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Mechanism of interferon therapy of Multiple Sclerosis

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Title:
Mechanism of interferon therapy of Multiple Sclerosis studies in an animal model
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Mujtaba, Mustafa Ghulam, 1973-
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English
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viii, 78 leaves : ill. ; 29 cm.

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Antibodies ( jstor )
B lymphocytes ( jstor )
Cells ( jstor )
Cytokines ( jstor )
Diseases ( jstor )
Experimental autoimmune encephalomyelitis ( jstor )
Interferons ( jstor )
Lymphocytes ( jstor )
Multiple sclerosis ( jstor )
Spleen cells ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF ( lcsh )
Interferon -- Therapeutic use ( lcsh )
Microbiology and Cell Science thesis, Ph.D ( lcsh )
Multiple sclerosis -- Animal models ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1999.
Bibliography:
Includes bibliographical references (leaves 66-77).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Mustafa Ghulam Mujtaba.

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MECHANISM OF INTERFERON THERAPY OF MULTIPLE SCLEROSIS:
STUDIES IN AN ANIMAL MODEL












By

MUSTAFA GHULAM MUJTABA










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 1999















ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Howard M. Johnson, for taking me into his laboratory and guiding me through my studies. He has always been knowledgeable and logical, which make him a great mentor. I must also express my thanks to my committee members -- Dr. Edward Hoffman, Dr. Jake Streit, Dr. Janet Yamamoto, and Dr. Julie Maupin -- for their time, patience, and effort. Many thanks to my fellow graduate students and labmates both past and present, including Barbara, Jeanne, Brian, Prem, Taishi, Martez, Pedro, Amy, George, Joe, Kendra, Scott, Karrie, Wiggins, and Tim. They make the lab more "interesting" and fun, at least at certain times.

Finally, I thank my family for their support through my graduate school

studies. My parents, Ghulam and Uzra Mujtaba, have always been there for me. I thank my brother and sisters and their families for their help and encouragement. Many thanks go to my Afghan cousins whom are always a great break from my graduate studies. I owe a great debt of gratitude to my wonderful family and friends.

















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TABLE OF CONTENTS



ACKNOWLEDGMENTS ............................................................. ii

LIST OF TABLES ........................................ v

LIST OF FIGURES ................. .................................................. vi

ABSTRACT vii

CHAPTERS

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

Overview ..................................................................... 1
Multiple Sclerosis ..................................... ................... 2
Experimental Allergic Encephalomyelitis ................................ 5
Interferons ....................................................................... 8

2 IFNt INDUCES STABLE REMISSION IN CHRONIC EAE ......... 11

Introduction .................................................................. 11
Materials and Methods ....................................................... 12
IFN ..................................... .................................. 12
Induction of EAE ....................................................... 12
Administration of IFN ................................................ 13
Histological Evaluation .......... ....................................... 13
Proliferation Assay and Isolation of T Cells and B Cells .......... 14 ELISA for MBP-Specific Antibodies ................................. 15
Results ......................................................... 15
IFNt Blocks Further Relapses into Paralysis of EAEAfflicted Mice ........................................................ 15
Reduction and Prevention of Lymphocytic Infiltrates in
EAE Mice by IFN ................................... ...... ......... 17
Deactivation of Microglia Following IFNt Treatment .............. 17
IFNt Treated Mice Have a Lower Antibody Level Against
MBP than Control Mice ............................................. 22
IFNt Inhibited MBP-Specific B Cell Proliferation ...............22
IFNt Inhibited MBP-Specific T Cell Proliferation .................. 29
Discussion ..................................................................34

3 CD4 T SUPPRESSOR CELLS MEDIATE IFNT PROTECTION AGAINST EAE ......................................................... 35



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Introduction ...................................................................35
Materials and Methods ...................................... .......36
IFNs ......................................................36
Antibodies and Cytokines ............................................36
Interferon Induction of Suppressor Cells ................. ..... 37
Induction of EAE Cells .............................................37
CD4 T Cell Isolation and Depletion .................................38
Production of Suppressor Factor ................................ 38
Proliferation Assay ....................................... .....38
Results ......................................................... 39
IFNt--Treated Spleen Cells Inhibit MBP-Specific T Cell
Proliferation ..........................................................39
IFNt Induction of Suppressor Cells is Dose-Dependent ............39
IFNt Suppressor Cells Protect Mice Against EAE ...............44
IFNt-Induced Suppressor Cells are CD4 T cells .................44
Suppressor Cells Produce Soluble Suppressor Factor(s) ..........49 IFNt-Induced Suppressor Cells Produce IL-10 and TGF ........49
IL-10 and TGFp Act Synergistically to Inhibit MBPSpecific T Cell Responses ..........................................54
Discussion ........................................54

4 CONCLUSION ............................................................... 58

REFERENCES .................................................. .. ..... 66

BIOGRAPHICAL SKETCH ........................................................78






























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LIST OF TABLES

Table Page


1. Treatment of SJL mice with IFNt after induction of EAE ............... 16

2. IFN inhibition of MBP-sensitized spleen cells ............................ 65







































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LIST OF FIGURES

Figures Page

1. Histological evaluation of EAE mice before and after treatment with PBS and IFN t ...................................................... 19

2. IFNt causes deactivation of microglia .................................. 21

3. Inhibition of anti-MBP antibody production in the chronic form of EAE by IFN ........................ ................................. 24

4. Inhibition of anti-MBP antibody production in the acute form of EAE by IFN ............................................................. 26

5. IFN- inhibits MBP-specific proliferation of B cells from chronic EAE mice .............. .............................................. 28

6. IFNr inhibits MBP-specific proliferation of B cells from acute EA E m ice .................................................................. 31

7. IFNt inhibits MBP-specific T cell proliferation .................. ........ 33

8. IFNr-treated spleen cells inhibit MBP-specific T cell proliferation .... 41 9. IFNt induction of suppressor cells is dose-dependent ................ 43

10. IFNt-induced suppressor cells can delay the onset of EAE in mice ... 46 11. IFNt-induced suppressor cells are CD4 T cells .......................... 48

12. IFNT-induced CD4 T suppressor cells produce soluble suppressor
factors(s) .............................................. .................... 51

13. Blockage of IFN-induced suppressor cell and suppressor supernatant
effects on MBP stimulation of sensitized EAE mouse spleen cells
with monoclonal antibodies to IL-10 and TGF3 ...................... 53

14. IL-10 and TGFp act synergistically to inhibit MBP-specific T cell
responses ........................... ........ ............................ 56

15. IFNt induction of CD4 suppressor T cells inhibit SEA stimulation
of naive spleen cells ....................................................... 61

16. IFNt-treated CD4 T cells produce suppressor factor(s) which
inhibit SEB stimulation of naive spleen cells ........................ 63


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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

MECHANISM OF INTERFERON THERAPY OF MULTIPLE SCLEROSIS: STUDIES IN AN ANIMAL MODEL




By

Mustafa Ghulam Mujtaba

August 1999



Chairperson: Howard M. Johnson
Major Department: Microbiology and Cell Science



Interferon (IFN) tau is a type I IFN that was originally identified as a

pregnancy recognition hormone produced by trophoblast cells. It is as potent an antiviral agent as IFNa and IFN, but lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in animal studies. Previously it has been shown that interferon - pretreatment inhibits the development of both acute and chronic mouse experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). Here, we show that IFNr induced remission in SJL/J mice that had ongoing chronic active EAE disease, and protected mice against secondary relapses. IFNt treatment reversed lymphocyte infiltration and microglial activation in the central nervous system. Mice that were treated with IFNr had lower levels of anti-MBP (myelin basic protein) antibodies than untreated mice in both chronic and acute forms of EAE.


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MBP induced proliferation in B cells from EAE mice, but treatment with IFNt either in vivo or in vitro blocked this activation. Furthermore, IFNt inhibited MBP activation of T cells from EAE mice. Thus, IFNt inhibits the humoral arm as well as the cellular arm of the autoimmune disease EAE. IFNt prevents EAE in mice by induction of suppressor cells and suppressor factors. Suppressor cells can be induced by IFNt in tissue culture and in vivo by either intraperitoneal injection or by oral administration to mice. Incubation of suppressor cells with MBP-sensitized T cells blocked or delayed the MBP-induced proliferation. Further, intraperitoneal injection of suppressor cells into mice blocked induction of EAE by MBP. Suppressor cells possessed the CD4 T cell phenotype, and produced soluble suppressor factors that inhibited MBP activation of T cells from EAE mice. The suppressor factors were found to be IL-10 and TGFP, which acted synergistically to inhibit the MBP activation of T cells from EAE mice. These findings are important for understanding the mechanism(s) by which type I IFNs protect against autoimmune disease.

























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CHAPTER 1
INTRODUCTION


Overview


Interferons (IFNs) are a group of glycoproteins that play important modulatory roles in the vertebrate immune system. Originally discovered in 1957 for their ability to interfere with viral replication in cells (Isaac and Lindenmann, 1957), they are now known to also have antiproliferative effects on a variety of cell types. For this reason, IFNs have been used for the treatment of autoimmune diseases, viral infections, and several types of cancers (Gutterman et al., 1994).Interferon-tau (IFNT) is a type I IFN that was originally identified as a pregnancy recognition hormone produced by trophoblast cells in sheep. It is as potent an antiviral agent as IFNa and IFN3, but lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in vivo (Bazer et al., 1989; Pontzer et al., 1991; Soos et al., 1995a; Soos et al., 1995b).

Multiple Sclerosis (MS) is one of the most common disease of the central nervous system (CNS). In MS, the loss of myelin is accompanied by a disruption in the ability of the nerves to conduct electrical impulses to and from the brain, and this produces the various debilitating symptoms of MS. Experimental allergic encephalomyelitis (EAE) is a murine model useful for studying the demyelinating disease MS. Myelin basic protein (MBP) has been shown to be one of the primary CNS antigens responsible for induction of autoimmunity in the EAE model (Zamvil et al., 1990). Immunization of mice with MBP results in tail and limb paralysis due to lymphocytic infiltration and demyelination in the CNS (Zamvil et al., 1990). MBP-specific antibodies and autoreactive MBP-specific T cells are also thought to contribute to the exacerbation of EAE and MS (Sun, 1993; Warren et


I





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al., 1993; Gerritse et al., 1994; Saoudi et al., 1995; Wang et al., 1995). Various inflammatory cytokines like tumor necrosis factor-alpha (TNFa) and IFNy contribute to the break down of myelin on nerve cells in the CNS. Furthermore, microglia, the resident macrophage of the CNS, are thought to play a key role in recruiting lymphocytes and function as antigen-presenting cell in MS and EAE (Benveniste, 1997; Streit et al., 1995; Hickey et al., 1988).

Currently, the type I IFN, IFNI3, is the only FDA approved cytokine treatment of MS. Previously, it has been shown that administration of IFNr to mice at the time of immunization with MBP blocked the development of EAE in mice without associated toxicity; however the mechanism of such action has not been fully elucidated (Soos et al., 1995a). Thus, the purpose of this study is to determine the mechanism by which IFNt suppresses autoimmune responses in EAE.



Multiple Sclerosis


Multiple Sclerosis (MS) is a chronic, demyelinating, inflammatory disease of the CNS. MS commonly affects young adults and mostly women (Ebers et al. 1986). MS is commonly found in Canada, the United States, South America, and Europe; near the equator MS is unknown (Ebers et al., 1986). In this disease there is an inflammation of myelin, which is fatty insulation or covering of nerve cell extensions, known as axons (Lassmann, 1998). Messages are sent along axons to other nerve cells in a kind of electrical signals. Myelin insulates the axons to help get these electrical impulses through. It stops currents from flowing between the individual axons. It also helps to speed the conduction of the electrical signal. This enables people to move almost without thinking. When myelin is affected in MS, impulses travel over the axons very slowly, if at all, and there is some electrical interference between axons. Messages are not sent efficiently and can fail to get through so that a person loses the ability to make smooth, rapid, and






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coordinated movements (McFarland, 1998). Inflammation in the CNS destroys myelin and the oligodendrocytes, the myelin-producing cells (Mews et al., 1998). After tissue destruction a scar or hardening area forms. These areas are multiple within the CNS, thus the term multiple sclerosis.

The clinical course is variable, but the most common form is characterized by

relapsing neurological deficits. Early MS lesions are characterized by local accumulation of activated T cells around small venules (Hauser et al., 1983). Later myelin degeneration occurs associated with perivascular inflammation consisting of T cells, B cells, plasma cells, and macrophages (Prineas et al., 1975; Prineas et al., 1978). T cells are also found at the leading edge of plaques, and they extend into the surrounding normal appearing white matter. The T cells express activation molecules on their cell surface, such as IL-2 receptors and class II major histocompatibility complex (MHC) antigens (Hoffman et al., 1986). In addition, class II MHC expression can also be detected on infiltrating macrophages and resident CNS cells, including microglia, astrocytes, and brain capillary endothelial cells (Hoffman et al., 1986). Class II MHC expression in the CNS is presumably induced by IFNy secreted by activated T cells. These findings indicate that in early acute MS lesion, demyelination occurs in the face of an active immune response within the CNS. Gliosis is also a prominent feature of MS; this process is characterized by astrocyte proliferation and hypertrophy (Bignami et al., 1972). This reaction eventually leads to the formation of dense glial scars in the CNS, which can contribute to motor and sensory impairment. TNFa, a proinflammatory cytokine, appears to contribute to this process (Salmaj et al., 1990).

The primary demyelination observed in MS results from damage to the myelin

sheath or to the myelin-producing cells, the oligodendrocytes. Because myelin is critical for saltatory excitation along axons, demyelination leads to loss of neurological function. Recent findings with magnetic resonance imaging (MRI) indicate that considerable subclinical disease occurs, and that there is breakdown of the blood-brain barrier (BBB)





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early in lesion development (Thomson et al., 1992; Gay et al., 1991), which may be a crucial event in the pathogenisis of new lesions in MS. A large body of experimental evidence implicates immune mediated processes in activation and progression of MS. In addition, both genetic and environmental factors contribute to disease (Ebers et al., 1986; Martin et al., 1992). Although the pathological lesions, or plaques, are confined to the white matter of the brain and spinal cord, studies of cerebrospinal fluid and peripheral blood lymphocytes provide evidence for both local and systemic activation of the immune system (Hafler and Weiner, 1989).

IFNy and TNFa and P have been implicated in exacerbating MS. The ability of

TNFa to mediate myelin and oligodendrocyte damage in vitro (Selmaj et al., 1988), and its ability to cause cell death of oligodendrocytes in vitro (Robbins et al., 1987) may contribute directly to myelin damage or the demyelination process observed in MS. Both TNFa and TNF can cause death of oligodendrocytes, the myelin-producing cells of the CNS (Robbins et al., 1987; Paul and Ruddle, 1988).

MS is characterized by migration of inflammatory cells from blood into the brain

and subsequent invasion of the extravascular tissue (Cross et al., 1990; Raine et al., 1990). Recent studies have shown that up-regulation of adhesion molecules, such as intercellular adhesion molecules (ICAM-1) on brain endothelial cells by exposure to proinflammatory cytokines such as TNFa and IFNy mediate leukocyte adhesion to endothelium (McCarron et al., 1993; Fabry et al., 1992; Wong et al., 1992). The presence of ICAM-1 and other adhesion molecules in the vessel walls as well as on astrocytes may guide inflammatory leukocytes into and through the brain, thereby contributing to impairment of the blood brain barrier and the neuropathology of MS.

IFNy is considered the most potent inducer of class II MHC antigen expression on most cell types, including astrocytes and microglia (Cogswell et al., 1991; Wong et al., 1984; Fierz et al., 1985; Fontana et al., 1984; Pulver et al., 1987; Suzumura et al., 1987). There is little or no expression of class II molecules in normal brain; however IFNy






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induces them on astrocytes and endothelial cells, which can then present myelin antigens to T cells (Fierz et al., 1985; McCarron et al., 1986). Other function of IFNy that may be important in MS include activation of macrophages, which act as effector cells in demyelination (Bever and Whitaker, 1985) and induction of adhesion molecules, which mediated homing of lymphocytes to sites of inflammation and may facilitate their entry into the CNS (Male et al., 1990). Furthermore, IFNy-positive cells have been detected in the CNS of patients with MS (Hofman et al., 1991; Traugott and Lebon, 1988). Recent studies on the association of IFNy with MS demonstrated that peripheral blood lymphocytes from patients with MS produce significantly more IFNy than those of normal lymphocytes (Beck et al., 1988; Hirsh et al., 1985).

Accumulating evidence supports the notion that MS is an autoimmune disorder mediated by T cells. This evidence includes the pathology of MS lesion, immunological abnormalities in both the periphery and CNS of patients with MS, immunoglobulin synthesis within the CNS, exacerbation of disease after treatment with IFNy, putative autoantigens such as myelin basic protein (MBP), and involvement of cytokine networks (Martin et al., 1992). Studies have also shown the presence of autoantibodies specifically bound to disintegrating myelin around axons in lesions of acute MS (Genain et al., 1999). Furthermore, peripheral blood T lymphocytes from patients with MS were activated by various MBP peptides (Baxenvanis et al., 1989). Thus, many factors are involved in the pathogenesis of MS.



