions. The EGF
receptor has three major domains: (1) an extracellular domain which is
responsible for the signal transduction after ligand binding; (2) a
transmembrane region; and (3) the intracellular region which contains the
tyrosine kinase domain responsible for the signal transduction after
ligand binding onto the receptor (57,100). Signal transduction is
mediated through autophosphoralation of the EGF receptor at tyrosine
1173.
The EGF-related peptides TGFa and VGF are structurally related to
EGF and compete with this ligand for its receptor (57,74-76). They also
share known biological functions that are mediated through the EGF
41


CHAPTER 2
MATERIALS AND METHODS
Mice
C57B1/6 female mice, 8 to 12 week old, are obtained from the Jackson
Laboratory, Bar Harbor, ME.
Reagents
The T cell mitogen SEA is purchased from Toxin Technology, Madison,
WI. Ultrapure mouse submaxillary gland EGF is obtained from Toyobo Co.,
Ltd., New York, NY. Recombinant human EGF is obtained from Scott
Laboratories, Fiskeville, RI. Highly purified synthetic rat TGFa is
obtained from Peninsula Laboratories, Inc., Belmont, CA. Recombinant VGF
is a gift from Dr. Gregory Bruce of Oncogen, Inc., Seattle, WA.
Recombinant c-sis PDGF (PDGF B) is from AMGen Biologicals, Thousand Oaks,
CA. -'-^-Iodide as a sodium salt (15 mCi/pg) and tritiated thymidine (21
mCi./mg) are obtained from Amersham Corp., Arlington Heights, IL.
Monoclonal anti-Lyt 1.2 antibody and anti-Lyt 2.2 antibody are obtained
from New England Nuclear, Boston, MA. The source of complement is serum
from New Zealand white rabbits. Endotoxin-free fetal bovine serum (FBS)
is obtained from HyClone Laboratories, Logan, UT. Trifluoroacetic acid
and radioimmunoassay grade bovine serum albumin (BSA) are obtained from
Sigma, St. Louis, MO. Dibutyl phthalate (gold label) and dioctyl
phthalate are obtained from Aldrich Chemical Co., Milwaukee, WI. Rabbit
anti-mouse EGF antibody (IgG fraction) is obtained from Collaborative
17


13
Figure 1-1. IFNY production is regulated by the dynamic interaction
between helper (T^j), suppressor (T3), and IFNy producer cells. Helper
cells provide IL-2. Suppressor ceils absorb IL-2. Arrows: ( ),
positive signal; ( ), negative signal (3).


6
The EGF gene encodes a large precursor molecule containing EGF and 7
related peptides (63,64). The mature EGF molecule is contained in the
carboxyl terminal region of the precursor molecule and is obtained by
processing of the polyprotein. The functions of the other EGF-related
peptides in the polyprotein are currently unknown.
As indicated above, the submaxillary gland is a major source of EGF
in the mouse, whereas platelets are a major source of EGF in humans
(65,66). The submandibular and Brunners glands are also thought to be
sources of EGF in humans (67). Studies are in progress to further
determine the roles of various hematopoietic cells as sources of EGF and
related growth factors (68,69).
Functional Roles of EGF
Purified EGF has a wide range of biological effects. These include
(1) augmentation of ectodermal and mesodermal cell (e.g. fibroblast)
proliferation (56); (2) regulation of spermatogenesis (70,71); (3)
inhibition of gastric acid secretion (72); and (4) regulation of immune
functions such as IFNY induction (22,23).
With regard to the first point, the proliferative effect of EGF on
ectodermal and mesodermal cells types in vitro suggests that it may play
a role in wound healing. In controlled experiments EGF does indeed aid
in the wound healing process (73). With regard to the second point, EGF
apparently plays a major role in spermatogenesis. Male mice which have
been treated so as to not produce EGF (by submaxillary gland removal)
have low sperm and spermatid counts, while the addition of exogenous EGF
to such mice reverses this condition. Humans also have an EGF-like
molecule in their seminal plasma. Thus, EGF is probably a differenti-


15
Table 1-1
Classification of I.FN's
Former
Interferon Cellular Source
Inducer
Nomenclature
IFN-cx
Lymphocytes (B, null,
Viruses, polyribonucleo-
Leukocyte,
and T), macrophage?
tides, tumors, chemicals
Type I
IFN-3
Fibroblast, epithelial,
Viruses, polyribonucleo-
Fibroblast,
macrophage
tides, chemicals
Type 1
IFN-Y
T lymphocyte
Antigens, T cell mitogens
Immune,
Type II
Reprinted from Lymphokines 1J^:33, 1985. Copyright (c) 1985 by Academic
Press, Inc. (3)


43
that purified mouse submaxillary gland EGF (Toyobo) and recombinant human
EGF (Scott Labs) were both capable of inducing the production of IFNy at
1 nM. Thus, unlike the relatively high EGF concentrations required to
induce IFNY in the previous study, the IFNY inducing concentrations of
these EGF preparations approximated those concentrations required to
detect mitogenic effects of EGF on fibroblasts. The initial observation
that EGF can provide the helper signal for IFNY production, then,
apparently involved the use of EGF that was not fully active. The EGF
preparations that we used in these studies were radioiodinated, eluted
from a reverse phase column and only one peak was found in each sample,
indicating only one major species in each sample. These data excluded
the possibility of there being a contaminant in these preparations that
was responsible for the induction of IFNy* Thus, the data presented here
show EGF to provide the help for IFNy production at concentrations
similar to those for its other biological effects. Also anti-EGF
antibodies inhibited EGF induction of IFNY in a specific manner.
The above data demonstrate that EGF can regulate lymphokine
production. As indicated earlier, lymphocytes do not constitutively
express an EGF receptor. Although EGF receptors are generally thought
not to be expressed on hematopoietic cells there is one preliminary
report of induction of an EGF receptor on B lymphoblastoid cells (121).
The B lymphoblastoid lines RPMI 1788 and RPMI 8392 were both shown to
bind -*-25i_egf after treatment with the B cell mitogen staphylococcal
B cell mitogen (STM). RPMI 1788 cells expressed between 500 to 1,000 EGF
receptors while RPMI 8392 expressed between 2,000 to 4,000 EGF receptors.
Normal lymphocytes from blood or spleen could also be induced to express