Experimental Allergic Encephalomyelitis


The best-characterized experimental model for MS is experimental allergic

encephalomyelitis (EAE). Similarities shared between EAE and MS are relapsing and chronic paralysis, CNS demyelination, linkage to MHC class II, CD4 T cells present in perivascular inflammatory lesions, and similar autoantigens. MS is a spontaneous disease






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in humans, but EAE is induced by injection of spinal cord components such as myelin basic protein (MBP) with adjuvant or transfer of encephalitogenic MBP-specific T cells to naive recipients (Swanborg, 1995; Paterson and Swanborg, 1988; Tabira, 1988, Pettinelli and McFarlin, 1981). MBP is a predominant protein present in myelin in the CNS. Transgenic mice have been constructed that mimics MS in its spontaneous induction and pathology (Goveram et al., 1993). EAE can be induced in a number of species including mice, rats, guineas pigs, monkeys, sheep, dogs, and chickens (Sturart and Krikorian, 1928). Clinical signs of EAE include dramatic weigh loss, weakness of the tail and hind limbs, and ascending paralysis. The earliest clinical signs of CNS dysfunction are closely associated with formation of perivasuclar cellular infiltrates and edema with the CNS (Raine et al., 1984; Leibowitz and Kennedy, 1972; Claudio et al., 1990; Cross et al., 1990; Raine et al, 1990; D'Amelio et al., 1990). Demyelination appears to be a later event (Raine et al., 1990; D'Amelio et al., 1990) and may account for chronic neurological dysfunction. EAE is characterized by inflammatory infiltration of the CNS by activated T cells and macrophages, de-myelination, and acute, chronic, or chronic-relapsing paralysis. The mediators of this disease are CNS antigen reactive CD4 T cells that are class II MHCrestricted (Zamvil and Steinman, 1990). Lymphocytes, as mediators of EAE, were first implicated by experiments in which anti-lymphocyte antibodies inhibited induction of EAE (Waksman et al., 1961). Further evidence that T cells were involved stemmed from the observation that thymocytes are required for EAE induction (Arnason et al., 1962). Furthermore CD4 T cells are present in inflammatory EAE lesions in the CNS (Traugott et al., 1986). Most encephalitogenic T cells are of the Thl subtype, which secrete IFNy, IL2, TNFa, and TNFP (Mosmann and Coffman, 1989). It has been suggested that TNFa and TNFP secretion by MBP-specific T cell clones correlates with their encephalitogenic potential (Powell et al., 1990). Expression of adhesion molecules also influences the pathogenicity of encephalitogenic T cells (Baron et al., 1993; Kuchroo et al., 1993). Susceptibility to EAE appears to be linked to MHC alleles, although non MHC genes may





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have a small role in contributing to EAE (Gasser et al., 1973). Although EAE in animals is initiated by T cells that recognize myelin antigens in the context of class II MHC molecules (Wekerle et al., 1986), some studies have suggested that B-cell activation and antibody responses are necessary for the full development of EAE (Brosnan and Raine, 1996; Willenborg and Prowse, 1983; Piddlesden et al., 1993), and earlier studies on immunemediated demyelination using myelinated cultures of CNS tissue have indicated that humoral factors are effector mechanisms (Raine and Bornstein, 1970; Raine et al., 1973). Autoantibodies against CNS antigen myelin/oligodendrocyte glycoproteins were identified to bind disintegrating myelin around axons in lesion of acute MS and the marmoset model of EAE (Genain et al., 1999).

Cytokines have been implicated in contributing to EAE disease progression, as well as mediating recovery from disease. Inflammatory cytokines released within the CNS may contribute to the disease process by influencing vascular permeability, inflammatory cell extravasation, and antigen presentation (Mantovani and Dejana, 1989; Martiney et al., 1990; Fierz et al., 1985). Cytokines released within the CNS in response to acute inflammation may also contribute to chronic damage associated with reactive gliosis of astrocytes (Giulian and Lachman, 1985; Selmaj et al., 1990; Yong et al., 1991) and the destruction of oligodendrocytes and myelin (Selmaj and Raine, 1988; Selmaj et al., 1991). The cytokines, IL-1, TNFa, and TNFP, contribute to the initiation and/or disease progression of EAE (Mannie et. al., 1987; Symons et al., 1987; Jacobs et al., 1991; Ruddle et al., 1990; Selmaj et al., 1991; Kuroda et al., 1991). Studies involving the role of IFNy show conflicting results. Some studies show that IFNy plays a protective role in different models of EAE (Billiau et al., 1988; Duong et al., 1992; Voorthuis et al., 1990). Other studies show the detection of IFNy before the onset of clinical disease (Kennedy et al., 1992; Merril et al., 1992; Stoll et al., 1993) and during the acute phase of disease (Khoury et al., 1992; Baker et al., 1991; Kennedy et al., 1992). Other cytokines such as IL-10, TGFP, and IL-4 have a protective effect on EAE. TGF can inhibit IFNy-induced






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class II MHC antigen expression on both human astroglioma and rat astrocyte cells (Zuber et al., 1988; Schluesener, 1990) and can act as a chemotactic agent for both rat astrocytes and microglia (Morganti-Kossmann et al., 1992; Yao et al., 1990). Microglia, the resident macrophages of the brain, are thought to play a key role in recruiting lymphocytes and function as antigen-presenting cells in MS and EAE (Benveniste et al., 1997; Streit et al., 1995; Hickey et al., 1988). TGFp inhibits the production of TNFa by microglia (Suzumura et al., 1993) and astrocytes (Benveniste et al., 1994), and TGFP has also been shown to be an important mediator of oligodendrocyte differentiation (McKinnon et al., 1993). Also, IL-10 has been detected in the CNS of SJL/J mice during disease recovery (Kennedy et al., 1992). IL-4, IL-10, and TGF3 share some similar biological activities because they are all capable of inhibiting secretion of proinflammatory cytokines (Bogdan et al., 1992; Chao et al., 1993).

The EAE model has been used in several novel immunotherapy experiments including anti-TCR antibodies, anti-MHC antibody, anti-CD4 antibody, peptide and interferon therapies, and T cell vaccination (Steinman et al., 1983; Brostoff and Mason, 1984; Howell et al., 1989; Bandenbark et al., 1989; Soos et al., 1997). Thus, the development and testing of a safe therapy for EAE and understanding the mechanism(s) of the therapeutic are the first steps toward identifying potential therapies for MS.



Interferons


Interferons (IFN) are glycoproteins that are produced and released from virally

infected cells; they were originally characterized for their antiviral properties (Pestka et al., 1987), and they were first described in 1957 by Issacs and Lindemann (Issacs and Lindeman, 1957). IFNs have been found in all higher vertebrates including humans. They have molecular weights ranging from 15 to 30 KDa (Gastle and Huber, 1988). The idea that IFNs alter the pathogenesis of natural virus infections was supported by observations





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that virus-infected animals injected with antibody prepared against IFNs succumbe more rapidly to disease than when virus was given alone (Gresser et al., 1976). IFNs inhibit the growth of viruses of all type in vivo and in vitro. Some viruses are more sensitive to IFN action than others (Grossberg, 1972). Besides their antiviral activities, IFNs possess many other activities including those which are antimicrobial, antitumor, and immunomodulatory (Pestka et al., 1987). IFNs are currently approved by the Food and Drug Administration for a number of diseases such as hairy cell leukemia, condyloma acuminatum, acquired immune deficiency syndrome (AIDS) related Kaposis's sarcoma, chronic hepatitis B and C, gebutak warts caused by papillomavirus, chronic granulomatous disease, and MS (Dorr, 1993; Johnson et al., 1994).

Different IFNs are distinguishable based upon their cellular source, immunological reactivity, and induction of biological responses. There are two main types of IFN, type I and type II. The type I IFNs include IFNa, IFNp, IFNw, and IFNt. The type II IFN refers to IFNy. IFNa and IFNo are produced by leukocytes, while IFN3 is produced by fibroblasts, and IFNt is primarily produced by the trophoblast cells of the conceptus. IFNy is produced by T cells and natural killer cells (Baron et al., 1991). IFNy appears to have a dominant immunoregulatory role while IFNa and IFN tend to mediate more antiviral activity. Both type I and type II IFNs have potent antiproliferative effects, while only IFNy, and not the type I IFNs, can upregulate MHC class II (Houghton et al., 1984, Schwartz et al., 1985). Twenty-six IFNa genes with common structures have been identified, and they encode for at least 22 distinct proteins consisting of 20KDa single polypeptide chains (Zoon et al., 1992). These different IFNas mediate distinct biological activities in different cells. In contrast, there is only a single form of IFNP encoded by a distinct gene located next to the IFNa locus in both human and mice (Farrar et al., 1993). Also, a single gene has been identified and described for IFNyr whereas, several genes for IFNm and IFNc have been identified (Sen and lengyel, 1992; Bazer and Johnson, 1991).






10


The type I IFNs bind to a common receptor, whereas IFNy binds to a different receptor (Langer and Pestka, 1988). IFNy is considerably more active as an immunomodulator than other classes of IFNs, but its antiviral activity is lower than these other classes. IFNy is produced by all CD8+ T cells populations and by the Thl and ThO subsets of CD4+ T cells following antigenic or T cell mitogen stimulation (Farrar et al. 1993). IFNy can upregulate the surface expression of MHC class I and class II antigens on a variety of cell types both in murine and human (Baron et al., 1991; Sen and Lengyel ,1992). IFNy production from T cells or NK cells is stimulated by IL-12, which can act synergistically with alloantigens, mitogens, or IL-2 (Stem et al., 1990). In contrast, IL-10 inhibits IFNy production by T cells and NK cells (Fiorentino et al., 1989).

IFNt has only been recently described. IFNT was discovered originally as a pregnancy recognition hormone that is essential for establishment of pregnancy in ruminants such as sheep and cows (Bazer and Johnson, 1991). It was identified as a member of the type I IFN family when it was shown to share significant amino acid sequence homology with the type I IFNs, IFNa and IFNo (Imakawa et al., 1987). IFNt possesse antiviral, antiproliferative, and immunomodulatory activities similar to the other type I IFNs (Pontzer et al., 1988; Bazer et al., 1989; Soos and Johnson, 1995b). IFNr differs from the type I IFNs in that it has been shown to be remarkably nontoxic when used at high concentrations in culture and animal studies (Bazer et al., 1989; Soos et al., 1995a; Pontzer et al., 1991), not readily induced by virus and dsRNA (Roberts et al., 1992), and is under genetic regulation different from other type I IFNs (Cross and Roberts, 1991). However, IFNP, which has been shown to ameliorate relapsing-remitting nature of MS (IFNP Multiple Sclerosis Study Group, 1993), is currently the only cytokine approved by the FDA for treatment in MS. The mechanism of this amelioration is not fully understood. Therefore, the data recounted here on the mechanism of therapy of EAE by IFNt may serve as the basis for understanding how type I IFNs may exert therapeutic effects in autoimmune diseases such as MS.















CHAPTER 2
IFNt INDUCES STABLE REMISSION IN CHRONIC EAE


Introduction

Interferon-tau (IFNt) is a member of the type I IFN family but unlike IFNa and IFNP, IFNt lacks toxicity at high concentrations in vitro and when used in vivo in animal studies (Bazer et al., 1989; Pontzer et al., 1991; Soos and Johnson, 1995b, Soos et al., 1995b). Like the type I IFNs, IFNT is acid stable and exerts antiviral, antiproliferative and immunomodulatory activities (Bazer et al., 1989; Pontzer et al., 1991; Soos and Johnson, 1995b). Previously, we have shown that IFNT blocks the development of acute and chronic experimental allergic encephalomyelitis (EAE) in mice without any associated toxicity (Soos and Johnson, 1995b). EAE is a murine model used to study the human demyelinating disease multiple sclerosis (MS) (Zamvil and Steinman, 1990). In the EAE model, immunization of mice with central nervous system (CNS) antigens such as myelin basic protein (MBP) and proteolipid protein (PLP) results in tail and limb paralysis due to lymphocytic infiltration and demyelination in the CNS (Zamvil and Steinman, 1990).

Treatment of mice with IFNt before, during, and shortly after immunization with MBP prevents the onset of EAE (Soos et al., 1997). This protection is mediated by IFNt through induction of CD4 T cells to produce IL-10 and TGFP which act synergistically to inhibit activation of MBP-sensitized T cells from NZW mice (Mujtaba et al., 1997). Here we demonstrate that treatment of SJL/J mice with IFNt after the induction of chronic EAE induces them into stable remission. Furthermore, the histological features of the CNS normally associated with EAE such as lymphocyte infiltration and activation of microglia could not be found in the IFNt treated mice. Microglia, the resident macrophages of the brain, are thought to play a key role in recruiting lymphocytes and function as antigen


11






12


presenting cells in MS and EAE (Benveniste, 1997; Streit and Kincaid-Colton, 1995; Hickey and Kimura, 1988). Microglia are process-bearing when they are in the inactivated resting state, but they undergo cell hypertrophy upon activation, and they transform into rounded brain macrophages after neuronal degeneration (Streit et al., 1988). Furthermore, since MBP-specific antibodies are also thought to contribute to the exacerbation of EAE and MS (Sun, 1993; Warren and Catz, 1993; Gerritse et al., 1994) along with autoreactive MBP-specific T cells (Zamvil and Steinman, 1990; Zamvil et al., 1986; Ando et al., 1989; Lemire et al., 1986; Hashim and Brewen, 1985), we analyzed MBP-specific antibody levels in vivo and MBP-specific B cell and T cell activation in vitro on IFNt treated and control mice. Overall, these findings serve as the basis for understanding how type I IFNs exert therapeutic effects in autoimmune diseases.


Materials and Methods



IFN1

The ovine IFNt gene was expressed in Pichia pastoris using a synthetic gene construct, which was kindly provided by Dr. Gino Van Heeke, Ciba Pharmaceuticals, London, England (Heeke et al., 1996). IFNT was secreted into the medium and was purified by successive DEAE-cellulose and hydroxylapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining analysis (Heeke et al, 1996). The purified protein had a specific activity of 2.9 to 4.4 x 107 U/mg protein as measured by antiviral activity using a standard viral microplaque reduction assay on MDBK (Pontzer et al., 1991).


Induction of EAE

For induction of EAE, 300 gg of bovine MBP (MBP) were emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml H37Ra (Mycobacterium tuberculosis, Difco,






13


Detroit, MI) and injected into two sites at the base of the tails of NZW mice (Jackson Laboratory, Bar Harbor, Maine). On the day of immunization and 48 h later, 400 ng of pertussis toxin (List Biologicals, Campbell, CA) were also injected. For induction of EAE in SJL/J mice (Jackson Laboratory, Bar Harbor, Maine), the same protocol was used as described except mice were immunized again 7 days after the initial immunization. Mice were clinically examined daily for signs of EAE, and severity of disease was graded using the following scale: 1, loss of tail tone; 2, hind limb weakness, 3, paraparesis, 4, paraplegia; 5, moribund/death.


Administration of IFN.

Mice were orally fed using feeding needles from Fisher Scientific (Norcross, GA) and injected intraperitoneally (i.p.) with 100 uL of 6 X 105 U total of IFNr with PBS used as the vehicle for administration. Administration of IFNt to SJL/J mice was started after the onset of EAE and was administered every 48 h thereafter. NZW mice were administered with IFNT 48 h before, during, and 48 h after immunization with MBP.


Histological Evaluation

SJL/J Mice were perfused transcardially with 0.9% saline followed by Bouin's fixative solution. The vertebral columns were removed, and spinal cords were dissected out and post-fixed for an additional 48h in Bouin's fixative. Thereafter, the tissue was dehydrated and embedded in paraffin. Sections were cut at 7pm and mounted onto subbed slides. Cresyl violet staining was used to visualize inflammatory infiltrates.

For detection of microglia, SJL/J mice were first perfused with saline and 4%

paraformaldehyde, and after removing the spinal cords and post fixing them for 1-2 h in 4% paraformaldehyde, they were then transferred to 30% sucrose solution for cryoprotection on at 4 oC. Spinal cord lumbar regions were embedded in OCT compound and 25 pm sections were cut on a cryostat and mounted onto subbed slides. Microglia were






14


detected by immunohistochemically staining for CD1 lb (CR3) with the monoclonal antimouse CD1 lb (CR3) antibody (SeroTec Inc., NC). A 1:300 dilution of monoclonal antimouse CDl lb antibody was incubated with the sections overnight at 4 oC. Then, a 1:400 dilution of the secondary biotinylated anti-rat IgG antibody was incubated with the section for lh at 25 OC followed by lh incubations of avidin and horse radish peroxidase. Then, 3 3'-diaminobenzidine (DAB) (0.5 mg/ml) was applied to the sections for 10 minutes and finally washed with phosphate buffered saline (PBS), after which sections were counterstained with cresyl violet.


Proliferation assay and isolation of T cells and B cells

Spleen cells from IFNt treated, PBS treated, or untreated MBP-immunized NZW or SJL/J mice (5.0 x 10' cells/well) were cocultured with IFNT (10,000 - 30,000 U/ml), MBP (30 jg/ml), or media for 48 h. Spleen cells were washed, and B cells or T cells were isolated from each treated group using an immunoaffinity column from the cellect-plus mouse B cell kit or the cellect-plus mouse T cell kit (Biotex Laboratories, Alberta), respectively. B cell and T cell preparations were then incubated (5.0 x 0I cells/well) in RPMI 1640 media containing 5% FBS for 72 h. Some B cell and T cell cultures were incubated with 200 ng/ml of staphylococcol enterotoxin B (SEB) (Toxin Technology, FL), 50 jig/ml of lipopolysacharide (LPS) (Sigma chemical co., St. Louis, MO), or a combination of 20 ng/ml Phorbol 12-myristate 13-acetate (PMA) and 200 ng/ml ionomycin (Sigma Chemical Co., St. Louis, MO). The cultures were pulsed with [3H]-thymidine (1.0 gpCi/well)(Amersham, Indianapolis, IN) 18 h before harvesting onto filter paper discs using a cell harvester. Cell-associated radioactivity was quantified using a 1-scintillation counter and activity is reported in CPM.






15


ELISA for MBP-Specific Antibodies

Bovine MBP was resuspended in binding buffer (0.1 M carbonate/bicarbonate, pH 9.6), adsorbed to the flat bottoms of plastic 96 well tissue culture wells overnight at 4 oC at a concentration of 600 ng/well, and evaporated to dryness. The plates were treated with blocking buffer, 5% powdered milk (Carnation) in PBS, for 2 h in order to block nonspecific binding and then washed 3 times with PBS containing 0.05% Tween 20. Various dilution of sera from NZW or SJL/J mice which were untreated, IFNt-treated by i.p. injection, and IFNt-treated by oral feeding, and PBS treated were added to the wells and incubated for 3h at room temperature. Binding was assessed with the secondary antibody, goat anti-mouse immunoglobulin (IgG) coupled to alkaline phosphatase. After the substrate solution, p-nitrophenyl phosphate (5mg/ml) was added and the reaction terminated with 3 N NaOH activity was monitored by color development at 405nm in an ELISA plate reader (Bio-Rad, Richmond, CA


Results



IFNt Blocks Further Relapses into Paralysis of EAE-Afflicted Mice

The chronic form of EAE was induced in SJL/J mice with MBP, and these mice

were treated with IFNt (6 x 105 U) both orally and i.p. starting at the time of paralysis and at 48 h intervals thereafter. Both oral feeding and i.p. injection of IFNt protected mice against further relapses of disease following a reduced primary relapse; protection against primary relapse was 80% for i.p. injected IFNt, 65% via oral administration of IFNt, and 17% for the PBS treated control group (Table 1). No further relapses were seen for the IFN-treated groups while 8 of 18 mice had secondary relapses in the PBS group during the treatment period. Thus oral administration and i.p. injection of IFNT blocked further relapses into paralysis in mice afflicted with chronic EAE.












Table 1. Treatment of SJL mice with IFNt after induction of EAEa



Before treatment During treatment (64 days)

Disease Mean day Mean Primary relapsing Mean day Mean Secondary relapsing Treatment incidence of onset severity incidence of relapse severity %Protected incidenceb IFNt-orally 20/20 17.9 � 0.4 3.4 7/20 37.4 � 7.8 2.9 65 0/20 IFNt-i.p. 19/20 18.0 � 0.5 3.5 4/20 40.3 � 8.0 3.3 80 0/20 PBS 17/18 17.9 � 0.7 3.2 15/18 34.0 � 3.3 3.5 17 8/18


aSJL mice were immunized with MBP for induction of EAE. After the onset of disease (19 days after immunization), mice were injected intraperitoneally (i.p.) or orally fed with 6 x 10' U total of IFNt every 48 h for 64 days. Mean severity and incidence of disease were recorded throughout the study for mice which relapsed into disease. Mice were clinically examined daily for signs of EAE, and severity of disease was graded using the following scale: 1, loss of tail tone; 2, hind limb weakness, 3, paraparesis, 4, paraplegia; 5, moribund/death. Protection of mice from relapse by oral administration (p<0.005) and i.p. injections (p<0.001) of IFNt was statistically significant when compared to mice treated with PBS as determined by the X2 test.
bNo further relapses into paralysis were seen in mice treated with IFN.






17


Reduction and Prevention of Lymphocytic Infiltrates in EAE Mice by IFNt

Histology was performed to determine the extent of lymphocyte infiltration into the CNS of MBP immunized SJL/J mice afflicted with EAE before treatment and after treatment with IFNt both i.p. and orally. Sections of the lumbar spinal cord were evaluated for lymphocyte infiltration after staining with cresyl violet. As shown in Figure lA, perivascular accumulations of lymphocytes were present in the mouse spinal cord white matter prior to IFNt treatment. Then, after 38 days in the absence of IFNT, mice had lymphocytic infiltrates in their spinal cords (Figure 1B). In contrast, no lymphocytic infiltrates could be detected in mice treated with IFNt by oral feeding (Figure 1C) or i.p. injection (Figure ID) after 38 days. Thus, IFNt reduced and prevented further lymphocytic infiltration into the CNS, demonstrating that the protective effect of IFNr is associated with inhibition and reversal of lymphocyte infiltration of the CNS.



Deactivation of Microglia Following IFNt Treatment

Spinal cord sections from SJL/J mice with EAE (Figure 2A) were marked by

widespread and maximal microglial activation. This was evidenced by the fact that nearly all microglial cells within a given section had lost their ramified (branched) morphology and had transformed into brain macrophages appearing as enlarged rounded cells (Figure 2A). Following both oral and i.p. treatments with IFNT, sections immunostained with anti-CD1 lb antibody showed only ramified microglia (Figure 2B, Figure 2C). Rounded brain macrophages were no longer detectable. The processes of ramified cells present after treatment did stain strongly with anti-CD1 lb antibody, and they were somewhat thicker than those of resting cells in CNS tissue from naive animals. Thus, it is likely that while microglia were de-activated by IFNt treatment, they had not completely reverted back to the resting state, but had maintained low levels of activation.





















Figure 1. Histological evaluation of EAE mice before and after treatment with PBS and IFNt. SJL/J mice were immunized with MBP for induction of EAE and then treated with PBS or IFNt, as per Table 1. SJL/J treatment groups evaluated included mice before any treatment (A), PBS treatment (B), IFNt treatment by oral feeding (a normal blood vessel with no lymphocyte infiltration is presented for comparison) (C), and IFNr treatment by i.p. injection (D). Histology was evaluated after the induction of EAE 19 days after immunization in A, and 38 days after the initiation of the treatments (57 days after immunization) in B-D. Two mice per group from separate experiments were examined. Sections of spinal cord were stained with cresyl violet and presented at a final magnification of 400X.




















a V. s
It.mo
4.~iiii zz ~~ii
.4i~iiiiiii iiiii !!! i iii~ !i!i ii !
























Figure 2. IFNt causes de-activation of microglia. Spinal cord section from PBS treated SJL/J mouse with EAE shows fully activated microglial cells which exist as rounded brain macrophages (arrows in A). Oral feeding (B) and i.p. injection
(C) of IFNt caused microglia to assume a process-bearing (arrows) morphology indicating a reversion to the resting state. Sections were taken 38 days after initiation of the treatments (57 days after immunization), and two mice per group from separate experiments were examined. Sections of spinal cord were immunohistochemically stained for microglia and counterstained with cresyl violet and are presented at a final magnification of 1000X. Mice were from the PBS and oral and i.p. IFNt treated groups from Table 1.





21

















Ni,.














'IK
/ ski





22


IFNr Treated Mice Have a Lower Antibody Level Against MBP than Control Mice

During the course of IFNt treatment of SJI/J mice for chronic relapsing EAE, mice were bled and sera were examined for the presence of MBP-specific antibodies. As shown in Figure 3, mice that received i.p. injections of IFNr had the lowest anti-MBP antibody level followed by the orally fed mice. Untreated and PBS treated mice had the highest antiMBP antibody levels. Similarly, in the acute form of EAE, which was induced in NZW mice, anti-MBP antibody levels were also lower in the IFNt treated group than in the PBS treated group (Figure 4). Thus, IFNt inhibition of antibody production against MBP may be a contributing mechanism by which IFNt prevents development or inhibits further relapses of EAE in the acute and chronic forms of the disease, respectively.


IFNt Inhibited MBP-Specific B Cell Proliferation

Related to antibody production, we addressed the question of whether IFNt had any effect on B cell activation in response to MBP in culture. Splenocytes obtained from SJL/J mice that had been immunized with MBP for induction of chronic EAE were incubated with media or IFNt in the presence or absence of MBP for 48 hr. B cells were isolated using an immunity affinity column and then incubated with media alone for 72 h, after which proliferation was measured. As shown in Figure 5, B cells isolated from IFNt-treated SJL/J splenocyte cultures did not proliferate in the presence of MBP, while the media treated B cells did respond to MBP significantly. B cells that were isolated with the immunoaffinity column did not proliferate in response to the T cell superantigen staphylococcal enterotoxin B (SEB), but did proliferate in the presence of lipopolysacharide (LPS), a polyclonal B cell stimulator (Figure 5 inset). Thus, the proliferation seen in Figure 5 is from B cells and not from T cell contamination. Furthermore, splenocytes obtained from IFNt and PBS-treated NZW mice, which developed the acute form of EAE, were incubated in media alone or with IFNT in the presence or absence of MBP for 48 h, after which B cells were isolated and incubated with media alone for 72 h and proliferation























Figure 3. Inhibition of anti-MBP antibody production in the chronic form of EAE by IFNT. Sera from SJL/J mice were obtained before MBP immunization (normal mouse serum (NMS)), at the time of paralysis (before treatment), and 38 days after the initiation of treatment with PBS and IFNt i.p. and orally as per Table 1. Direct ELISA was performed to detect anti-MBP antibodies. Four mice per group were used, and average absorbance is shown. Statistical significance for the inhibition of anti-MBP antibody production was shown by Student's t-test at the 100 dilution factor for oral IFNT treatment (p<0.01) and i.p. IFNr treatment (p<0.001) as compared to the PBS treatment.








1.20
. Before treatment

1 .00 - - PBS
- - Oral IFNr 0.80 . I.P. IFNt


0
0.60



0.40



0.20



0.00
10 100 1000 10000 Dilution factor























Figure 4. Inhibition of anti-MBP antibody production in the acute form of EAE by IFNt. NZW mice were immunized with MBP for induction of acute EAE. Mice were given IFNt (6 X 10' U/treatment time) or PBS by i.p. injection at the time of, 48 h before, and 48 h after immunization. Sera were obtained from mice before immunization with MBP (normal mouse serum (NMS)), and three weeks after immunization from the PBS and IFNt treated groups. Direct ELISA was performed to detect anti-MBP antibodies. Pooled sera from four mice per group were used and average absorbance is shown. Statistical significance for the inhibition of antiMBP antibody production was shown by Student's t-test at 1000 and 3000 dilution factors between IFNt and PBS treatments (p<0.001).










3.0

*PBS
2.5
IFNr e 2.0 NIVMS


0 1.5


1.0


0.5 0.0
1000 3000 6000 10000 30000

Dilution factor

















Figure 5. IFNr inhibits MBP-specific proliferation of B cells from chronic EAE mice. SJL/J mice were immunized with MBP for induction of EAE. After three weeks, spleen cells were taken and treated with IFNT (3.0 X 104 U/ml) or media in presence or absence of MBP for 48 h in culture. B cells were isolated and reincubated (5 x 10 cell/well) in media alone for another 48 h. B cells from media treated splenocytes were also incubated with 200 ng/ml SEB or 50 jg/ml LPS for negative and positive controls of B cell isolation, respectively (figure inset). All cultures were pulsed with tritiated thymidine, and cell-associated radioactivity was quantified 18 h later, and data from one of three respective experiment are presented as mean CPM of quadruplicate wells � SD. IFNt's inhibition of MBP-specific B cell proliferation was statistically significant as compared to the MBP-specific B cell proliferation of MBP sensitized media treated cells as shown by the Student's t-test (p<0.001).











2500
80006000
2000
0 40001500- 2000 0
00
1000_ Media SEB LPS



500





Media Media+MBP IFNt IFNr+MBP Treatment






29


measured. As shown in Figure 6, EAE B cells isolated from MBP-treated splenocytes that were taken from a PBS-treated NZW mouse had a stimulation index greater than 11 relative to media control, while EAE B cells isolated from IFNt-treated splenocytes did not proliferate in response to MBP. Furthermore, EAE B cells isolated from MBP-treated splenocytes that were taken from an IFNt-treated NZW mouse did not proliferate either. Thus, IFNT can inhibit activation of B cells, taken from mice having either acute or chronic EAE, in response to MBP both in vivo and in vitro, consistent with reduction of antibody production to MBP in IFNt treated EAE mice.



IFNT Inhibited MBP-Specific T Cell Proliferation

Previously, we have shown that IFNe inhibits MBP-specific splenocyte proliferation (4) by inducing CD4 T cell to produce IL-10 and TGF3 which act synergistically to inhibit EAE splenocyte activation (Mujtaba et al., 1997). Here we determine the effect of IFNT on proliferation of T cells isolated from MBP-sensitized SJL/J mouse splenocytes stimulated with MBP and treated with IFNT or media in culture. At both IFNt concentrations, MBP activation of MBP-specific T cell were inhibited, and at 30,000 U/ml, IFNt reduced levels of proliferation to less than 50% of that observed in response to MBP alone (Figure 7). The T cells that were isolated with the immunoaffinity columns did not proliferate in response to LPS, but did proliferate in presence of PMA and ionomycin, which are T cell activators when used in combination (Figure 7 inset). Therefore, the proliferation seen in Figure 7 is from T cells and not from B cell contamination. Thus, IFNt inhibits the activation of MBP-specific effector T cells of chronic EAE mice.

















Figure 6. IFNt inhibits MBP-specific proliferation of B cells from acute EAE mice. NZW mice were immunized with MBP for induction of acute EAE. Mice were given IFNt (6 X 105 U/treatment time) or PBS by i.p. injection at the time of, 48 h before, and 48 h after immunization. After three weeks, splenocytes were taken from the PBS and IFNt groups and treated with IFNt (3.0 X 104 U/ml) or media in the presence or absence of MBP for 48 h in culture. B cells were then isolated and reincubated (5 x 10' cell/well) in media alone for another 48 h after which the cultures were pulsed with tritiated thymidine. Cell-associated radioactivity was quantified 18 h later using a 1-scintillation counter, and data from one of three respective experiment are presented as mean CPM of quadruplicate wells � SD. Inhibition of B cell proliferation with both in vitro and in vivo IFNT treatments were statistically significant when compared to the MBP treatment alone as shown by Student's t-test (p<0.001).











7000 60005000


4000O.

3000


20001000



EAE mice treated with: PBS PBS PBS IFN In vitro treatment for a cell proliferation: Media Media + MBP IFNt + MBP Media + MBP



















Figure 7. IFNt inhibits MBP-specific T cell proliferation. SJL/J mice were immunized with MBP for induction of EAE. After three weeks, spleen cells were taken and treated with IFN (3.0 X 104 U/ml) or media in the presence or absence of MBP for 48 h in culture. T cells were isolated and reincubated (5 x 105 cell/well) in media alone for another 48 h. T cells from media treated splenocytes were also incubated with 50 gag/ml LPS or with 20 ng/ml PMA and 200 ng/ml ionomycin for negative and positive controls of T cell isolation, respectively (figure inset). All cultures were pulsed with tritiated thymidine, and cell-associated radioactivity was quantified 18 h later, and data from one of three respective experiment are presented as mean CPM of quadruplicate wells � SD. MBP-specific T cell proliferation of both IFNt concentrations were statistically significant from the MBP-specific T cell proliferation of MBP treated cells alone as shown by the Student's t-test (p<0.001).










20000
800002 60000 16000- 0000
40000 20000 12000Media LPS lonomycin 8000- +PMA


4000


0
Media MBP MBP+IFNr MBP+IFNt 15,000U/ml 30,000U/ml Treatment






34



Discussion


The findings reported here clearly show that ongoing chronic, relapsing EAE in

SJL/J mice can be significantly alleviated by either parenteral or oral administration of IFNt on a continuous basis. Although some mice in both IFNT treatment groups had primary relapsing incidence of paralysis during treatment, further relapses were not detected unlike the PBS treated group. Parenteral administration of IFNt was slightly more effective in alleviating chronic EAE than oral administration in that only 4 of 20 mice had relapses while 7 of 20 mice had primary relapses in the orally treated group. Furthermore, untreated EAE mice showed lymphocyte infiltration of the CNS and activation of microglia, whereas, IFN, treatment of mice with active EAE reversed these cellular effects. This suggests that IFN, treatment results in relief or permanent remission of the observed EAE symptoms.

Although considerable attention is usually given to T-helper cell-mediated events in EAE, we show that anti-MBP antibody and MBP-specific B cell effects, like the T cell effects, are inhibited by IFN in both chronic and acute forms of EAE. MBP antibody production in EAE mice was significantly inhibited by IFN-t, with i.p. administration of IFN, more effective than oral, and MBP induced proliferation of sensitized B cells was also blocked. IFNt inhibition of B cell proliferation probably plays a central role in inhibition of anti-MBP antibody production. We have shown that IFNt and other type I IFNs induce terminal differentiation of Daudi B cells to plasma cells (Subramaniam et al., 1998). Thus, IFNT inhibition of B cell clonal expansion by induction of terminal differentiation, which results in an overall lower plasma cell number, probably plays a central role in IFNt inhibition of production of antibodies to MBP in EAE mice. Overall, we have demonstrated that IFNt is an effective therapy for ongoing EAE and as such should have potential for treatment of MS in humans.














CHAPTER 3
CD4 T SUPPRESSOR CELLS MEDIATE IFNt PROTECTION AGAINST EAE


Introduction


Previously, we showed that interferon tau (IFNz) blocks the development of

experimental allergic encephalomyelitis (EAE) in mice without associated toxicity; however the mechanism of such action has not been fully elucidated (Soos et al., 1995a). EAE is a murine model useful for studying the demyelinating disease multiple sclerosis (MS) (Zamvil and Steinman, 1990). Myelin basic protein (MBP) has been shown to be one of the primary central nervous system antigens responsible for induction of autoimmunity in the EAE model. Upon immunization with MBP, mice develop clinically observable tail and limb paralysis due to lymphocyte infiltration into the central nervous system accompanied by acute demyelination (Zamvil and Steinman, 1990).

The type I IFNs, a and p, have previously been shown to induce suppressor cells that block in vitro antibody production (Johnson and Blalock, 1980). Further, when type I IFN-induced suppressor cells were cultured in vitro, they were shown to produce a soluble factor that mediated immunosuppression. Past studies by others suggested that "classic" T suppressor cells bear the CD8 phenotype. Here we demonstrate that IFNr-induced suppressor cells bear the CD4 phenotype, and these cells mediate the amelioration of EAE by IFNr. In addition, IFNT-induced suppressor cell function occurs via a mechanism similar to that originally observed for type I IFNa and P inhibition of antibody production in vitro. A suppressor mechanism shared by the type I IFNs is the induction of soluble suppressor factors, which we demonstrate in this study. These findings serve as the basis





35






36


for understanding how type I IFNs exert therapeutic effects in autoimmune diseases such as MS.