3
adsorption or sequestration of IL-2. The IL-2 helper signal for induc
cin of IFN Yis dissociated from its ability to stimulate T-cell pro
liferation (3).
The question arose as to whether nonhematopoietic growth factors
were also able to stimulate IFNY production. Epidermal growth factor
(EGF), platelet-derived growth factor (PDGF), and fibroblast growth
factor (FGF), were shown not to stimulate lymphocyte proliferation
(23,24). It was demonstrated that these growth factors induce the
production of IFNY in conjunction with a T-cell mitogen signal (23,24).
By comparison, neither rat growth hormone nor human growth hormone are
capable of inducing IFNY production.
Immunoregulatorv Functions of IFNY
Interest in the immunoregulatory functions of IFNs began with the
observation that murine IFNa and IFN(3 suppressed antibody production m
in vitro assays (25,26). Concurrent studies showed that IFN could also
regulate cellular immunity and delayed type hypersensivity (27,28). Once
it was established that IFN enhanced natural killer (NK) cytotoxic
activity, the immunology community began a more rigorous study of the
immunomodulatory effects of the IFNs. It became apparent that the scope
of the immunoregulatory effects of the IFNs went far beyond the initial
observations, as shown in Table 1-2 (25-50).
Some of the immunoregulatory properties of IFNY are described herein.
Class I MHC molecules are up-regulated upon the addition of IFNY (44,45).
This surface antigen is important for the efficient killing of virally
infected cells. Class II MHC antigens are also up-regulated by IFNY
(43). The class II molecules are required for the cellular interactions
important for immune function such as antigen presentation. The expres-


iriiy (ii/iiin
29
GROWTH FAC i OR (fir!)
Figure 3-3. Ability of growth factors to restore competence for IFN y
production by Lyt L-, 2"*" spleen cells. EGF iO)< TGFa (A), and VGF (Q)
were incubated with Lyt 1-depleted spleen cells in the presence of the T-
cell mitogen SEA. IFNYtiters are from day 3 of culture. Reprinted from
Progress in Leukocyte Biology 8:159, 1988. Copyright (c) by Alan R.
Liss, Inc. (113).


30
0-
!
0.001
1 i
0,01 0,1
GROWTH FACTOR (nil)
~ I
10 30
Figure 3-4. Relative mitogenic activity of growth factors on Balb/c 3T3
cells. Cells were incubated for 24 hr in SDMEM containing 10% FBS,
washed extensively, and growth factors were added at the indicated
concentrations. After 24 hr at 37C in the presence of 5% CC>2, JH-
thymidine was added for 2 hr. Cells were then harvested and counted on a
beta scintillation counter. Symbols: EGF (A); TGFa (Q)? VGF ().


This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December, 1988
Dean, College of Medicine
Dean, Graduate School


25
EGF, TGFa, and VGF compete similarly for the EGF receptor on 3T3
cells (69,75,76). We confirmed this in competitive binding experiments
with -'-^-I-EGF and the EGF, TGFa, and VGF that were used in the functional
studies above (Figure 3-6). PDGF does not bind to the EGF receptor, but
does slightly down regulate the expression of high affinity receptors
(97-99). Thus, ^^^I-EGF binding to 3T3 cells was reduced by PDGF
treatment, but not nearly as much as by the ligands that compete for the
receptor. Thus, the data in Figure 3-6 are consistent with and confirm
previous observations on the properties of the EGF receptor on 3T3
fibroblasts.
As indicated, the functional data presented suggest that EGF exerts
its effects on lymphocytes by binding to a novel receptor, since TGFa and
VGF could not provide the helper signal for IFNy production, and could
not functionally block the help of EGF. The binding competition data of
Figure 3-7, where cold submaxillary gland EGF blocked ^^I-EGF binding to
lymphocytes, but TGFa at the same concentrations was without effect is
further evidence that the EGF receptor on lymphocytes is novel. We
currently do not know why relatively high concentrations of cold EGF are
required in receptor competition experiments with lymphocytes, but the
relatively low specific binding to total binding may play a role. Also,
it is possible that the binding studies using submaxillary gland -^JI-EGF
could involve a contaminant in the EGF preparation, but this is unlikely
since purified recombinant human EGF inhibited submaxillary gland iiJI-
EGF binding as effectively as did cold submaxillary gland EGF
(Table 3-6). Similar competitive binding patterns have been observed in
other systems such as the adrenergic receptors (114).