Materials and Methods

IFNs

The ovine IFNt (IFNt) gene was expressed in Pichia pastoris using a synthetic

gene construct, which was kindly provided by Dr. Gino Van Heeke, Ciba Pharmaceuticals, London, England (Heeke et al., 1996). IFNr was secreted into the medium and was purified by successive DEAE-cellulose and hydroxylapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining analysis. The purified protein had a specific activity of 2.9 to 4.4 x 10' U/mg protein as measured by antiviral activity using a standard viral microplaque reduction assay on MDBK cells (Pontzer et al, 1991). MulFNP (specific activity 4.1 x 10' U/mg) was obtained from Lee Biomolecular (San Diego, CA).



Antibodies and Cytokines

Monoclonal rat anti-mouse IL-10, recombinant mouse IL-10, and monoclonal mouse anti-TGF-p11,-,,-23 were obtained from Genzyme, Cambridge, MA. Ultrapure natural human TGFD,, which shows cross-reactivity in most mammalian cell types, was also obtained from Genzyme. A 1:10 dilution of HL100, a specific monoclonal antibody for IFNt, was used to neutralize 5000 U/ml of IFNr prior to usage. The HL100 monoclonal antibody was kindly provided by Dr. Carol Pontzer, University of Maryland, College Park, MD. All antibodies and cytokines were used in proliferation assays described below.






37

Interferon Induction of Suppressor Cells

Suppressor cells were induced both in vitro and in vivo. For in vitro induction,

NZW mouse spleen cells (5.0 x 107/ml) were incubated with 5000 U/ml of IFNt for 24 h at 370C, after which the cells were washed twice prior to use. In vivo induction of suppressor cells in naive NZW mice involved administration of a single dose of IFNT (10 U) either intraperitoneally (i.p.) or by oral feeding with PBS used as the vehicle for administration. After 24 h, mice were sacrificed and the spleens removed. Spleen cells were washed and resuspended in RPMI 1640 medium supplemented with 2% fetal bovine serum and used as described below.


Induction of EAE

For induction of EAE, 300 jtg of bovine MBP (MBP) were emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml H37Ra (Mycobacterium tuberculosis, Difco, Detroit, MI) and injected into two sites at the base of the tails of NZW mice. On the day of immunization and 48 h later, 400 ng of pertussis toxin (List Biologicals, Campbell, CA) were also injected. Mice were clinically examined daily for signs of EAE, and severity of disease was graded using the following scale: 1, loss of tail tone; 2, hind limb weakness, 3, paraparesis, 4, paraplegia; 5, moribund/death.


Adoptive Transfer of Suppressor Cells

Suppressor cells were induced in vitro with IFNt as described above and resuspended in phosphate buffered saline (PBS). NZW mice were injected intraperitoneally with 100 pl. of PBS containing 5 x 106 suppressor cells 48 h before, on the day of, and 48 h after immunization with MBP for induction of EAE. Mice were examined daily for signs of EAE, and the severity of disease was graded as noted above.






38


CD4 T Cell Isolation and Depletion

CD4 T cells effects were examined using both positive and negative CD4 T cell selection processes. The Cellectplus mouse CD4 kit (Biotex Laboratories, Inc., Alberta, Canada), an immuno affinity column, was used to isolate CD4 cells from NZW mouse spleen lymphocyte cultures treated with media or IFNt. Depletion of CD4 T cells from mouse spleen lymphocyte cultures treated with IFNt or media was carried out using rat anti-mouse L3/T4 CD4 monoclonal antibody (Biosource International, Camarillo, CA) and Low-Toxic-M rabbit complement (Accurate Chemical and Scientific Corporation, Westbury, NY). Lymphocytes from NZW mouse spleen were resuspended at 10' cells/mi in RPMI 1640 medium and incubated with 1:10 dilution of anti-mouse L3/T4 CD4 antibody for 1 h at 4o C. Cells were then centrifuged and resuspended in 1:10 dilution of rabbit complement in RPMI 1640 medium for 1 h at 370C. The cultures were washed and used for further experimentation.



Production of Suppressor Factor

Suppressor cells were generated in vitro by incubating spleen cells with 5000 U/ml of IFNt for 24 h at 370C as described above. Cells were then washed and resuspended at 108 cells/ml in fresh culture medium. After incubating for an additional 2 h at 370C, clarified supernatants were collected and tested for suppressor activity.


Proliferation Assay

Spleen cells from MBP-immunized NZW mice (2.5 - 5.0 x 10' cells/well) were

cocultured with IFNr- or IFNp-induced suppressor cells (1.0-5.0 x 10'/well), suppressor cell supernatants, or IL-10 and TGF in the presence of 30 or 100 gg/ml of MBP. Suppressor cell supernatants were also pretreated for 2 h with either anti-IL 10 antibody (25 gg/ml) or anti-TGF antibody (25 jgg/ml ) and then cultured with MBP-specific cells in the presence of MBP. Cultures were incubated for 96 h at 370 C. The cultures were then






39


pulsed with [3H]-thymidine (1.0 gCi/well; Amersham, Indianapolis, IN) 18 h before harvesting onto filter paper discs using a cell harvester. Cell-associated radioactivity was quantified using a P-scintillation counter. Stimulation index was determined by dividing experimental CPM by control (unstimulated) CPM.


Results


IFNt-Treated Spleen Cells Inhibit MBP-Specific T Cell Proliferation

We first addressed the question of whether IFNt can suppress MBP-specific T cell proliferation by induction of suppressor cells in NZW mouse spleen cells. Spleen cells were treated with IFN in tissue culture or were obtained from mice injected intraperitoneally (i.p.) with IFNt, or from mice treated orally with IFNt. IFNt-treated spleen cells from all three sources inhibited MBP induced proliferation of spleen cells from EAE mice by as much as 80% relative to the control response (Figure 8). Similar to type I IFN induction of suppressor cells for antibody production (Johnson and Blalock, 1980), IFNt suppressed MBP-specific immune response via induction of suppressor cells.


IFNt Induction of Suppressor Cells are Dose-Dependent

IFNt and IFNP were compared at various concentrations for induction of

suppressor cells in spleen cell cultures for inhibition of MBP stimulation of sensitized cells from EAE mice (Figure 9). For both IFNs, the induction of suppressor cells was dose dependent. IFNP was slightly more effective at induction of suppressor cells, but the slopes of the dose response curves for the two IFNs were similar. Thus, Type I IFN induction of suppressor cells is dose-dependent.





















Figure 8. IFNt-treated spleen cells inhibit MBP-specific T cell proliferation. Suppressor cells were induced with IFNt both in vitro and in vivo. IFNt-treated spleen cells were washed and cocultured at 1.0 x 105 cells/well with MBP-specific mouse spleen cells (2.5 x 105 cells/well) and MBP protein at 100 gg/ml for 96 h. Media-treated spleen cells served as controls. Data from one of three representative experiments are presented as mean CPM � S.D. of quadruplicate cultures. Proliferation was measured by [ H]-thymidine incorporation. IFNt suppressor cells induced by all three methods showed significant suppression by X2 test with p < 0.001 relative to MBP-stimulated controls.












12000100008000


. 600040002000



None None Tissue culture Intraperitoneal Oral feeding

MBP: - + + + + IFNt treatment





















Figure 9. IFNt induction of suppressor cells is dose-dependent. NZW spleen cells (5 x 107 cell/ml) were treated with IFNt and IFNI3 at various concentrations for 24 h in vitro. IFN-treated cells (5 x 105 cells/well) were then cocultured with MBP-specific cells (2.5 x 105 cells/well) in the presence of 100 jgg/ml of MBP protein. Proliferation was measured by [3H]-thymidine incorporation. Data from one of three representative experiments are presented as mean stimulation index � S.D. of quadruplicate cultures. Coculture of MBP-specific cells and media-treated cells had a stimulation index of 7.5 � 3.3. The CPM for unstimulated cells were 405 � 97.













5.0


--0-- IFNr treated cells
4.0
4.0- ...... ....... IFNp treated Cells



3.0



2.0 1.0 0.0
10 100 1000 10000 IFN Concentration (U/ml)





44




IFNz Suppressor Cells Protect Mice Against EAE

Adoptive transfer of IFNt-induced suppressor cells to NZW mice immunized with bovine MBP was carried out in order to determine if the suppressor cells protected the mice from development of EAE. NZW mice have previously been shown to be susceptible to development of EAE after immunization with either rat MBP (Zamvil et al., 1994; Kumar et al., 1994) or bovine MBP (J. Schiffenbauer, unpublished observation). Others have shown the transfer of peripheral cells from orally administered IFN donor mice to recipient mice causes suppression of white blood cells (Fleischmann et al., 1992). Suppressor cells induced in culture with IFNt were injected i.p. 48 h before, at the time of, and 48 h after immunization of mice with MBP. Suppressor cell-treated mice showed delayed onset of EAE (34.3 days) compared to untreated controls (19.6 days), and the incidence of EAE was 3 of 5 with lower severity of disease for suppressor cell-treated mice compared to 5 of 5 with higher severity of disease for untreated mice (Figure 10). Thus, adoptive transfer of IFNt induced suppressor cells significantly protected mice against EAE.



IFNT-Induced Suppressor Cells are CD4 T Cells

We next determined the phenotype of the suppressor cells by using antibody

affinity columns to purify CD4 T cells and using specific CD4 antibody and complement to deplete CD4 T cells from IFNt-treated cultures (Figure 11). CD4 T cells purified from an IFNt-treated spleen cell preparation inhibited MBP-specific T cell responses by almost 50%, while non-CD4 T cultures from IFNt-treated spleen cells were without effect. The non-CD4 T cell preparations consisted of CD8 T cells, macrophages, and other cells. Thus, the suppressor cell appears to be a CD4 T cell.




















Figure 10. IFNT-induced suppressor cells can delay the onset of EAE in mice. NZW mice were injected i.p. with IFNt-treated whole spleen cells (5x106) 48 h before, on the day of, and 48 h after immunization with bovine MBP for induction of EAE. Mice were followed daily for signs of EAE, and mean severity of paralysis for each group was graded based on the scale mentioned in the materials and methods. Control mice had a average severity of 2.8, while adoptive transferred mice had a severity of 2.0. Mean day of onset of paralysis for the control and suppressor celltreated mice were 19.6 � 2.6 and 34.3 � 2.3 days, respectively. The delay of onset of paralysis was statistically significant as shown by student's t-test (p < 0.001).














3.0
----- No treatment ....... ....... Suppressor cell treated


h 2.0Incidence=5/5

Incidence=3/5
1.0



0.0.


10 20 30 40 50 60 70 Days after treatment





















Figure 11. IFNT-induced suppressor cells are CD4 T cells. NZW mouse spleen cells were treated with media or IFNt for 24 h in vitro. CD4 T cells were isolated from IFNt-treated cell cultures using an immunoaffinity column. CD4 T cells were depleted from a second set of IFNT-treated cells using anti-CD4 antibody plus complement. Whole and fractionated spleen cells (2.5 x 10) were cocultured with MBP-specific cells (2.5 x IO cell/well) in the presence of 30 gg/ml of MBP. Proliferation was measured by ['H]-thymidine incorporation. Data from from one of two representative experiments are presented as mean stimulation index � S.D. of quadruplicate cultures. Cocultures of MBP-specific cells and fractionated media-treated cells had similar stimulation indices as those with whole unfractionated cells. Statistical significance by x2 test for CD4 T cell suppression was p<0.001 relative to the media treated spleen cell control. The CPM for unstimulated cells were 1003 � 183.













3.0 2.5



a 2.0



1.5 00 | 1.0



0.5



0.0 N
Media treated IFNT treated IFNr treated IFNr treated spleen cells spleen cells Non-CD4 T cells CD4 T cells





49



Suppressor Cells Produce Soluble Suppressor Factor(s)

We previously showed that type I IFN-treated cells produced a suppressor factor for production of antibody to sheep red blood cells (Johnson and Blalock, 1980). IFNttreated cells were thus examined for production of a suppressor factor. As shown in Figure 12A, supernatants from IFNt-treated spleen cells that had been incubated for 2 h at 370 C inhibited MBP-specific T cell responses. Inhibitory supernatants were not produced by cells treated with IFNt that had been neutralized with specific antibody prior to treatment of cells. Further, the antibodies did not inhibit suppressor cell activity when added to cells after treatment with IFN. Consistent with the CD4 T cell phenotype of the suppressor cell, supernatants from IFNt-treated CD4 T cells suppressed the MBP-specific responses (Figure 12B). Thus, the IFNt-induced CD4 suppressor T cell produces soluble suppressor factor(s).


IFN',-Induced Suppressor Cells Produce IL-10 and TGF

We next characterized the suppressor factors that IFNt induced in spleen cells using antibodies to IL-10 and TGFO. As shown in Figure 13A, both monoclonal anti-IL-10 and monoclonal anti-TGFP antibodies blocked the suppressive activity of the suppressor cells on MBP-specific T cell responses. Similarly, both anti-IL-10 and anti-TGFP antibodies neutralized the suppressive activity of supernatants from IFNT-induced suppressor cells on the MBP-specific T cell responses (Figure 13B). Not unexpectedly, the antibodies to IL10 and TGFP combined showed complete recovery as did each antibody showed separately (data not shown). Also, addition of the corresponding cytokines in excess reversed the blockage of suppression by the antibodies. The control monoclonal anti-IFNt antibody had no effect on the suppressor activity of the suppressor cells or their supernatant. Thus, both anti-IL-10 and anti-TGFp antibodies restored the MBP-induced response to that of the




















Figure 12. IFNt-induced CD4 T suppressor cells produce soluble suppressor factor(s). NZW mouse spleen cells were treated with media or with 5000 U/mI IFNt in the presence and absence of neutralizing antibody to IFNt (mAb HL-100) (panel A). CD4 T cells were isolated from media and IFNt-treated cultures (panel B). After washing, the cells were incubated for 2 h in media, and supernatants were collected. Supernatants were incubated with MBPspecific mouse spleen cells (5.0 x 105 cells/well) in the presence of 30 gg/ml of MBP protein. Proliferation was measured by [3H]-thymidine incorporation. Data from one of two representative experiments are presented as mean stimulation index + S.D. of quadruplicate cultures. In panel A, statistical significance by x2 test for suppressive activity of supernatants from cells pretreated with IFNt was p < 0.001. In panel B, statistical significance by X2 test for suppressive activity of supernatants from CD4 T cells pretreated with IFNt was p < 0.001. The CPM for the unstimulated cells were 138 � 36 in panel A and 262 + 52 in panel B.












7.0 - 3.5 A B
6.0- - 3.0


5.0- - 2.5


S4.0- -2.0


S3.0- 1.5 2.0- - 1.0


1.0- - 0.5


0.0 0.0
Media Ab-neutralized IFNc ' Media IFNt Media IFNr treated IFNt treated pretreated treated treated treated treated
spleen treated spleen cells spleen cells spleen cells spleen cells CD4 T cells CD4 T cells
cells spleen cells +anti-IFNe
Source of supernatants



















Figure 13. Blockage of IFNt-induced suppressor cell and suppressor supernatant effects on MBP stimulation of sensitized EAE mouse spleen cells with monoclonal antibodies to IL-10 and TGFP. Media-treated spleen cells and suppressor spleen cells (3.0 x 10' cells/well) induced in vitro with IFNt (panel A) and their supernatants (panel B) were each cocultured with MBP-sensitized spleen cells (5 x 105 cells/well) in the presence of 30 rtg/ml of MBP. AntiIL-10 (25 jgg/ml) and anti-TGFP (25 jig/ml) antibodies were added at initiation of cultures. Proliferation was assessed after 96 h by [3H]-thymidine incorporation. Data from one of two representative experiment are presented as mean stimulation index � S.D. of quadruplicate cultures. The effects of anti-IL-10 and anti-TGF3 antibodies on media treated cells were similar to those of media controls shown. In panel A, blockage of the suppressive activity of the suppressor cells by both antibodies was statistically significant (p < 0.001) as determined by the x2 test. In panel B, blockage of the suppressive activity of the suppressor cell supernatant by both antibodies was statistically significant (p < 0.001) as determined by the X2 test. The CPM for the unstimulated cells were 4843 � 130 in panel A and 2716 + 18 in panel B.













6.0- 16.0

A B 14.0
5.0

- 12.0

4.0
0- 10.0


3.0- - 8.0


- 6.0
2.0

4.0

1.0
2.0


0.0 0.0
Media Suppressor Suppressor Suppressor Media Suppressor Suppressor Suppressor treated cells cells cells supernatant supernatant supernatant
cells + anti-IL-10 + anti-TGF3 + anti-IL-10 + anti-TGF8






54


control, which suggests a synergistic interaction between IL-10 and TGFP in inducing suppression.


IL-10 and TGF5 Act Synergistically to Inhibit MBP-Specific T Cell Responses

We next evaluated the possible synergistic interaction between IL-10 and TGFP on suppression of MBP-induced mouse spleen cell proliferation. As shown in Figure 14, both IL-10 and TGFP suppressed MBP-specific T cell responses individually, but 1-10 and TGFP together enhanced the suppression of MBP-sensitized spleen cell proliferation in response to MBP. Both IL-10 and TGFp at a concentration of 8 ng/ml each, greatly reduced MBP-specific responses, compared to that obtained at 16 ng/ml of each factor. Thus, the combined effects of IL-10 and TGFP are apparently not additive. These data suggest that 1L-10 and TGFP act synergistically at certain concentrations to inhibit MBPinduced EAE spleen cell proliferation.