ACKNOWLEDGEMENTS
Allah, God the Creator, has asked people to study creation and to
find and use its hidden treasures to help mankind. This dissertation is
dedicated to that end. The last revelation of Allah, the Holy Qur'an,
is unparalleled in its prophetic scientific description of natural
phenomena. Allah is the greatest (A1 Kabir), the most wise (A1 Hakim),
and the most kind (A1 Latif).
I give a special thanks to Howard M. Johnson, Ph.D., as my mentor
and guide through the "real world of scientific research. His piercing
insights have helped me enormously in maturing as a scientist. I thank
the supervisory committee members Donna H. Duckworth, Ph.D., Lindsey M.
Hutt-Fletcher, Ph.D., and Arthur K. Kimura, Ph.D., for their many
suggestions and scientific insights in the preparation of this manu
script. Another titan that helped me at the bench, in writing and
through discussions was Barbara A. Torres, M.S. She took precious time
away from running the lab to instruct me on many new techniques and the
nuances and quirks of each of them. The two postdocotral scientists
Carol Hanlon-Pontzer, Ph.D., and Jeffry K. Russell, Ph.D., and the
graduate students Myron 0. Downs, D.V.M., Michael A. Jarpe, B.S., and
Harold I. Magazine, B.S., were great peers and counsel to interact with
in many areas of research and allowed me to look at a given problem from
different prospectives. Miss Sarah A. Hunt was most helpful in ordering
ii


procedure using radiolabeled EGF was carried out with 48 hr 5EA-
stimulated lymphocytes. The detected bands migrated at 130,000 daltons,
100,000 daltons, and 50,000 daltons upon autoradiographic exposure
(Figure A-2). The 100,000 dalton and 50,000 dalton bands could represent
degradation products of the 130,000 dalton band or the 3 bands could
represent a complex EGF receptor on lymphocytes. Further studies need to
be conducted to better delineate the nature of the induced lymphocyte EGF
receptor.


ABBREVIATIONS
BSA, bovine serum albumin, radioimmunoassay grade from Sigma
CPM (cpm), counts per minute
EBSS, Earle's balanced salt solution
EGF, epidermal growth factor
FBS, fetal bovine serum
FGF, fibroblast growth factor
Hepes, N-2-Hydroxyethyl piperazine-N'-2-ethanesulfonic acid
HMEM, minimal essential medium modified with Earle's salts, penicillin,
streptomycin and 1 mM glutamine
HPLC, high performance liquid chromatography
hr, hours
IFN, interferon
M, molar
MAF, macrophage activation factor
pM, micromolar
mM, millimolar
nM, nanamolar
min, minutes
NaN^, sodium azide
PBS, phosphate buffered saline
PDGF, platelet-derived growth factor
Pen, penicillin 100 U/ml final concentration
v


100
Figure 3-3. Surface profile of EGF-related proteins. Composite surface profiles of mouse EGF (A),
human EGF (B), rat TGFot (C), and VGF (D) were developed as described by using a computer program
i'118) This program takes into account HPLC mobility, accessibility, and segmental mo i lty l
values) of amino acids in model proteins and peptides. Residues with high composite values are most
likely to reside on the surface of a protein molecule. The abscissa equals the surface value. e
ordinate equals the residue number.


27
Figure 3-1. HPLC profile of murine EGF. Murine submaxillarv gland EGF
(Toyobo) was radioiodinated and passed over a reverse phase HPLC
column. Fractions are eluted and counted in a gamma counter.


REGULATION OE MURINE INTERFERON GAMMA PRODUCTION
BY EPIDERMAL GROWTH FACTOR
By
Na'eem Adib Abdullah
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988


23
As indicated, the growth factors TGFa and VGF are related to EGF
(Figure 1-2). They are in the family of EGF-like peptides as determined
by structural similarity, the placement of cysteine residues within the
molecules, and, most importantly, they exert their mitogenic effects on
fibroblasts via the EGF receptor (57,74-77). Thus, synthetic rat TGFa
(Peninsula Labs) and recombinant VGF (Oncogen) were examined for their
ability to provide the helper signal for IFNYproduction. Unlike EGF,
neither TGFa nor VGF could restore competence for IFNy production by
mouse C57B1/6 spleen cell cultures that were depleted of helper cell
function (Figure 3-3) (113). Both growth factors were as competent as
EGF in stimulating proliferation of 3T3 fibroblasts (Figure 3-4), so
their inability to provide the helper signal for IFNy production was not
due to factors such as lack of biological activity. Rather, it is
possible that the EGF receptor on lymphocytes is different from that on
3T3 cells and thus novel, or that TGFa and VGF can bind to the EGF
receptor on lymphocytes, but cannot trigger the signal for IFNy
induction. If the former were true, then TGFa and VGF should not
functionally compete with EGF for the receptor and thus block its helper
signal, whereas if the latter were true, then they should block the EGF
helper signal for IFNy production. As shown in Table 3-3, neither TGFa
nor VGF in molar excess blocked the helper signal of EGF for production
of IFNy by lymphocytes (113). Additionally, neither growth factor
blocked IFNy production by spleen cells that were not depleted of helper
cell function (Table 3-4). Thus, the functional data suggest that EGF
provides its helper signal for IFNy production by interaction with a
novel receptor on lymphocytes.


36
Table 3-2
Ability of anti-EGF antibodies to block EGF
helper signal for IFNy production
by Lyt 2+ spleen cells
Anti-EGF Antibodies3
(Mg/ml)
IFN y(U/ml + S.D. )
EGF 5nM PDGF 5nM
0
120 28
105 21
0.5
80 14
220 28
5
<3
85 21
50
<3
135 21
aSampies were from day 3 of culture. Whole spleen cells had 80 28
IFNYunits. Anti-Lyt 1.2 + C control cells had <3 IFNyunits.


11
which subsequently converts it to the oncogenic form called neu
(105,106). The neu oncogene product of c-erb B-2 differs from its wild
type counterpart by this single point mutation which drastically alters
the function of the gene product. Another related oncogene is the v-erb
B gene product from the avian erythroblastosis virus (107). It is 68,000
daltons and has approximately 95% homology to the EGF receptor within a
stretch of 400 cytoplasmic domain residues (57). The amino terminal of
this molecule is truncated and thus does not bind EGF and there is also a
small truncation at the carboxyl terminus. The v-erb B oncogene has
autophosphorylation activity (108,109).
Thus, the interaction of EGF with its receptor initiates a complex
series of events which are responsible for EGE induced physiological cell
functions.
Specific Aims
The objective of the work outlined in this dissertation is to
characterize the EGF helper signal for induction of IFNy in a C57B1/6
murine splenic T cell system. We will focus on the functional and
receptor binding properties of EGF on mouse splenic lymphocytes as
compared to the interactions of EGF with its classic receptor on murine
3T3 fibroblast cells. We propose to achieve the objective through the
following specific aims: (1) definitively determine if EGF can provide
the helper signal for IFNy induction by using both EGF purified to
homogeneity and recombinant EGF in IFNy production studies; (2) determine
if the EGF related growth factors TGFa and VGF have the ability to
provide the helper signal for IFNy production and/or modulate the EGF
helper signal through competition for the receptor. Such studies should


24
Hematopoietic cells have been reported to lack EGF receptors (57),
thus EGF receptor binding studies were performed with untreated splenic
lymphocytes cultured for 48 hr and splenic lymphocytes that were stimu
lated for 48 hr with the T-cell mitogen SEA. Exogenous SEA is required
for several aspects of IFNY production (3). First, SEA acts as a T cell
mitogen by inducing IL-2 production by Ty- cells (3). Second, SEA is a
required second signal, along with IL-2, to initiate T cell production of
IFNY.
As shown in Table 3-5, specific binding of -"^I-EGF (125 pCi/ug
protein) was observed only in cultures that were stimulated with SEA for
48 hr (113). Specific binding was generally 25 to 50 percent of total
binding. The data indicate that the EGF receptor is induced on
lymphocytes and not constitutively expressed as in 3T3 fibroblasts.
Similar data have been obtained in at least 3 separate experiments. A
saturation binding curve for ^-I-EGF on splenic lymphocytes is
presented in Figure 3-5, and indicates a of 5 to 10 nM. Since the
cultures were depleted of adherent cells, it is unlikely that the binding
observed was due to macrophages, but it cannot be ruled out that small
amounts of contaminating macrophages may play some role in EGF binding
to lymphocytes. The question has arisen that the novel EGF receptor
described above may actually be the IL-2 receptor on lymphocytes, since
the high affinity IL-2 receptor is also induced by T-cell mitogens. It
has previously been shown, however, that EGF did not compete with IL-2
for the IL-2 receptor on lymphocytes (86). Therefore, the novel EGF
receptor on lymphocytes is different from the IL-2 receptor on
lymphocytes.


-EOF (CPM)
32
:,800
2,400-

o-
i
0
ih
[
0.1
I I
1 10
GROWTH FACTOR CnM)
Figure 3-6. Competitive binding of ^^I-EGF to Baib/c 3T3 cells in the
presence of growth factors. Confluent monolayers were incubated for 15
min with cold EGF (.), TGFa {Q), VGF (A), PDGF B (Q), or buffer (A).
125i-EGF was then added at a final concentration of 5 nM. After 1 hr
incubation at room temperature, cells were harvested and counted on a
gamma counter.


14
4 O
VGF
.**>
. 1
! J
: p
a :
TG Fa
V
v s
H F
m t G F
N
5 Y
hEGF
N
S 0
50
R L
C
G P E G 0 G
Y C
N K
C
P 0 S H T Q
Y C
P G
C
P S S Y 0 G
Y C
S E
£.
P L 5 H 0 G
Y C
6 0
H -
G
T
c
[HARO
K -
G
7
c
R F L V Q
N G
G
V
c
M H I E S
K 0
s|
V
c
M Y I E A
70
80
VGF I D G Y
TGFo E E K P
mEGF LOS Y
hEGF L D K _Y.
Q T R D
R C Q Y R D
90
V D Y Q R
L A
R W W E L R
l|k W W E L R
Figure 1-2. Alignment of the VGF, rTGFa (rat), mEGF (mouse), and hEGF
(human). The sequences of the mature peptide growth factors are shown in
their entirety, numbered from the N-terminus of the precursor VGF.
Residues conserved in all four sequences are boxed. The VGF sequence
represents the recombinant subcloned VGF used in the present studies and
was provided by Oncogen Inc., Seattle, WA. Adapted from Brown et al.
Nature 313:491 (1985) Reprinted from Nature, Vol. 313, No. 6002, pp.
491-492. Copyright (c) 1985 by Macmillan Journals Limited (77).