Discussion



Data presented here demonstrate that IFNT induces CD4 T cells to become

suppressor cells in NZW mice by oral administration or intraperitoneal injection of IFNt, and by treatment of mouse spleen cells with IFNt in tissue culture. The suppressor cells inhibit MBP stimulation of spleen cells from MBP-immunized mice, and protect mice against induction of EAE. Also, the CD4 T suppressor cells produce both 1L-10 and TGF3, which act synergistically to inhibit MBP-specific T cell proliferation. Induction of suppressor cells can be blocked by pretreatment but not posttreatment of IFNt with neutralizing antibodies, thus establishing that induction of suppressor cells is specific for IFNt, but is not itself IFNt. Therefore, IFNt inhibition of EAE appears to occur via induction of suppressor cells and their suppressor factors such as IL-10 and TGFB3.




















Figure 14. IL-10 and TGF3 act synergistically to inhibit MBP-specific T cell responses. Varying concentrations of IL-10 and TGFP individually and together were preincubated with MBP-sensitized mouse spleen cells (5 x 105 cells). After 2 h, the cells were stimulated with 30 gg/ml of MBP and incubated for 96 h. Proliferation was assessed by ['H]thymidine incorporation. Data from one of two representative experiments are presented as CPM � S.D. of quadruplicate cultures. The CPM for unstimulated media treated cells were 6310 � 911.













100000

---I---IL-10

TGF3

-0- TGFD+IL-10 1000,
E




1000






1 0 0 *.*. . . . * *. . . ..* , * . . . ..* * * . . . . . .*
0.0 0.1 1.0 10.0 100.0


Concentration (ng/ml)






57


These finding are consistant with our previous observation that orally administered IFNr protected mice against EAE in the absence of detectable IFNT in the circulation (Soos et al., 1997).

The induction of suppressor cells is not unique to IFNT, as IFNP also induced suppressor cells in spleen cell cultures. Further, the dose response curves for the two IFNs were similar. Also, these suppressor cells produce suppressor factors that inhibit MBP stimulation of EAE spleen cells. Thus, it is quite likely, then, that type I IFNs in general protect against autoimmune diseases such as MS by induction of suppressor cells and suppressor factors.

As indicated above, IFNt protected mice against EAE when administered orally

even though relatively little IFNt was found in the circulation (Soos et al., 1997). The gut is lined with over half of the cells of the immune system. The suppressor cells induced by oral IFNt administration must be mobile, since the autoreactive MBP-specific T cells that are inhibited are themselves mobile, and in fact migrate to the central nervous system to cause EAE in the absence of IFNt treatment. We have shown that IFN--treated mice that are immunized with MBP show little or no lymphocyte infiltration of the CNS (Soos et al., 1997).

The CD4 suppressor T cell produced both IL-10 and TGFP that acted

synergistically to inhibit MBP stimulation of spleen cells from EAE mice. IL-10 and TGF3 have previously been shown to inhibit events associated with autoimmune disease (Chaouat et al., 1995; Rott et al., 1994; Stevens et al., 1994; Johns et al., 1991; Schluesener and Lider, 1989). We have shown that IL-10 can be detected in sera of mice which received prolonged i.p. injections or prolonged oral feeding of IFNt (Soos et al., submitted). Here we have also demonstrated that IFNt-induced suppressor cells produce 1L-10 and TGFp to synergistically inhibit MBP-specific T cell proliferation. Thus, the findings here serve as the basis for understanding how type I IFNs exert therapeutic effects in autoimmune neuropathies.














CHAPTER 4
CONCLUSION



IFNt, a recently discovered IFN, is a type I IFN that has pregnancy recognition hormone activity in ruminants. It possesses similar activities observed for the other type I IFNs, IFNa and IFND, but in contrast, IFNt lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in animal studies. Considering the positive therapeutic value of the related IFNP for treatment of MS, IFNt has been examined for its ability to prevent the development of EAE, the animal model for MS. IFNt has been previously shown to prevent the development and superantigen-induced exacerbation of EAE in the absence of toxicity (Soos et al., 1995a). These studies of IFNt protection against EAE involved initiation of IFN treatment before MBP immunization or before disease development. IFNt protected against both acute and chronic, relapsing EAE in mice, however, administration of IFN did not block sensitization, since cessation of treatment resulted in development of EAE (Soos et al., 1997). In order to have potential for treatment of MS in humans, the IFNt must be effective in treatment of active EAE.

In this study we show that both oral administration and ip injection of IFNT induced remission in SJL/J mice that had ongoing chronic active EAE disease and protected mice against secondary relapses. IFNt treatment reversed lymphocyte infiltration and microglial activation in the CNS. IFNt inhibition of antibody production against MBP may be a contributing mechanism by which IFNt inhibits further relapses of EAE. proliferation of effector B cells and T cells of EAE mice are inhibited by IFNt in both chronic and acute forms of EAE. Inhibition of MBP-specific T cell clones and reduced B cell responses could contribute to the reversal of disease and histopathological changes shown. Furthermore, IFNt can prevent EAE by induction of suppressor cells. Injection of these


58






59


suppressor cells into mice delayed the onset of EAE. The suppressor cells were found to produce the inhibitory cytokine IL-10 and TGF, which acted synergistically to inhibit MBP activation of T cells from EAE mice. This CD4 T suppressor cell is most likely the Th2 type based on the detection of TGFp and IL-10 in suppressor cell supernatants. Further, since this suppressor cell is induced by IFNt and probably also by other type I IFNs in the absence of MBP, it is most likely to be antigen-nonspecific in its effect. In fact, preliminary data suggest that suppressor cell supernatant inhibits mitogen stimulation of mouse spleen cells, and superantigen induced effects were similarly suppressed by CD4 T suppressor cells (Figure 15) and their supernatant (Figure 16) via IL-10 and TGF3. There was no evidence that non-CD4 T cells, including CD8 cells, possessed suppressor cell activity. This observation is in contrast to some other studies on suppressor cells (Nouri et al., 1991; Mukasa et al., 1994; Blank et al., 1995; Castedo et al., 1993). Other studies have also shown that the immune response is suppressed by antigen-specific CD4 Th2 cells (Karpus and Swanborg, 1991; Nabozny et al., 1991; Martinotti et al., 1995; Smith et al., 1991).

Additional potential mechanisms for IFNT prevention of EAE could include altered cell migration into the CNS, as well as downregulation of MHC I and II molecules and certain co-stimulatory/adhesion molecules. We have previously shown that type I IFNs inhibit the progression of Daudi B cells through the G 1 phase of the cell cycle (Subramaniam et al., 1998). Therefore, the inhibition of the cell cycle of cells in G 1 may be a mechanism by which IFNT could inhibit MBP-specific antibody production directly. Thus, other lines of investigation remain to be explored to completely understand the mechanism by which IFNt can prevent EAE.

Others have seen similar inhibition of clinical disease and reversal of

histopathological changes with IFNp used as the treatment for EAE mice (Yu et al., 1996). Furthermore, treatment of MS patients with IFNB in vivo as well as treatment of T cells




















Figure 15. IFNt induction of CD4 suppressor T cells inhibit SEA stimulation of naive spleen cells. Mouse spleen cell preparations were either pretreated with media or IFNt for 24 h. (B) CD4 T cells were separated from both media and IFNr-treated cultures using an affinity immunocolumn. (C) Also, the CD4 T cells were deleted from other media and IFNt-treated spleen cell prepartions using specific CD4 monoclonal antibody and complement. Naive mouse spleen cells were incubated with superantigen and the media or IFN-treated unfractionated cell preparation, the CD4 T cell fraction, or the non-CD4 T cell fraction. Proliferation was measured in CPM 114h later.














200 30 50
A: Unfractionated cells 1B: CD4 T cell fraction C: Non-CD4 T cell fraction


Media
25
E2 IFNr 40150

20
0
O
0 30
X
100- 15.
o 20 10

50
10 5M




0 0- 0
Media SEA Media SEA Media SEA




















Figure 16. IFNr treated CD4 T cells produce supreesor factor(s) which inhibit SEB stimulation of naive spleen cells. Mouse spleen cell preparations were either pretreated with media or IFNT for 24 h. CD4 T cells were separated from both media and IFNt-treated cultures using an affinity immunocolumn. Also, the CD4 T cells were deleted from other media and IFNt-treated spleen cell prepartions using specific CD4 monoclonal antibody and complement. Cells were culture in media for 24h and supernatants were collected from various prepations. Naive mouse spleen cells were incubated with superantigen and the media or IFNz-treated unfractionated cell supernatant preparation, the CD4 T cell supernatant fraction, or the non-CD4 T cell supernatant fraction. Proliferation was measured in CPM 114h later.












40000 35000U Media-treated 30000- El IFNT-treated 25000

20000 15000 10000

5000


Media CD 4 T cells Non-CD 4 T cells
SEB: . .

Source of supernatant






64


in vitro resulted in an inhibition of T cell activation (Noronha et al., 1993; Rudick et al., 1993). But, it was previously shown that the type I IFNs murine IFNa and murine IFNP induced toxic side effects manifested as flu-like symptoms, fever, nausea and malaise when used as a therapeutic in humans (Degre, 1974; Fent and Zbinden, 1987). Although we have not focused on the lack of toxicity of IFN' for the mice in these studies, we have previously shown such lack of toxicity in tissue culture and in mice by monitoring for weight loss and bone marrow suppression (Soos et al., 1995a; Soos et al., 1997).

The study here involved the use of ovine IFNT. There has not been a successful expression of a human IFNt with similar properties like those of ovine IFNt. Therefore, current studies are focused on "humanizing" the ovine IFNt by construction and expression of an ovinelFNt/human IFNaD chimeric. The chimeric is made up of residues 1-27 of the ovine IFNt and residues 28-166 of the human IFNaD, and differs from human IFNaD by 15 residues. The chimeric was constructed based on studies showing that the N-terminus of type I IFNs played a central role as to their toxicity or lack thereof (Pontzer et al., 1994; Subramaniam et al., 1995). These recent studies show that the IFNt/IFNaD chimeric lacks the toxicity associated with IFNaD with human PBMC and mouse splenocytes (Mujtaba et al. 1999). Preliminary data show that the IFNt/IFNaD chimeric also inhibits MBP stimulation of MBP-sensitized spleen cell (Table 2). The IFNt/IFNaD chimeric suppressed proliferation more effectively than IFNT but not as effectively as IFNaD. Viabilities were determined and showed that the IFNaD was the most toxic as compared to IFNt and the IFNt/IFNaD chimeric. Thus, the IFNt/IFNaD chimeric may be a better therapeutic for use in human diseases. Overall, the finding reported here indicates that IFNt is an effective treatment for ongoing active EAE, and this amelioration of disease is mediated by suppressor cells and their synergistically acting suppressor factors such as IL-10 and TGFp.






65












Table 2. IFN inhibition of MBP-sensitized spleen cells


IFN MBP-induced Proliferation Cell Viability (U/ml) (% Inhibition) (%)

IFNT (7000 U/ml) 21 � 6.6 91 IFNt (15000 U/ml) 42 � 2.0 89 IFNt (3000 U/ml) 57 � 6.1 85



IFNt/IFNaD chimeric (7000 U/ml) 57 � 2.4 86 IFN't/IFNaD chimeric (15000 U/ml) 58 � 1.1 81 IFNTIJFNaD chimeric (30000 U/ml) 73 � 1.3 81



IFNaD (7000 U/ml) 60 + 2.4 84 IFNaD (15000 U/ml) 75 � 2.3 83 IFNaD (30000 U/ml) 76 + 4.6 76 CPM and cell viability values for media-treated spleen cell cultures were 2710 + 77 and 93%, respectively.















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BIOGRAPHICAL SKETCH



Mustafa Ghulam Mujtaba was born in Kabul, Afghanistan, on September 18,

1973, to Ghulam and Uzra Mujtaba. During the war with the communist Soviet Union, the Mujtaba family left Afghanistan for the neighboring country, Pakistan, in 1983 and then to Cape Coral, Florida in June 1984. Mustafa completed his middle school in Cape Coral. The Mujtaba family then moved to Lake City, Florida in 1988 after his father had a job transfer. He graduated from Columbia High School in 1992. Mustafa attended college at the University of Florida majoring in Microbiology and Cell Science. In 1995, he received his Bachelor of Science degree in Microbiology and was accepted by the same department as a graduate student. He was kindly taken into the laboratory of Dr. Howard Johnson. After completion of his doctoral program, Mustafa plans to pursue research in a similar area as a postdoctoral fellow.























78











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. Johnsonhair Graduate Research Pofessor of Microbiology and ell Science


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.



Edward M. f
Professor of icr bi logy and Cell Science


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.



Juli Maupin
As isant Professor f Miefobiology and Cell Science

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.



Jft K.Yamamot o Associate Professtfr of Veterinary 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 qu ity, as a dissertation for the degree of Doctor of Philosophy. /


Wolfgang J. Streit
Professor of Neuroscience




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


August 1999 , Dean, College of Agriculture



Dean, Graduate School




Full Text

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MECHANISM OF INTERFERON THERAPY OF MULTIPLE SCLEROSIS: STUDIES IN AN ANIMAL MODEL By MUSTAFA GHULAM MUJTABA 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 1999

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ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Howard M. Johnson, for taking me into his laboratory and guiding me through my studies. He has always been knowledgeable and logical, which make him a great mentor. I must also express my thanks to my committee members Dr. Edward Hoffman, Dr. Jake Streit, Dr. Janet Yamamoto, and Dr. Julie Maupin for their time, patience, and effort. Many thanks to my fellow graduate students and labmates both past and present, including Barbara. Jeanne, Brian, Prem, Taishi, Martez, Pedro, Amy, George, Joe, Kendra, Scott, Karrie, Wiggins, and Tim. They make the lab more "interesting" and fun, at least at certain times. Finally, I thank my family for their support through my graduate school studies. My parents, Ghulam and Uzra Mujtaba, have always been there for me. I thank my brother and sisters and their families for their help and encouragement. Many thanks go to my Afghan cousins whom are always a great break from my graduate studies. I owe a great debt of gratitude to my wonderful family and friends. ii

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TABLE OF CONTENTS ACKNOWLEDGMENTS ii LIST OF TABLES v LIST OF FIGURES vi ABSTRACT vii CHAPTERS 1 INTRODUCTION 1 Overview 1 Multiple Sclerosis 2 Experimental Allergic Encephalomyelitis 5 Interferons 8 2 IFNt INDUCES STABLE REMISSION IN CHRONIC EAE 11 Introduction 11 Materials and Methods 12 IFNt 12 Induction of EAE 12 Administration of IFNt 13 Histological Evaluation 13 Proliferation Assay and Isolation of T Cells and B Cells 14 ELISA for MBP-Specific Antibodies 15 Results 15 IFNt Blocks Further Relapses into Paralysis of EAEAfflicted Mice ". 15 Reduction and Prevention of Lymphocytic Infiltrates in EAE Mice by IFNt 17 Deactivation of Microglia Following IFNt Treatment 17 IFNt Treated Mice Have a Lower Antibody Level Against MBP than Control Mice 22 IFNt Inhibited MBP-Specific B Cell Proliferation 22 IFNt Inhibited MBP-Specific T Cell Proliferation 29 Discussion 34 3 CD4 T SUPPRESSOR CELLS MEDIATE IFNt PROTECTION AGAINST EAE 35 iii

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Introduction 35 Materials and Methods 36 IFNs 36 Antibodies and Cytokines 36 Interferon Induction of Suppressor Cells 37 Induction of EAE Cells 37 CD4 T Cell Isolation and Depletion 38 Production of Suppressor Factor 38 Proliferation Assay 38 Results 39 IFNc--Treated Spleen Cells Inhibit MBP-Specific T Cell Proliferation 39 IFNt Induction of Suppressor Cells is Dose-Dependent 39 EFN-c Suppressor Cells Protect Mice Against EAE 44 IFNc-Induced Suppressor Cells are CD4 T cells 44 Suppressor Cells Produce Soluble Suppressor Factor(s) 49 IFNt-Induced Suppressor Cells Produce IL-10 and TGFp 49 IL-10 and TGFp Act Synergistically to Inhibit MBPSpecific T Cell Responses 54 Discussion 54 4 CONCLUSION 58 REFERENCES 66 BIOGRAPHICAL SKETCH 78 iv

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LIST OF TABLES Tabl e Page 1 . Treatment of SJL mice with IFNx after induction of EAE 16 2 . IFN inhibition of MBP-sensitized spleen cells 65 v

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LIST OF FIGURES Fi gures Page 1 . Histological evaluation of EAE mice before and after treatment with PBS and IFNt 19 2. IFNx causes deactivation of microglia 21 3 . Inhibition of anti-MBP antibody production in the chronic form of EAE by IFNt 24 4. Inhibition of anti-MBP antibody production in the acute form of EAE by IFNt 26 5 . IFNt inhibits MBP-specific proliferation of B cells from chronic EAE mice 28 6 . IFNt inhibits MBP-specific proliferation of B cells from acute EAE mice 31 7 . IFNt inhibits MBP-specific T cell proliferation 33 8 . IFNc-treated spleen cells inhibit MBP-specific T cell proliferation .... 4 1 9. IFNt induction of suppressor cells is dose-dependent 43 10. IFNt-induced suppressor cells can delay the onset of EAE in mice ... 46 1 1 . IFNt-induced suppressor cells are CD4 T cells 48 12. IFNt-induced CD4 T suppressor cells produce soluble suppressor factors(s) 51 1 3 . Blockage of IFNt-induced suppressor ceil and suppressor supernatant effects on MBP stimulation of sensitized EAE mouse spleen cells with monoclonal antibodies to IL-10 and TGFp 53 14. IL-10 and TGFp act synergistically to inhibit MBP-specific T cell responses 56 1 5 . IFNt induction of CD4 suppressor T cells inhibit SEA stimulation of naive spleen cells 61 1 6 . IFNt-treated CD4 T cells produce suppressor factor(s) which inhibit SEB stimulation of naive spleen cells 63 vi