2
containing IFN 3 genes are called IFNf$2 (7). IFNfio has a number of
interesting properties in addition to its antiviral properties (8-13).
While IFNP2 was initially identified as an antiviral protein isolated
from fibroblasts, others have found that IFNf^ from T cells acted as a B-
cell differentiation factor (BSF-2) that can enhance antibody secretion
and induce B-cell proliferation (8,10). IFNB2 is also called inter
leukin-6 (IL-6) (13).
IFN y has been cloned from the human (14,15), mouse (16), and bovine
(17) genomes. Only one gene has been found in all 3 species and it
contains 3 introns. The precursor polypeptide is processed and glyco
sylated. Based on the cDNA structure of the human and mouse genes the
mature IFNY molecule is composed of 133 amino acids and has a mass of
1.7,000 daltons (14,16). The molecular weight of natural IFNy varies from
20,000 to 25,000 daltons by SDS-PAGE due to glycosylation of the mature
molecule (18). Gel filtration studies have assigned IFN Ya mass of
40,000 to 50,000 daltons suggesting a natural multimer formed by the
mature molecule (19).
Regulation of IFNY Production
The cellular requirements for IFNY production have been studied in
detail in both the human and mouse systems (20-22). IFNY production in
the C57BL/6 mouse spleen cell system is regulated by a dynamic inter
action between helper cells (Lyt l+,2-), suppressor cells (Lyt l+,2+),
and IFNy-producing cells (Lyt 1",2+) (Figure 1-1) (3). Helper cells aid
IFN Y production via production of interleukin-2 (IL-2), which acts in
concert with a T-cell mitogen, staphylococcal enterotoxin A (SEA), to
induce IFNY- Suppressor cells inhibit or block IFN Y production by


42
receptor. These include mitogenic effects on fibroblasts (57,69,76) and
wound healing (81). VGF can also augment the production of viral progeny
from infected cells (84). As indicated in the Results EGF, TGFa, and VGF
molecules are structurally and functionally related to each other,
suggesting evolution from a common gene (78,79). The third disulfide
loop, a region that is most common to all three ligands in sequence and
structure, is believed to be the ligand binding region which is
recognized by the classic EGF receptor (115). In the data presented here
we have confirmed that TGFa and VGF, like EGF, can stimulate the
proliferation of 3T3 fibroblasts as well as compete for binding to the
EGF receptor on these cells using ^JI-labeled EGF. The question arises
as to whether purified and recombinant EGF from other sources and whether
TGFa and VGF can similarly provide the helper signal for induction of
IFN y .
In the course of studies on the regulation of murine IFNY production
by various growth factors it was found that EGF was capable of inducing
IFNY production in spleen cell cultures depleted of Tg cells (23,24).
The mouse submaxillary gland EGF preparation used in these studies (from
Collaborative Research) was electrophoretically pure. This preparation
was found, however, to restore the production of IFNYat 17 nM. This
concentration is higher than that required for the proliferative
functions of EGF for fibroblasts. Thus, the question was raised that a
possible contaminant of the EGF preparation was responsible for the
helper signal for IFNY production. To address this question and to
further extend our knowledge of the effects of EGF in the production of
IFNY, we obtained EGF from several other sources. We have shown here


8
to be a growth factor encoded in the genome of several species. The TGFa
gene encodes a messenger RNA (mRNA) that synthesizes a precursor poly
peptide, but unlike EGF there is no evidence of TGFa related peptides in
the precursor molecule. The mature TGFa molecule is a 6,000 dalton
protein of 50 amino acids (7A,75) and like EGF it is not glycosylated.
Biological properties that TGFa has demonstrated are mitogenic effects on
fibroblasts (57,69), and wound healing by inducing keratinocyte prolifer
ation and migration (81). TGFa is synthesized by both macrophages and
keratinocytes and thus may contribute directly to wound healing in vivo
as both cells are found in such lesions (68,82,83).
VGF, like EGF and TGFa, is synthesized as a precursor polypeptide.
The mature VGF molecule has 77 amino acids, is glycosylated, and has a
molecular mass of 23,000 daltons as determined by SDS polyacrylamide gel
electrophoresis (76). The structural relationship of EGF, TGFa, and VGF
is shown in Figure 1-2.
VGF has several EGF-like and other biological activities which
include: (1) mitogenic activity for fibroblast cells in vitro (57); (2)
initiation of mitogenesis and migratory activities for keratinocytes to
aid in wound healing (81); (3) augmentation of production of viral
progeny from infected cells (84). This latter property suggests that VGF
aids the lifecycle of the vaccinia virus through its cellular prolifera
tive properties.
Again, it should be emphasized that all of the known effects of TGFa
and VGF are mediated through the EGF receptor.
The EGF Receptor
The EGF receptor, also called c-erb B-l (57,85), has been found on
almost all tissues but has not been shown to be constitutiveiy produced


38
Table 3-4
Failure of TGFa and VGF to block IFN Y production
in spleen cells
SEA-stimulated cultures3
IFN Y (U/ml SD)
TGFa
275 35
VGF
350 71
Control
275 106
aSamples are from day 3 of culture. Concentration of
factors: TGFa, 14 nM; VGF, 17 nM.


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interferon, -J?roc. R. Soc. London Ser. B, 147 :258, 1957.
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Vilcek, J.T., Youngner, J.S. and Zoon, K.C. Interferon
nomenclature, J. Immunol. 125, 2353, 1980.
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6. Chen, L.K., Tourvieille, B., Burns, G.F., Bach, F.H., Mathieu-
Mahul, D.. Sasportes, M. and Bensussan, A. Interferon: A
cytotoxic T lymphocyte differentiation signal. Eur. J. Immunol.
_16:767, 1986.
7. Dervnck, R., Content, J., DeClercq, E., Volckaert, G., Tavernier,
J., Devos, R. and Fiers, W. Isolation and structure of a human
fibroblast interferon gene. Nature 285:542, 1980.
8. Sebgal, P.B., May, L.T., Tamm, I., and Vilcek, J. Human
interferon and B-cell differentiation factor BSF-2 are identical.
Science 235:731, 1987.
9.Yasukawa, K., Hirano, T., Watanabe, Y., Muratani, K., Matsuda, T.,
Nakai, S. and Kishimoto, T. Structure and expression of human B
cell stimulatory factor-2 (BSF-2/IL-6) gene. The EMBO J. 6:2939,
1987.
52