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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 MECHANISM OF INTERFERON THERAPY OF MULTIPLE SCLEROSIS: STUDIES IN AN ANIMAL MODEL By Mustafa Ghulam Mujtaba August 1999 Chairperson: Howard M. Johnson Major Department: Microbiology and Cell Science Interferon (JFN) tau is a type I IFN that was originally identified as a pregnancy recognition hormone produced by trophoblast cells. It is as potent an antiviral agent as IFNcc and IFNp, but lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in animal studies. Previously it has been shown that interferon x pretreatment inhibits the development of both acute and chronic mouse experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). Here, we show that IFNt induced remission in SJL/J mice that had ongoing chronic active EAE disease, and protected mice against secondary relapses. IFNt treatment reversed lymphocyte infiltration and microglial activation in the central nervous system. Mice that were treated with IFNt had lower levels of anti-MBP (myelin basic protein) antibodies than untreated mice in both chronic and acute forms of EAE. vii

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MBP induced proliferation in B cells from EAE mice, but treatment with IFNt either in vivo or in vitro blocked this activation. Furthermore, IFNt inhibited MBP activation of T cells from EAE mice. Thus, IFNt inhibits the humoral arm as well as the cellular arm of the autoimmune disease EAE. IFNt prevents EAE in mice by induction of suppressor cells and suppressor factors. Suppressor cells can be induced by IFNt in tissue culture and in vivo by either intraperitoneal injection or by oral administration to mice. Incubation of suppressor cells with MBP-sensitized T cells blocked or delayed the MBP-induced proliferation. Further, intraperitoneal injection of suppressor cells into mice blocked induction of EAE by MBP. Suppressor cells possessed the CD4 T cell phenotype, and produced soluble suppressor factors that inhibited MBP activation of T cells from EAE mice. The suppressor factors were found to be IL-10 and TGFp, which acted synergistically to inhibit the MBP activation of T cells from EAE mice. These findings are important for understanding the mechanism(s) by which type I IFNs protect against autoimmune disease. viii

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CHAPTER 1 INTRODUCTION Overview Interferons (IFNs) are a group of glycoproteins that play important modulatory roles in the vertebrate immune system. Originally discovered in 1957 for their ability to interfere with viral replication in cells (Isaac and Lindenmann, 1957), they are now known to also have antiproliferative effects on a variety of cell types. For this reason, IFNs have been used for the treatment of autoimmune diseases, viral infections, and several types of cancers (Gutterman et al, 1994).Interferon-tau (IFNx) is a type I IFN that was originally identified as a pregnancy recognition hormone produced by trophoblast cells in sheep. It is as potent an antiviral agent as IFNoc and IFNp, but lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in vivo (Bazer et al, 1989; Pontzer et al, 1991; Soos etal, 1995a; Soos et al, 1995b). Multiple Sclerosis (MS) is one of the most common disease of the central nervous system (CNS). In MS, the loss of myelin is accompanied by a disruption in the ability of the nerves to conduct electrical impulses to and from the brain, and this produces the various debilitating symptoms of MS. Experimental allergic encephalomyelitis (EAE) is a murine model useful for studying the demyelinating disease MS. Myelin basic protein (MBP) has been shown to be one of the primary CNS antigens responsible for induction of autoimmunity in the EAE model (Zamvil et al, 1990). Immunization of mice with MBP results in tail and limb paralysis due to lymphocytic infiltration and demyelination in the CNS (Zamvil et al, 1990). MBP-specific antibodies and autoreactive MBP-specific T cells are also thought to contribute to the exacerbation of EAE and MS (Sun, 1993; Warren et 1

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al, 1993; Gerritse etal, 1994; Saoudi etal, 1995; Wang etal, 1995). Various inflammatory cytokines like tumor necrosis factor-alpha (TNFa) and IFNy contribute to the break down of myelin on nerve cells in the CNS. Furthermore, microglia, the resident macrophage of the CNS, are thought to play a key role in recruiting lymphocytes and function as antigen-presenting cell in MS and EAE (Benveniste, 1997; Streit et al, 1995; Hickey etal, 1988). Currently, the type I IFN, IFNp, is the only FDA approved cytokine treatment of MS. Previously, it has been shown that administration of IFNt to mice at the time of immunization with MBP blocked the development of EAE in mice without associated toxicity; however the mechanism of such action has not been fully elucidated (Soos et al, 1995a). Thus, the purpose of this study is to determine the mechanism by which IFNt suppresses autoimmune responses in EAE. Multiple Sclerosis Multiple Sclerosis (MS) is a chronic, demyelinating, inflammatory disease of the CNS. MS commonly affects young adults and mostly women (Ebers et al. 1986). MS is commonly found in Canada, the United States, South America, and Europe; near the equator MS is unknown (Ebers et al, 1986). In this disease there is an inflammation of myelin, which is fatty insulation or covering of nerve cell extensions, known as axons (Lassmann, 1998). Messages are sent along axons to other nerve cells in a kind of electrical signals. Myelin insulates the axons to help get these electrical impulses through. It stops currents from flowing between the individual axons. It also helps to speed the conduction of the electrical signal. This enables people to move almost without thinking. When myelin is affected in MS, impulses travel over the axons very slowly, if at all, and there is some electrical interference between axons. Messages are not sent efficiendy and can fail to get through so that a person loses the ability to make smooth, rapid, and

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coordinated movements (McFarland, 1998). Inflammation in the CNS destroys myelin and the oligodendrocytes, the myelin-producing cells (Mews et ai, 1998). After tissue destruction a scar or hardening area forms. These areas are multiple within the CNS, thus the term multiple sclerosis. The clinical course is variable, but the most common form is characterized by relapsing neurological deficits. Early MS lesions are characterized by local accumulation of activated T cells around small venules (Hauser et ai, 1983). Later myelin degeneration occurs associated with perivascular inflammation consisting of T cells, B cells, plasma cells, and macrophages (Prineas et ai, 1975; Prineas et ai, 1978). T cells are also found at the leading edge of plaques, and they extend into the surrounding normal appearing white matter. The T cells express activation molecules on their cell surface, such as IL-2 receptors and class II major histocompatibility complex (MHC) antigens (Hoffman et ai, 1986). In addition, class II MHC expression can also be detected on infiltrating macrophages and resident CNS cells, including microglia, astrocytes, and brain capillary endothelial cells (Hoffman et al., 1986). Class II MHC expression in the CNS is presumably induced by IFNy secreted by activated T cells. These findings indicate that in early acute MS lesion, demyelination occurs in the face of an active immune response within the CNS. Gliosis is also a prominent feature of MS; this process is characterized by astrocyte proliferation and hypertrophy (Bignami et ai, 1972). This reaction eventually leads to the formation of dense glial scars in the CNS, which can contribute to motor and sensory impairment. TNFa, a proinflammatory cytokine, appears to contribute to this process (Salmaj etal, 1990). The primary demyelination observed in MS results from damage to the myelin sheath or to the myelin-producing cells, the oligodendrocytes. Because myelin is critical for saltatory excitation along axons, demyelination leads to loss of neurological function. Recent findings with magnetic resonance imaging (MRI) indicate that considerable subclinical disease occurs, and that there is breakdown of the blood-brain barrier (BBB)

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4 early in lesion development (Thomson et al, 1992; Gay et al, 1991), which may be a crucial event in the pathogenisis of new lesions in MS. A large body of experimental evidence implicates immune mediated processes in activation and progression of MS. In addition, both genetic and environmental factors contribute to disease (Ebers et al, 1986; Martin et al, 1992). Although the pathological lesions, or plaques, are confined to the white matter of the brain and spinal cord, studies of cerebrospinal fluid and peripheral blood lymphocytes provide evidence for both local and systemic activation of the immune system (Hafler and Weiner, 1989). EFNy and TNFcc and p have been implicated in exacerbating MS. The ability of TNFoc to mediate myelin and oligodendrocyte damage in vitro (Selmaj et al, 1988), and its ability to cause cell death of oligodendrocytes in vitro (Robbins et al, 1987) may contribute directly to myelin damage or the demyelination process observed in MS. Both TNFoc and TNFp can cause death of oligodendrocytes, the myelin-producing cells of the CNS (Robbins et al, 1987; Paul and Ruddle, 1988). MS is characterized by migration of inflammatory cells from blood into the brain and subsequent invasion of the extravascular tissue (Cross et al, 1990; Raine et al, 1990). Recent studies have shown that up-regulation of adhesion molecules, such as intercellular adhesion molecules (ICAM-1) on brain endothelial cells by exposure to proinflammatory cytokines such as TNFcc and IFNy mediate leukocyte adhesion to endothelium (McCarron et al, 1993; Fabry et al, 1992; Wong et al, 1992). The presence of ICAM-1 and other adhesion molecules in the vessel walls as well as on astrocytes may guide inflammatory leukocytes into and through the brain, thereby contributing to impairment of the blood brain barrier and the neuropathology of MS. IFNy is considered the most potent inducer of class II MHC antigen expression on most cell types, including astrocytes and microglia (Cogswell et al, 1991; Wong et al, 1984; Fierz etal, 1985; Fontana etal, 1984; Pulver etal, 1987; Suzumura etal, 1987). There is little or no expression of class II molecules in normal brain; however IFNy

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5 induces them on astrocytes and endothelial cells, which can then present myelin antigens to T cells (Fierz et al, 1985; McCarron et al, 1986). Other function of IFNy that may be important in MS include activation of macrophages, which act as effector cells in demyelination (Bever and Whitaker, 1985) and induction of adhesion molecules, which mediated homing of lymphocytes to sites of inflammation and may facilitate their entry into the CNS (Male et al, 1990). Furthermore, IFNy-positive cells have been detected in the CNS of patients with MS (Hofman et al., 1991; Traugott and Lebon, 1988). Recent studies on the association of IFNy with MS demonstrated that peripheral blood lymphocytes from patients with MS produce significantly more IFNy than those of normal lymphocytes (Beck et al, 1988; Hirsh et al, 1985). Accumulating evidence supports the notion that MS is an autoimmune disorder mediated by T cells. This evidence includes the pathology of MS lesion, immunological abnormalities in both the periphery and CNS of patients with MS, immunoglobulin synthesis within the CNS, exacerbation of disease after treatment with IFNy, putative autoantigens such as myelin basic protein (MBP), and involvement of cytokine networks (Martin et al, 1992). Studies have also shown the presence of autoantibodies specifically bound to disintegrating myelin around axons in lesions of acute MS (Genain et al, 1999). Furthermore, peripheral blood T lymphocytes from patients with MS were activated by various MBP peptides (Baxenvanis et al, 1989). Thus, many factors are involved in the pathogenesis of MS . Experimental Allergic Encephalomyelitis The best-characterized experimental model for MS is experimental allergic encephalomyelitis (EAE). Similarities shared between EAE and MS are relapsing and chronic paralysis, CNS demyelination, linkage to MHC class II, CD4 T cells present in perivascular inflammatory lesions, and similar autoantigens. MS is a spontaneous disease

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in humans, but EAE is induced by injection of spinal cord components such as myelin basic protein (MBP) with adjuvant or transfer of encephalitogenic MBP-specific T cells to naive recipients (Swanborg, 1995; Paterson and Swanborg, 1988; Tabira, 1988, Pettinelli and McFarlin, 1981). MBP is a predominant protein present in myelin in the CNS. Transgenic mice have been constracted that mimics MS in its spontaneous induction and pathology (Governam et ai, 1993). EAE can be induced in a number of species including mice, rats, guineas pigs, monkeys, sheep, dogs, and chickens (Sturart and Krikorian, 1928). Clinical signs of EAE include dramatic weigh loss, weakness of the tail and hind limbs, and ascending paralysis. The earliest clinical signs of CNS dysfunction are closely associated with formation of perivasuclar cellular infiltrates and edema with the CNS (Raine et ai, 1984; Leibowitz and Kennedy, 1972; Claudio et ai, 1990; Cross et ai, 1990; Raine et ai 1990; D' Amelio et ai, 1990). Demyelination appears to be a later event (Raine et ai, 1990; D' Amelio et ai, 1990) and may account for chronic neurological dysfunction. EAE is characterized by inflammatory infiltration of the CNS by activated T cells and macrophages, de-myelination, and acute, chronic, or chronic -relapsing paralysis. The mediators of this disease are CNS antigen reactive CD4 T cells that are class II MHCrestricted (Zamvil and Steinman, 1990). Lymphocytes, as mediators of EAE, were first implicated by experiments in which anti-lymphocyte antibodies inhibited induction of EAE (Waksman etal., 1961). Further evidence that T cells were involved stemmed from the observation that thymocytes are required for EAE induction (Arnason et ai, 1962). Furthermore CD4 T cells are present in inflammatory EAE lesions in the CNS (Traugott et al, 1986). Most encephalitogenic T cells are of the Thl subtype, which secrete IFNy, IL2, TNFa, and TNFp (Mosmann and Coffman, 1989). It has been suggested that TNFoc and TNFp secretion by MBP-specific T cell clones correlates with their encephalitogenic potential (Powell etal., 1990). Expression of adhesion molecules also influences the pathogenicity of encephalitogenic T cells (Baron etal., 1993; Kuchroo etal., 1993). Susceptibility to EAE appears to be iinked to MHC alleles, although non MHC genes may

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have a small role in contributing to EAE (Gasser et al, 1973). Although EAE in animals is initiated by T cells that recognize myelin antigens in the context of class II MHC molecules (Wekerle et al., 1986), some studies have suggested that B-cell activation and antibody responses are necessary for the full development of EAE (Brosnan and Raine, 1996; Willenborg and Prowse, 1983; Piddlesden etai, 1993), and earlier studies on immunemediated demyelination using myelinated cultures of CNS tissue have indicated that humoral factors are effector mechanisms (Raine and Bornstein, 1970; Raine et al., 1973). Autoantibodies against CNS antigen myelin/oligodendrocyte glycoproteins were identified to bind disintegrating myelin around axons in lesion of acute MS and the marmoset model of EAE (Genain et al, 1999). Cytokines have been implicated in contributing to EAE disease progression, as well as mediating recovery from disease. Inflammatory cytokines released within the CNS may contribute to the disease process by influencing vascular permeability, inflammatory cell extravasation, and antigen presentation (Mantovani and Dejana, 1989; Martiney etai, 1990; Fierz et al, 1985). Cytokines released within the CNS in response to acute inflammation may also contribute to chronic damage associated with reactive gliosis of astrocytes (Giulian and Lachman, 1985; Selmaj et al., 1990; Yong et al., 1991) and the destruction of oligodendrocytes and myelin (Selmaj and Raine, 1988; Selmaj etai, 1991). The cytokines, IL-1, TNFoc, and TNF(3, contribute to the initiation and/or disease progression of EAE (Mannie et. al., 1987; Symons et al., 1987; Jacobs et al., 1991; Ruddle et ai, 1990; Selmaj et ai, 1991; Kuroda et al., 1991). Studies involving the role of IFNy show conflicting results. Some studies show that IFNy plays a protective role in different models of EAE (Billiau et al., 1988; Duong et al., 1992; Voorthuis et al., 1990). Other studies show the detection of IFNy before the onset of clinical disease (Kennedy et al., 1992; Merril etai., 1992; Stoll et al., 1993) and during the acute phase of disease (Khoury et al., 1992; Baker et al., 1991 ; Kennedy et al., 1992). Other cytokines such as IL-10, TGFp, and IL-4 have a protective effect on EAE. TGFp can inhibit UNy-induced

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class II MHC antigen expression on both human astroglioma and rat astrocyte cells (Zuber et al, 1988; Schluesener, 1990) and can act as a chemotactic agent for both rat astrocytes and microglia (Morganti-Kossmann et al, 1992; Yao et al, 1990). Microglia, the resident macrophages of the brain, are thought to play a key role in recruiting lymphocytes and function as antigen-presenting cells in MS and EAE (Benveniste et al, 1997; Streit et al, 1995; Hickey et al, 1988). TGFp inhibits the production of TNFa by microglia (Suzumura et al, 1993) and astrocytes (Benveniste et al, 1994), and TGFp has also been shown to be an important mediator of oligodendrocyte differentiation (McKinnon et al, 1993). Also, IL-10 has been detected in the CNS of SJL/J mice during disease recovery (Kennedy et al, 1 992). EL-4, IL-10, and TGFB share some similar biological activities because they are all capable of inhibiting secretion of proinflammatory cytokines (Bogdan et al, 1992; Chao et al, 1993). The EAE model has been used in several novel immunotherapy experiments including anti-TCR antibodies, anti-MHC antibody, anti-CD4 antibody, peptide and interferon therapies, and T cell vaccination (Steinman et al, 1983; Brostoff and Mason, 1984; Howell etal, 1989; Bandenbark et al, 1989; SoosetaL, 1997). Thus, the development and testing of a safe therapy for EAE and understanding the mechanism(s) of the therapeutic are the first steps toward identifying potential therapies for MS. Interferons Interferons (IFN) are glycoproteins that are produced and released from virally infected cells; they were originally characterized for their antiviral properties (Pestka et al, 1987), and they were first described in 1957 by Issacs and Lindemann (Issacs and Lindeman, 1957). IFNs have been found in all higher vertebrates including humans. They have molecular weights ranging from 15 to 30 KDa (Gastle and Huber, 1988). The idea that IFNs alter the pathogenesis of natural virus infections was supported by observations

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9 that virus-infected animals injected with antibody prepared against IFNs succumbe more rapidly to disease than when virus was given alone (Gresser et al, 1976). IFNs inhibit the growth of viruses of all type in vivo and in vitro. Some viruses are more sensitive to IFN action than others (Grossberg, 1972). Besides their antiviral activities, IFNs possess many other activities including those which are antimicrobial, antitumor, and immunomodulatory (Pestka et al, 1987). IFNs are currently approved by the Food and Drug Administration for a number of diseases such as hairy cell leukemia, condyloma acuminatum, acquired immune deficiency syndrome (AIDS) related Kaposis's sarcoma, chronic hepatitis B and C, gebutak warts caused by papillomavirus, chronic granulomatous disease, and MS (Dorr, 1993; Johnson et al, 1994). Different IFNs are distinguishable based upon their cellular source, immunological reactivity, and induction of biological responses. There are two main types of IFN, type I and type II. The type I IFNs include IFNa, IFNp, IFNco, and IFNt. The type II IFN refers to IFNy. IFNa and IFNco are produced by leukocytes, while IFNp is produced by fibroblasts, and IFNx is primarily produced by the trophoblast cells of the conceptus. IFNy is produced by T cells and natural killer cells (Baron et al, 1991). IFNy appears to have a dominant immunoregulatory role while IFNa and IFNp tend to mediate more antiviral activity. Both type I and type II IFNs have potent antiproliferative effects, while only IFNy, and not the type I IFNs, can upregulate MHC class II (Houghton et al, 1984, Schwartz et al, 1985). Twenty-six IFNa genes with common structures have been identified, and they encode for at least 22 distinct proteins consisting of 20KDa single polypeptide chains (Zoon et al, 1992). These different IFNos mediate distinct biological activities in different cells. In contrast, there is only a single form of IFNp encoded by a distinct gene located next to the IFNa locus in both human and mice (Farrar et al, 1993). Also, a single gene has been identified and described for IFNy, whereas, several genes for IFNco and IFNt have been identified (Sen and lengyel, 1992; Bazer and Johnson, 1991).