37
Table 3-3
Failure of TGFa and VGF to block helper signal for
FNY production by mouse spleen cells
SEA-stimulated cultures3
IFNY (U/ml SD)
Ly t 1
> 2+
cells
<7
Lyt 1"
, 2+
cells +
EGF
270
42
Lyt 1"
, 2+
cells +
EGF
+ TGFa
290
14
Lyt 1-
, 2+
cells +
EGF
+ VGF
295
7
Whole
spleen cells
215
21
aSamples are from day 3 of culture. Concentration of
factors: EGF, 5 nM; TGFa, 14 nm; VGF, 17 nM.
Reprinted from Progress in Leukocyte Biology 8:159, 1988.
Copyright (c) 1988 by Alan R. Liss, Inc. (113)


19
with phosphate buffered saline (PBS), cells are solublized by treatment
with 0.1 M NaOH (0.1 ml/well). The contents of the wells are then
harvested and counted on a LKB beta liquid scintillation counter
(Gaithersburg, MD).
Anti-EGF Antibody functional Studies
Rabbit anti-mouse EGF antibodies are used to block EGF functional
interactions with EGF receptors on Balb/c 3T3 cells and on C57B1/6 mouse
spleen cells. Mouse EGF and anti-EGF are mixed and incubated for 1 hr at
37C prior to addition to either 3T3 cells or spleen cells. 3T3
proliferation and IFNy production assays are carried out as previously
described. Recombinant human c-sis PDGF is also incubated with anti
mouse EGF as described above and is added to spleen cell cultures.
Radioiodination of EGF
EGF is labeled with ^-^1 using a modification of Das et al. (112).
Briefly, 2 Mg in 10 pi of EGF (200 pg/ml in 0.4 M KP04, pH 7.5) is added
to a 1.5 ml Eppendorf test tube. Five pi of Na ^--"Iodide (500 pCi) and
10 pi of freshly dissolved chloramine-T (6.25 mg/ml) are added. After 1
min the reaction is quenched by the addition of 10 pi of Na
metabisulfide (12.5 mg/ml) and 10 pi of KI (80 mg/ml). Then 10 pi of BSA
(Sigma) (20 mg/ml) is added and the sample is run over a 7 ml Sephadex G-
10 column (Pharmacia) equilibrated with 50 mM KP04 (pH 7.5) containing
0.15 M NaCl and 2 mg BSA/ml. Fractions are counted and those with the
highest counts are pooled. The specific activity of the pooled sample is
generally from 100-125 pCi/pg. ^^I-EGF is aliquoted and stored at -70C
for no more than 3 weeks prior to use.


expression of the EGF receptor, analogous to the induction of the high
affinity receptor on lymphocytes for interleukin-2. Neither TGFa nor VGF
were capable of restoring competence for IFNy production by Tjj cell
depleted spleen cell cultures. Furthermore, neither TGFa nor VGF could
block the ability of EGF to restore competence for IFN yproduction,
suggesting that the induced EGF receptor on lymphocytes is different from
that which is expressed on nonhematopoietic cells such as mouse 3T3
fibroblasts.
Consistent with the functional data for a novel inducible EGF
receptor on lymphocytes, the receptor was also detected by binding
studies with ^-I-labeled EGF. The receptor has a dissociation constant
of about 5 nanomolar. The receptor is optimally expressed after 48 hr
treatment of lymphocytes by T-cell mitogen. TGFa did not compete with
l^I-EGF for the lymphocyte EGF receptor. Both TGFa and VGF induced
proliferation of mouse 3T3 cells. Furthermore, consistent with previous
studies, both mitogens competed with EGF in receptor binding studies on
3T3 cells. Thus, the failure of TGFa and VGF to functionally mimic the
EGF help in IFNyproduction, and the failure of TGFa to compete for
receptor on lymphocytes, is compatible with the hypothesis that lympho
cytes express a novel inducible EGF receptor that differs from the
classic receptor expressed on cells such as 3T3 fibroblasts.
viii


54
21. Torras, B.A., Yamamoto, J.K. and Johnson, H.M. Cellular regulation
of gamma interferon production: Lyt phenotype of the suppressor
cell. Infect. Immun. 35:770, 1982.
22. Kasahara, T., Hooks, J.J., Dougherty, S.F. and Oppenheim, J.J.
Interleukin 2-mediated immune interferon (IFN-Y) production by
human T cells and T cell subsets. J. Immunol. 130:1784, 1983.
23. Johnson, H.M. and Torres, B.A. Peptide growth factors PDGF, EGF,
and FGF regulate interferon-Y production. J. Immunol. 134:2824,
1985.
24. Johnson, H.M. and Torres, B.A. Lymphokine-like and interferon
regulatory activity of platelet-derived growth factor, epidermal
growth factor, and fibroblast growth factor. Lymphokines 14:253,
1987.
25. Gisler, R.H., Lindahl, P. and Gresser, I. Effects of interferon on
antibody synthesis in vitro. J. Immunol. 113:438, 1974.
26. Johnson, H.M., Smith, B.G. and Baron, S. Inhibition of the primary
in vitro antibody response by interferon preparations. J. Immunol.
114:403, 1975.
27.Lindahl-Magnusson, P., Leary, P. and Gresser, I. Interferon
Inhibits DNA synthesis induced in mouse lymphocyte suspensions by
phytohaemagglutinin or by allogeneic cells. Nature New Biol.
237:120, 1972.
28.De Maeyer, E., De Maeyer-Guignard, J. and Vandeputte, M.
Inhibition by interferon of delayed-type hypersensitivity in the
mouse. Proc. Natl. Acad. Sci. USA 72:1753. 1975.
29. Braun, W. and Levy, H.B. Interferon preparations as modifiers of
immune responses. Proc. Soc Exp. Bio. Med. 141:769, 1972.
30. Brodeur, B.R. and Merigan, T.C. Suppressive effect of interferon
on the humoral immune response to sheep red blood cells in mice.
J. Immunol. 113:1319, 1974.
31. Chester, T.J., Paucker, K. and Merigan, T.C. Suppression of mouse
antibody producing spleen cells by various interferon preparations.
Nature 246:92, 1973.
32. Cerottini, J.-C., Brunner, K.T., Lindahl, P. and Gresser, I.
Inhibitory effect of interferon preparations and inducers on the
multiplication of transplanted allogeneic spleen cells and
syngeneic bone marrow cells. Nature New Biol. 242:152, 1973.
33. Farrar, W.L., Johnson, H.M. and Farrar, J.J. Regulation of the
production of immune interferon and cytotoxic T lymphocytes by
interleukin 2. J. Immunol. 126:1120, 1981.