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10 The type I IFNs bind to a common receptor, whereas IFNy binds to a different receptor (Langer and Pestka, 1988). IFNy is considerably more active as an immunomodulator than other classes of IFNs, but its antiviral activity is lower than these other classes. IFNy is produced by all CD8+ T cells populations and by the Thl and ThO subsets of CD4+ T cells following antigenic or T cell mitogen stimulation (Farrar et al 1993). IFNy can upregulate the surface expression of MHC class I and class II antigens on a variety of cell types both in murine and human (Baron et al, 1991; Sen and Lengyel ,1992). IFNy production from T cells or NK cells is stimulated by IL-12, which can act synergistically with alloantigens, mitogens, or IL-2 (Stern et al, 1990). In contrast, IL-10 inhibits IFNy production by T cells and NK cells (Fiorentino et al, 1989). IFNt has only been recently described. IFNt was discovered originally as a pregnancy recognition hormone that is essential for establishment of pregnancy in ruminants such as sheep and cows (Bazer and Johnson, 1991). It was identified as a member of the type I IFN family when it was shown to share significant amino acid sequence homology with the type I IFNs, IFNa and IFNco (Imakawa et al, 1987). IFNt possesse antiviral, antiproliferative, and immunomodulatory activities similar to the other type I IFNs (Pontzer et al, 1988; Bazer et al, 1989; Soos and Johnson, 1995b). IFNt differs from the type I IFNs in that it has been shown to be remarkably nontoxic when used at high concentrations in culture and animal studies (Bazer et al, 1989; Soos et al, 1995a; Pontzer et al, 1991), not readily induced by virus and dsRNA (Roberts et al, 1992), and is under genetic regulation different from other type I IFNs (Cross and Roberts, 1991). However, IFNfJ, which has been shown to ameliorate relapsing-remitting nature of MS (IFNp Multiple Sclerosis Study Group, 1993), is currently the only cytokine approved by the FDA for treatment in MS. The mechanism of this amelioration is not fully understood. Therefore, the data recounted here on the mechanism of therapy of EAE by IFNt may serve as the basis for understanding how type I IFNs may exert therapeutic effects in autoimmune diseases such as MS.

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CHAPTER 2 IFNt INDUCES STABLE REMISSION IN CHRONIC EAE Introduction Interferon-tau (IFNt) is a member of the type I IFN family but unlike IFNa and IFNp, IFNt lacks toxicity at high concentrations in vitro and when used in vivo in animal studies (Bazer et al., 1989; Pontzer et al, 1991; Soos and Johnson, 1995b, Soos et al, 1995b). Like the type I IFNs, IFNt is acid stable and exerts antiviral, antiproliferative and immunomodulatory activities (Bazer et al, 1989; Pontzer et al, 1991; Soos and Johnson, 1995b). Previously, we have shown that IFNt blocks the development of acute and chronic experimental allergic encephalomyelitis (EAE) in mice without any associated toxicity (Soos and Johnson, 1995b). EAE is a murine model used to study the human demyelinating disease multiple sclerosis (MS) (Zamvil and Steinman, 1990). In the EAE model, immunization of mice with central nervous system (CNS) antigens such as myelin basic protein (MBP) and proteolipid protein (PLP) results in tail and limb paralysis due to lymphocytic infiltration and demyelination in the CNS (Zamvil and Steinman, 1990). Treatment of mice with IFNt before, during, and shortly after immunization with MBP prevents the onset of EAE (Soos et al, 1997). This protection is mediated by IFNt through induction of CD4 T cells to produce IL-10 and TGFp which act synergistically to inhibit activation of MBP-sensitized T cells from NZW mice (Mujtaba et al, 1997). Here we demonstrate that treatment of SJL/J mice with IFNt after the induction of chronic EAE induces them into stable remission. Furthermore, the histological features of the CNS normally associated with EAE such as lymphocyte infiltration and activation of microglia could not be found in the IFNt treated mice. Microglia, the resident macrophages of the brain, are thought to play a key role in recruiting lymphocytes and function as antigen 11

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12 presenting cells in MS and EAE (Benveniste, 1997; Streit and Kincaid-Colton, 1995; Hickey and Kimura, 1988). Microglia are process-bearing when they are in the inactivated resting state, but they undergo cell hypertrophy upon activation, and they transform into rounded brain macrophages after neuronal degeneration (Streit et al, 1988). Furthermore, since MBP-specific antibodies are also thought to contribute to the exacerbation of EAE and MS (Sun, 1993; Warren and Catz, 1993; Gerritse etal, 1994) along with autoreactive MBP-specific T cells (Zamvil and Steinman, 1990; Zamvil et al, 1986; Ando et al, 1989; Lemire et al, 1986; Hashim and Brewen, 1985), we analyzed MBP-specific antibody levels in vivo and MBP-specific B cell and T cell activation in vitro on IFNt treated and control mice. Overall, these findings serve as the basis for understanding how type I IFNs exert therapeutic effects in autoimmune diseases. Materials and Methods IFNt The ovine IFNt gene was expressed in Pichia pastoris using a synthetic gene construct, which was kindly provided by Dr. Gino Van Heeke, Ciba Pharmaceuticals, London, England (Heeke et al, 1996). IFNt was secreted into the medium and was purified by successive DEAE-cellulose and hydroxylapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining analysis (Heeke et al, 1996). The purified protein had a specific activity of 2.9 to 4.4 x 10 7 U/mg protein as measured by antiviral activity using a standard viral microplaque reduction assay onMDBK(Pontzerefa/., 1991). Induction of EAE For induction of EAE, 300 \ig of bovine MBP (MBP) were emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml H37Ra (Mycobacterium tuberculosis, Difco,

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13 Detroit, MI) and injected into two sites at the base of the tails of NZW mice (Jackson Laboratory, Bar Harbor, Maine). On the day of immunization and 48 h later, 400 ng of pertussis toxin (List Biologicals, Campbell, CA) were also injected. For induction of EAE in SJL/J mice (Jackson Laboratory , Bar Harbor, Maine), the same protocol was used as described except mice were immunized again 7 days after the initial immunization. Mice were clinically examined daily for signs of EAE, and severity of disease was graded using the following scale: 1, loss of tail tone; 2, hind limb weakness, 3, paraparesis, 4, paraplegia; 5, moribund/death. Administration of IFNt Mice were orally fed using feeding needles from Fisher Scientific (Norcross, GA) and injected intraperitoneally (i.p.) with 100 uL of 6 X 10 5 U total of IFNt with PBS used as the vehicle for administration. Administration of IFNt to SJL/J mice was started after the onset of EAE and was administered every 48 h thereafter. NZW mice were administered with IFNt 48 h before, during, and 48 h after immunization with MBP. Histological Evaluation SJL/J Mice were perfused transcardially with 0.9% saline followed by Bouin's fixative solution. The vertebral columns were removed, and spinal cords were dissected out and post-fixed for an additional 48h in Bouin's fixative. Thereafter, the tissue was dehydrated and embedded in paraffin. Sections were cut at 7um and mounted onto subbed slides. Cresyl violet staining was used to visualize inflammatory infiltrates. For detection of microglia, SJL/J mice were first perfused with saline and 4% paraformaldehyde, and after removing the spinal cords and post fixing them for 1-2 h in 4% paraformaldehyde, they were then transferred to 30% sucrose solution for cryoprotection on at 4 °C. Spinal cord lumbar regions were embedded in OCT compound and 25 urn sections were cut on a cryostat and mounted onto subbed slides. Microglia were

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14 detected by immunohistochemically staining for CD1 lb (CR3) with the monoclonal antimouse CD1 lb (CR3) antibody (SeroTec Inc., NC). A 1:300 dilution of monoclonal antimouse CD1 lb antibody was incubated with the sections overnight at 4 °C. Then, a 1 :400 dilution of the secondary biotinylated anti-rat IgG antibody was incubated with the section for lh at 25 °C followed by lh incubations of avidin and horse radish peroxidase. Then, 3 3'-diaminobenzidine (DAB) (0.5 mg/ml) was applied to the sections for 10 minutes and finally washed with phosphate buffered saline (PBS), after which sections were counterstained with cresyl violet. Proliferation assay and isolation of T cells and B cells Spleen cells from IFNt treated, PBS treated, or untreated MBP-immunized NZW or SJL/J mice (5.0 x 10 7 cells/well) were cocultured with IFNt (10,000 30,000 U/ml), MBP (30 ug/ml), or media for 48 h. Spleen cells were washed, and B cells or T cells were isolated from each treated group using an immunoaffinity column from the cellect-plus mouse B cell kit or the cellect-plus mouse T cell kit (Biotex Laboratories, Alberta), respectively. B cell and T cell preparations were then incubated (5.0 x 10 5 cells/well) in RPMI 1640 media containing 5% FBS for 72 h. Some B cell and T cell cultures were incubated with 200 ng/ml of staphylococcol enterotoxin B (SEB) (Toxin Technology, FL), 50 ng/ml of lipopolysacharide (LPS) (Sigma chemical co., St. Louis, MO), or a combination of 20 ng/ml Phorbol 12-myristate 13-acetate (PMA) and 200 ng/ml ionomycin (Sigma Chemical Co., St. Louis, MO). The cultures were pulsed with [ 3 H] -thymidine (1.0 uCi/well)(Amersham, Indianapolis, IN) 18 h before harvesting onto filter paper discs using a cell harvester. Cell-associated radioactivity was quantified using a (i-scintillation counter and activity is reported in CPM.

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15 EPS A for MBP-Specific Antibodies Bovine MBP was resuspended in binding buffer (0.1 M carbonate/bicarbonate, pH 9.6), adsorbed to the flat bottoms of plastic 96 well tissue culture wells overnight at 4 °C at a concentration of 600 ng/well, and evaporated to dryness. The plates were treated with blocking buffer, 5% powdered milk (Carnation) in PBS, for 2 h in order to block nonspecific binding and then washed 3 times with PBS containing 0.05% Tween 20. Various dilution of sera from NZW or SJL/J mice which were untreated, IFNt-treated by i.p. injection, and rFNx-treated by oral feeding, and PBS treated were added to the wells and incubated for 3h at room temperature. Binding was assessed with the secondary antibody, goat anti-mouse immunoglobulin (IgG) coupled to alkaline phosphatase. After the substrate solution, p-nitrophenyl phosphate (5mg/ml) was added and the reaction terminated with 3 N NaOH activity was monitored by color development at 405 nm in an EPSA plate reader (Bio-Rad, Richmond, CA Results IFNt Blocks Further Relapses into Paralysis of EAEAfflicted Mice The chronic form of EAE was induced in SJL/J mice with MBP, and these mice were treated with IFNt (6 x 10 5 U) both orally and i.p. starting at the time of paralysis and at 48 h intervals thereafter. Both oral feeding and i.p. injection of IFNt protected mice against further relapses of disease following a reduced primary relapse; protection against primary relapse was 80% for i.p. injected IFNt, 65% via oral administration of IFNt, and 17% for the PBS treated control group (Table 1). No further relapses were seen for the IPN-treated groups while 8 of 18 mice had secondary relapses in the PBS group during the treatment period. Thus oral administration and i.p. injection of IFNt blocked further relapses into paralysis in mice afflicted with chronic EAE.

PAGE 24

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17 Reduction and Prevention of Lymphocytic Infiltrates in EAE Mice by IFNt Histology was performed to determine the extent of lymphocyte infiltration into the CNS of MBP immunized SJL/J mice afflicted with EAE before treatment and after treatment with IFNt both i.p. and orally. Sections of the lumbar spinal cord were evaluated for lymphocyte infiltration after staining with cresyl violet. As shown in Figure 1 A, perivascular accumulations of lymphocytes were present in the mouse spinal cord white matter prior to IFNt treatment. Then, after 38 days in the absence of IFNt, mice had lymphocytic infiltrates in their spinal cords (Figure IB). In contrast, no lymphocytic infiltrates could be detected in mice treated with IFNt by oral feeding (Figure 1C) or i.p. injection (Figure ID) after 38 days. Thus, IFNt reduced and prevented further lymphocytic infiltration into the CNS, demonstrating that the protective effect of IFNt is associated with inhibition and reversal of lymphocyte infiltration of the CNS. Deactivation of Microglia Following IFNt Treatment Spinal cord sections from SJL/J mice with EAE (Figure 2A) were marked by widespread and maximal microglial activation. This was evidenced by the fact that nearly all microglial cells within a given section had lost their ramified (branched) morphology and had transformed into brain macrophages appealing as enlarged rounded cells (Figure 2A). Following both oral and i.p. treatments with IFNt, sections immunostained with anti-CD 1 lb antibody showed only ramified microglia (Figure 2B, Figure 2C). Rounded brain macrophages were no longer detectable. The processes of ramified cells present after treatment did stain strongly with anti-CD 1 lb antibody, and they were somewhat thicker than those of resting cells in CNS tissue from naive animals. Thus, it is likely that while microglia were de-activated by IFNt treatment, they had not completely reverted back to the resting state, but had maintained low levels of activation.

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Figure 2. IFNt causes de-activation of microglia. Spinal cord section from PBS treated SJL/J mouse with EAE shows fully activated microglial cells which exist as rounded brain macrophages (arrows in A). Oral feeding (B) and i.p. injection (C) of IFNt caused microglia to assume a process-bearing (arrows) morphology indicating a reversion to the resting state. Sections were taken 38 days after initiation of the treatments (57 days after immunization), and two mice per group from separate experiments were examined. Sections of spinal cord were immunohistochemically stained for microglia and counterstained with cresyl violet and are presented at a final magnification of 1000X. Mice were from the PBS and oral and i.p. IFNt treated groups from Table 1.

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22 IFNt Treated Mice Have a Lower Antibody Level Against MBP than Control Mice During the course of IFNt treatment of SJL/J mice for chronic relapsing EAE, mice were bled and sera were examined for the presence of MBP-specific antibodies. As shown in Figure 3, mice that received i.p. injections of IFNt had the lowest anti-MBP antibody level followed by the orally fed mice. Untreated and PBS treated mice had the highest antiMBP antibody levels. Similarly, in the acute form of EAE, which was induced in NZW mice, anti-MBP antibody levels were also lower in the IFNt treated group than in the PBS treated group (Figure 4). Thus, IFNt inhibition of antibody production against MBP may be a contributing mechanism by which IFNt prevents development or inhibits further relapses of EAE in the acute and chronic forms of the disease, respectively. IFNt Inhibited MBP-Specific B Cell Proliferation Related to antibody production, we addressed the question of whether IFNt had any effect on B cell activation in response to MBP in culture. Splenocytes obtained from SJL/J mice that had been immunized with MBP for induction of chronic EAE were incubated with media or IFNt in the presence or absence of MBP for 48 nr. B cells were isolated using an immunity affinity column and then incubated with media alone for 72 h, after which proliferation was measured. As shown in Figure 5, B cells isolated from EFNt-treated SJL/J splenocyte cultures did not proliferate in the presence of MBP, while the media treated B cells did respond to MBP significantly. B cells that were isolated with the immunoaffinity column did not proliferate in response to the T cell superantigen staphylococcal enterotoxin B (SEB), but did proliferate in the presence of lipopolysacharide (LPS), a polyclonal B cell stimulator (Figure 5 inset). Thus, the proliferation seen in Figure 5 is from B cells and not from T cell contamination. Furthermore, splenocytes obtained from IFNt and PBS-treated NZW mice, which developed the acute form of EAE, were incubated in media alone or with IFNt in the presence or absence of MBP for 48 h, after which B cells were isolated and incubated with media alone for 72 h and proliferation

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29 measured. As shown in Figure 6, EAE B cells isolated from MBP-treated splenocytes that were taken from a PBS-treated NZW mouse had a stimulation index greater than 1 1 relative to media control, while EAE B cells isolated from IFNT-treated splenocytes did not proliferate in response to MBP. Furthermore, EAE B cells isolated from MBP-treated splenocytes that were taken from an IFNT-treated NZW mouse did not proliferate either. Thus, IFNx can inhibit activation of B cells, taken from mice having either acute or chronic EAE, in response to MBP both in vivo and in vitro, consistent with reduction of antibody production to MBP in IFNt treated EAE mice. IFNx Inhibited MBP-Specific T Cell Proliferation Previously, we have shown that IFNt inhibits MBP-specific splenocyte proliferation (4) by inducing CD4 T cell to produce IL-10 and TGFji which act synergistically to inhibit EAE splenocyte activation (Mujtaba et al, 1997). Here we determine the effect of IFNt on proliferation of T cells isolated from MBP-sensitized SJL/J mouse splenocytes stimulated with MBP and treated with IFNt or media in culture. At both IFNt concentrations, MBP activation of MBP-specific T cell were inhibited, and at 30,000 U/ml, IFNt reduced levels of proliferation to less than 50% of that observed in response to MBP alone (Figure 7). The T cells that were isolated with the immunoaffinity columns did not proliferate in response to LPS, but did proliferate in presence of PMA and ionomycin, which are T cell activators when used in combination (Figure 7 inset). Therefore, the proliferation seen in Figure 7 is from T cells and not from B cell contamination. Thus, IFNt inhibits the activation of MBP-specific effector T cells of chronic EAE mice.