12
provide insight into the relationship of the EGF receptor on lymphocytes
to the well characterized EGF receptor on 3T3 fibroblasts to which all
three mitogens bind and induce cellular proliferation; (3) determine if
the EGF receptor on splenic lymphocytes is constitutively produced or is
induced upon stimulation with SEA, and determine the binding character
istics of EGF with this receptor; (A) determine if TGFa and/or VGF can
compete with EGF for the EGF receptor on splenic lymphocytes.


Table 1-2
Some Immunoregulatory Effects of IFN
Effects
References
Suppression and enhancement of antibody production
25,26,29-31
Suppression of antigen- and mitogen-induced
lymphocyte proliferation
27,28,32
Enhancement of specific cytotoxicity of T lymphocytes
33
Enhancement of NK cell cytotoxicity
34-37
Enhancement of antibody-dependent cell-mediated
cytotoxicity
38
Activation of macrophages for enhanced tumor ceil
killing
39-41
Modulation of expression of products of the major
histocompatibility complex on the cell membrane
42-45
Modulation of expression of Fc receptors on the
cell membrane
46-48
Modulation of expression of interleukin 2 receptors
on the cell membrane
49
Maturation of B cells for immunoglobulin production
and secretion
50
Reprinted from Lymphokines 11:33, 1985. Copyright (c) 1985 by Academic
Press, Inc. (3)


Table 3-1
Relative abilities of mouse EGF and recombinant human EGF
to provide the helper signal for IFNy production
by Lyt 2+ spleen cells
GFa
(nM)
IFNy(U/ml
moEGF
+ S.D. )
huEGF
0
<3
<3
0.1
<3
<3
1
65 7
55 + 7
10
60 .14
85 21
100
95 7
200 141
aSamples were from day 3 of culture. Whole spleen cells
had 95 35 IFN Yunits/ml.


9
in hematopoietic cells. Studies have not been carried out to determine
if the receptor is inducible in such cells as primary lymphocytes in a
manner analogous to the receptor for IL-2, where the high affinity IL-2
receptor is not constitutively expressed but is induced by lymphocyte
mitogens (86). The EGF receptor is a 170,000 dalton single chain
glycoprotein with intrinsic tyrosine kinase activity (87). The mature
receptor is composed of three major domains, which are: (1) a large
glycosylated extracellular domain, (2) a transmembrane hydrophilic region
of 23 amino acids, and (3) a cytoplasmic region containing the tyrosine
kinase domain (57) that is composed of residues that are characteristic
of the tyrosine kinase family (88). Binding of EGF to its receptor
results in activation of the protein tyrosine kinase and thus triggers
subsequent intracellular events (57,89). The binding site of EGF on its
receptor is between residues 294 to 543 (90). Signal transduction is
mediated through the autophosphorylation of tyrosine residue 1173 (91).
Binding experiments with radiolabeled EGF to its receptor demon
strate a stoichiometric ligand-receptor interaction of one-to-one (92).
Scatchard analysis of EGF binding to intact cells, with high receptor
numbers, suggests the presence of different receptor classes with
distinct affinities toward EGF. High affinity receptors comprise 5 to
10% of the total, while the remaining receptors are of low affinity (93).
In EGF receptor negative 3T3 cells the introduction of wild type EGF
receptor showed both high and low affinity binding (94). Thus, the basis
for the same EGF receptor showing different affinities is not known.
Cells which are treated with the tumor promoter phorbol myristate
acetate (PMA) or platelet-derived growth factor (PDGF) abolish the high-


5
Thus, IFNy is an immunoregulatory molecule which can modulate a wide
range of physiological states in a variety of cell types; its biological
functions augment both cellular and humoral defense systems as well as
identify potential targets for immune responses. An understanding of the
biological signals which regulate the production of IFNy will help in
understanding the initiation of the complex immune response. The work
presented here addresses the regulation of production of IFNy by EGF with
particular emphasis on receptor recognition.
Epidermal Growth Factor
The EGF Molecule
EGF is mitogenic for a wide variety of cell types. Studies on EGF
and its receptor have helped to increase our understanding of cell
growth, ligand-receptor interactions, receptor activation, and oncogenes
(56,57).
EGF was discovered in 1959 when it was observed that murine sub
maxillary gland extracts, when injected into newborn mice, induced early
eyelid opening and incisor eruption (56,58). Purification of the respon
sible substance showed that the active factor was EGF and not nerve
growth factor which is also present in extracts of murine submaxillary
glands (56). The purified EGF was also shown to stimulate proliferation
of epidermal cells and keratinocytes (59,60).
Mature murine submaxillary gland EGF is composed of 53 amino acids
with a molecular mass of 6,000 daltons (61). It has three disulfide
bridges which are required for biological activity. EGF is not glyco
sylated. There is 70% homology between mouse and human EGF and the two
molecules behave identically in cell and biochemical assays (56,57,62).


44
EGF receptors after treatment with B cell mitogens. Induction of
receptor was not observed when the cells were treated with the T-cell
mitogen Concanavalin A. Other T-cell mitogens were not tested. This EGF
receptor was not further characterized. Interestingly, in our studies we
found an EGF receptor on splenic lymphocytes that required induction by
the T-cell mitogen SEA. The splenic lymphocyte EGF receptor was
detectable after a 48 hr incubation in the presence of SEA.
Approximately 25% of total binding was specific, which would suggest the
presence of low receptor numbers. The splenic lymphocyte EGF receptor
has an apparent dissociation constant of 7 nM.
The IL-2 receptor is another example of an inducible lymphocyte
receptor (86). This receptor is induced by a T-cell mitogen and takes
several days before it can be detected on the surface of lymphocytes. The
induced IL-2 receptor does not recognize the EGF molecule as a
competitive ligand in the presence of labeled IL-2. Therefore, the
induced EGF receptor is different from the IL-2 receptor on lymphocytes.
Since we had shown that the EGF molecule itself, and not a
contaminant, regulated the production of the IFNY lymphokine, we then
determined if the EGF-related growth factors TGFa (synthetic) and VGF
(recombinant) also induced IFNy production. We were surprised to find
that, unlike EGF, neither TGFa nor VGF were able to induce the production
of IFNY(Figure 3-3). Moreover, neither peptide functionally blocked the
ability of EGF to induce IFNY; nor were these peptides able to suppress
the production of IFNY in whole spleen cells. Thus while TGFa and VGF
were biologically active in induction of 3T3 fibroblast proliferation
(Figure 3-4), unlike EGF. they were not capable of inducing IFNy in


AO
Table 3-6
Ability of recombinant human EGF to compete with
murine purified submaxillary lz,Jl-EGF for the
EGF spleen cell receptor3
rHuEGF
(nM)
MuEGF
(nM)
CPM
+
SD
-
-
1488
+
269
10
-
1424
+
197
100
-
1463
+
235
330
-
1189
+
183
1000
-
1184
+
97
3300
-
1100
+
108
-
3300
1152
+
78
aSpleen cells were stimulated with SEA for 48 hr
and washed extensively prior to binding with
-^-I-EGF at 5 nM final concentration.


20
Balb/c 3T3 Fibroblast Growth Factor Binding Assays
The 3T3 growth factor binding assay is performed using a
modification of Kobayashi et al. (ill). 3T3 fibroblasts are grown as
above and seeded at 6 x 10^ cells/weil in 0.2 ml volume. All the
peripheral wells are treated as above. After 24 hrs at 37C, 5% CO2
confluent monolayers are washed 3 times with Earle's balanced salt
solution (EBSS) lacking calcium (57) containing Pen/Strep and 20 mM NaNg,
2 mg BSA/ml and aspirated to dryness. Then 25 pi of EBSS or dilutions of
growth factors are added to wells in triplicate. After 15 min, 25 pi of
l^I-EGE is added. After 1 hr at room temperature the wells are washed 3
times with EBSS and NaOH (50 pi of 0.1 M) is added to each well to
solubilize the cells. The liquid is absorbed by cotton-tipped
applicators and counted on an LKB gamma counter.
Murine Spleen Lymphocyte Binding Assays
Spleen cells are resuspended at 5 x 10^ cells/ml in SDMEM containing
10% FBS in the presence or absence of 0.5 pg SEA/ml. The cells are
seeded at 25 ml per 150 cm^ Corning flask and incubated at 37C, 5% CO2
for 48 hr. Nonadherent cells are then collected and centrifuged at 1,000
rpm for 10 min. Red blood cells are lysed by the addition of cold
distilled water (1.5 ml per 3 spleens) to the completely dispersed
pellet. After 10 seconds, cold EBSS (5.5 ml per 3 spleens) lacking
calcium and containing 0.15 M NaCl, 15% FBS, 20 mM NaNg, and 2 mg/ml RIA-
BSA is added to the spleen cells. After 10 minutes on ice the cells are
placed into a 15 ml Corning test tube and centrifuged at 133 X g for 40
sec (adding 30 sec for each additional 5 ml). The supernatant is
aspirated and the cells are washed twice in cold EBSS containing 20 mM


lymphocytes. Finally, in competitive binding studies we demonstrated
that TGFa did not compete with ^-*I-EGF for the induced lymphocyte EGF
receptor while it did compete for receptor on 3T3 cells. We conclude
that there is a novel EGF receptor on lymphocytes which, unlike the
classic EGF receptor, does not functionally or physicochemically (in
binding studies) recognize the EGF-related peptides TGFa and VGF.
The induction of IFNy by EGF is a newly described function for this
nonhematopoietic growth factor. Its effect is mediated through a novel
EGF receptor. This EGF receptor differs from the classic EGF receptor in
that it is inducible by a T-cell mitogen, and fails to recognize the EGF-
related growth factors TGFa and VGF.