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34 Discussion The findings reported here clearly show that ongoing chronic, relapsing EAE in SJL/J mice can be significantly alleviated by either parenteral or oral administration of IFNt on a continuous basis. Although some mice in both IFNt treatment groups had primary relapsing incidence of paralysis during treatment, further relapses were not detected unlike the PBS treated group. Parenteral administration of IFNx was slightly more effective in alleviating chronic EAE than oral administration in that only 4 of 20 mice had relapses while 7 of 20 mice had primary relapses in the orally treated group. Furthermore, untreated EAE mice showed lymphocyte infiltration of the CNS and activation of microglia, whereas, IFNt treatment of mice with active EAE reversed these cellular effects. This suggests that IFNt treatment results in relief or permanent remission of the observed EAE symptoms. Although considerable attention is usually given to T-helper cell-mediated events in EAE, we show that anti-MBP antibody and MBP-specific B cell effects, like the T cell effects, are inhibited by IFNt in both chronic and acute forms of EAE. MBP antibody production in EAE mice was significantly inhibited by IFNt, with i.p. administration of IFNt more effective than oral, and MBP induced proliferation of sensitized B cells was also blocked. IFNt inhibition of B cell proliferation probably plays a central role in inhibition of anti-MBP antibody production. We have shown that IFNt and other type I IFNs induce terminal differentiation of Daudi B cells to plasma cells (Subramaniam et al., 1998). Thus, IFNt inhibition of B cell clonal expansion by induction of terminal differentiation, which results in an overall lower plasma cell number, probably plays a central role in IFNt inhibition of production of antibodies to MBP in EAE mice. Overall, we have demonstrated that IFNt is an effective therapy for ongoing EAE and as such should have potential for treatment of MS in humans.

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CHAPTER 3 CD4 T SUPPRESSOR CELLS MEDIATE IFNt PROTECTION AGAINST EAE Introduction Previously, we showed that interferon tau (IFNt) blocks the development of experimental allergic encephalomyelitis (EAE) in mice without associated toxicity; however the mechanism of such action has not been fully elucidated (Soos et al, 1995a). EAE is a murine model useful for studying the demyelinating disease multiple sclerosis (MS) (Zamvil and Steinman, 1990). Myelin basic protein (MBP) has been shown to be one of the primary central nervous system antigens responsible for induction of autoimmunity in the EAE model. Upon immunization with MBP. mice develop clinically observable tail and limb paralysis due to lymphocyte infiltration into the central nervous system accompanied by acute demyelination (Zamvil and Steinman, 1990). The type I IFNs, a and p, have previously been shown to induce suppressor cells that block in vitro antibody production (Johnson and Blalock, 1980). Further, when type I IFN-induced suppressor cells were cultured in vitro, they were shown to produce a soluble factor that mediated immunosuppression. Past studies by others suggested that "classic" T suppressor cells bear the CD8 phenotype. Here we demonstrate that IFNt-induced suppressor cells bear the CD4 phenotype, and these cells mediate the amelioration of EAE by IFNt. In addition, IFN-t-induced suppressor cell function occurs via a mechanism similar to that originally observed for type I IFNcc and p inhibition of antibody production in vitro. A suppressor mechanism shared by the type I IFNs is the induction of soluble suppressor factors, which we demonstrate in this study. These findings serve as the basis 35

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36 for understanding how type I IFNs exert therapeutic effects in autoimmune diseases such as MS. Materials and Methods IFNs The ovine IFNt (IFNx) gene was expressed in Pichia pastoris using a synthetic gene construct, which was kindly provided by Dr. Gino Van Heeke, Ciba Pharmaceuticals, London, England (Heeke et al., 1996). IFNt was secreted into the medium and was purified by successive DEAE-cellulose and hydroxylapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining analysis. The purified protein had a specific activity of 2.9 to 4.4 x 10 7 U/mg protein as measured by antiviral activity using a standard viral microplaque reduction assay on MDBK cells (Pontzer et al, 1991). MuIFN3 (specific activity 4. 1 x 10 7 U/mg) was obtained from Lee Biomolecular (San Diego, CA). Antibodies and Cytokines Monoclonal rat anti -mouse IL-10, recombinant mouse IL-10, and monoclonal mouse anti-TGF-p 1 ,-(3 2 ,-p 3 were obtained from Genzyme, Cambridge, MA. Ultrapure natural human TGFfr, which shows cross-reactivity in most mammalian cell types, was also obtained from Genzyme. ALIO dilution of HL 100, a specific monoclonal antibody for IFNx, was used to neutralize 5000 U/ml of IFNt prior to usage. The HL100 monoclonal antibody was kindly provided by Dr. Carol Pontzer, University of Maryland, College Park, MD. AH antibodies and cytokines were used in proliferation assays described below.

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37 Interferon Induction of Suppressor Cells Suppressor cells were induced both in vitro and in vivo. For in vitro induction, NZW mouse spleen cells (5.0 x 10 7 /ml) were incubated with 5000 U/ml of IFNt for 24 h at 37°C, after which the cells were washed twice prior to use. In vivo induction of suppressor cells in naive NZW mice involved administration of a single dose of IFNt (10 5 U) either intraperitoneally (i.p.) or by oral feeding with PBS used as the vehicle for administration. After 24 h, mice were sacrificed and the spleens removed. Spleen cells were washed and resuspended in RPMI 1640 medium supplemented with 2% fetal bovine serum and used as described below. Induction of EAE For induction of EAE, 300 ug of bovine MBP (MBP) were emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml H37Ra (Mycobacterium tuberculosis, Difco, Detroit, MI) and injected into two sites at the base of the tails of NZW mice. On the day of immunization and 48 h later, 400 ng of pertussis toxin (List Biologicals, Campbell, CA) were also injected. Mice were clinically examined daily for signs of EAE, and severity of disease was graded using the following scale: 1, loss of tail tone; 2, hind limb weakness, 3, paraparesis, 4, paraplegia; 5, moribund/death. Adoptive Transfer of Suppressor Cells Suppressor cells were induced in vitro with IFNt as described above and resuspended in phosphate buffered saline (PBS). NZW mice were injected intraperitoneally with 100 ixl of PBS containing 5 x 10 6 suppressor cells 48 h before, on the day of, and 48 h after immunization with MBP for induction of EAE. Mice were examined daily for signs of EAE, and the severity of disease was graded as noted above.

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38 CD4 T Cell Isolation and Depletion CD4 T cells effects were examined using both positive and negative CD4 T cell selection processes. The Cellectplus mouse CD4 kit (Biotex Laboratories, Inc., Alberta, Canada), an immuno affinity column, was used to isolate CD4 cells from NZW mouse spleen lymphocyte cultures treated with media or IFNx. Depletion of CD4 T cells from mouse spleen lymphocyte cultures treated with IFNx or media was carried out using rat anti-mouse L3/T4 CD4 monoclonal antibody (Biosource International, Camarillo, CA) and Low-Toxic-M rabbit complement (Accurate Chemical and Scientific Corporation, Westbury, NY). Lymphocytes from NZW mouse spleen were resuspended at 10 7 cells/ml in RPMI 1640 medium and incubated with 1:10 dilution of anti-mouse L3/T4 CD4 antibody for 1 h at 4° C. Cells were then centrifuged and resuspended in 1: 10 dilution of rabbit complement in RPMI 1640 medium for 1 h at 37°C. The cultures were washed and used for further experimentation. Production of Suppressor Factor Suppressor cells were generated in vitro by incubating spleen cells with 5000 U/ml of IFNt for 24 h at 37°C as described above. Cells were then washed and resuspended at 10 8 cells/ml in fresh culture medium. After incubating for an additional 2 h at 37°C, clarified supernatants were collected and tested for suppressor activity. Proliferation Assay Spleen cells from MBP-immunized NZW mice (2.5 5.0 x 10 5 cells/well) were cocultured with IFNtor IFN(3-induced suppressor cells (1.0-5.0 x 10 5 /well), suppressor cell supernatants, or IL-10 and TGFp in the presence of 30 or 100 ug/ml of MBP. Suppressor cell supernatants were also pretreated for 2 h with either anti-ILlO antibody (25 ug/ml) or anti-TGFp antibody (25 ug/ml ) and then cultured with MBP-specific cells in the presence of MBP. Cultures were incubated for 96 h at 37° C. The cultures were then

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39 pulsed with [ 3 H] -thymidine (1.0 uCi/well; Amersham, Indianapolis, IN) 18 h before harvesting onto filter paper discs using a cell harvester. Cell-associated radioactivity was quantified using a p-scintillation counter. Stimulation index was determined by dividing experimental CPM by control (unstimulated) CPM. Results IFNt -Treated Spleen Cells Inhibit MBP-Specific T Cell Proliferation We first addressed the question of whether IFNt can suppress MBP-specific T cell proliferation by induction of suppressor cells in NZW mouse spleen cells. Spleen cells were treated with IFNt in tissue culture or were obtained from mice injected intraperitoneally (i.p.) with IFNt, or from mice treated orally with IFNt. IFNT-treated spleen cells from all three sources inhibited MBP induced proliferation of spleen cells from EAE mice by as much as 80% relative to the control response (Figure 8). Similar to type I IFN induction of suppressor cells for antibody production (Johnson and Blalock, 1980), IFNt suppressed MBP-specific immune response via induction of suppressor cells. IFNt Induction of Suppressor Cells are Dose-Dependent IFNt and IFNp were compared at various concentrations for induction of suppressor cells in spleen cell cultures for inhibition of MBP stimulation of sensitized cells from EAE mice (Figure 9). For both IFNs, the induction of suppressor cells was dose dependent. IFNp was slightly more effective at induction of suppressor cells, but the slopes of the dose response curves for the two IFNs were similar. Thus, Type I IFN induction of suppressor cells is dose-dependent.

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44 IFN-t Suppressor Cells Protect Mice Against EAE Adoptive transfer of IFNt-induced suppressor cells to NZW mice immunized with bovine MBP was carried out in order to determine if the suppressor cells protected the mice from development of EAE. NZW mice have previously been shown to be susceptible to development of EAE after immunization with either rat MBP (Zamvil et ai, 1994; Kumar et ai, 1994) or bovine MBP (J. Schiffenbauer, unpublished observation). Others have shown the transfer of peripheral cells from orally administered IFN donor mice to recipient mice causes suppression of white blood cells (Fleischmann et ai, 1992). Suppressor cells induced in culture with IFNx were injected i.p. 48 h before, at the time of, and 48 h after immunization of mice with MBP. Suppressor cell-treated mice showed delayed onset of EAE (34.3 days) compared to untreated controls (19.6 days), and the incidence of EAE was 3 of 5 with lower severity of disease for suppressor cell-treated mice compared to 5 of 5 with higher severity of disease for untreated mice (Figure 10). Thus, adoptive transfer of IFNx induced suppressor cells significantly protected mice against EAE. IFNx-Induced Suppressor Cells are CD4 T Cells We next determined the phenotype of the suppressor cells by using antibody affinity columns to purify CD4 T cells and using specific CD4 antibody and complement to deplete CD4 T cells from rFNx-treated cultures (Figure 11). CD4 T cells purified from an IFNt-treated spleen cell preparation inhibited MBP-specific T cell responses by almost 50%, while non-CD4 T cultures from rFNt-treated spleen cells were without effect. The non-CD4 T cell preparations consisted of CD8 T cells, macrophages, and other cells. Thus, the suppressor cell appears to be a CD4 T cell.

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49 Suppressor Cells Produce Soluble Suppressor Factor(s) We previously showed that type I IFN-treated cells produced a suppressor factor for production of antibody to sheep red blood cells (Johnson and Blalock, 1980). EFNrtreated cells were thus examined for production of a suppressor factor. As shown in Figure 12 A, supematants from rFNx-treated spleen cells that had been incubated for 2 h at 37° C inhibited MBP-specific T cell responses. Inhibitory supematants were not produced by cells treated with IFNt that had been neutralized with specific antibody prior to treatment of cells. Further, the antibodies did not inhibit suppressor cell activity when added to cells after treatment with IFNt. Consistent with the CD4 T cell phenotype of the suppressor cell, supematants from EFNx-treated CD4 T cells suppressed the MBP-specific responses (Figure 12B). Thus, the IFNt-induced CD4 suppressor T cell produces soluble suppressor factor(s). IFNx-Induced Suppressor Cells Produce IL-10 and TGFft We next characterized the suppressor factors that IFNt induced in spleen cells using antibodies to IL-10 and TGFp. As shown in Figure 13 A, both monoclonal anti-EL-lO and monoclonal anti-TGF(3 antibodies blocked the suppressive activity of the suppressor cells on MBP-specific T cell responses. Similarly, both anti-EL-10 and anti-TGFp antibodies neutralized the suppressive activity of supematants from rFNx-induced suppressor cells on the MBP-specific T cell responses (Figure 13B). Not unexpectedly, the antibodies to IL10 and TGFp combined showed complete recovery as did each antibody showed separately (data not shown). Also, addition of the corresponding cytokines in excess reversed the blockage of suppression by the antibodies. The control monoclonal anti-IFNT antibody had no effect on the suppressor activity of the suppressor cells or their supernatant. Thus, both anti-IL-10 and anti-TGFp antibodies restored the MBP-induced response to that of the

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54 control, which suggests a synergistic interaction between IL-10 and TGFp in inducing suppression. IL-10 and TGFp Act Synergistically to Inhibit MBP-Specific T Cell Responses We next evaluated the possible synergistic interaction between EL10 and TGFp on suppression of MBP-induced mouse spleen cell proliferation. As shown in Figure 14, both EL10 and TGFp suppressed MBP-specific T cell responses individually, but EL10 and TGFp together enhanced the suppression of MBP-sensitized spleen cell proliferation in response to MBP. Both IL-10 and TGFp at a concentration of 8 ng/ml each, greatly reduced MBP-specific responses, compared to that obtained at 16 ng/ml of each factor. Thus, the combined effects of IL-10 and TGFp are apparently not additive. These data suggest that IL-10 and TGFp act synergistically at certain concentrations to inhibit MBPinduced EAE spleen cell proliferation. Discussion Data presented here demonstrate that IFNt induces CD4 T cells to become suppressor cells in NZW mice by oral administration or intraperitoneal injection of IFNt, and by treatment of mouse spleen cells with IFNt in tissue culture. The suppressor cells inhibit MBP stimulation of spleen cells from MBP-immunized mice, and protect mice against induction of EAE. Also, the CD4 T suppressor cells produce both IL-10 and TGFp, which act synergistically to inhibit MBP-specific T cell proliferation. Induction of suppressor cells can be blocked by pretreatment but not posttreatment of DrNx with neutralizing antibodies, thus establishing that induction of suppressor cells is specific for IFNt, but is not itself IFNt. Therefore, IFNt inhibition of EAE appears to occur via induction of suppressor cells and their suppressor factors such as IL-10 and TGFp.

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57 These finding are consistant with our previous observation that orally administered IFNt protected mice against EAE in the absence of detectable IFNt in the circulation (Soos et al, 1997). The induction of suppressor cells is not unique to IFNt, as IFNp also induced suppressor cells in spleen cell cultures. Further, the dose response curves for the two IFNs were similar. Also, these suppressor cells produce suppressor factors that inhibit MBP stimulation of EAE spleen cells. Thus, it is quite likely, then, that type I IFNs in general protect against autoimmune diseases such as MS by induction of suppressor cells and suppressor factors. As indicated above, IFNt protected mice against EAE when administered orally even though relatively little IFNt was found in the circulation (Soos et al, 1997). The gut is lined with over half of the cells of the immune system. The suppressor cells induced by oral IFNt administration must be mobile, since the autoreactive MBP-specific T cells that are inhibited are themselves mobile, and in fact migrate to the central nervous system to cause EAE in the absence of IFNt treatment. We have shown that IFNT-treated mice that are immunized with MBP show little or no lymphocyte infiltration of the CNS (Soos et al, 1997). The CD4 suppressor T cell produced both IL-10 and TGFp that acted synergistically to inhibit MBP stimulation of spleen cells from EAE mice. IL-10 and TGFp have previously been shown to inhibit events associated with autoimmune disease (Chaouat etal, 1995; Rotter al, 1994; Stevens et al, 1994; Johns etal, 1991; Schluesener and Lider, 1989). We have shown that IL-10 can be detected in sera of mice which received prolonged i.p. injections or prolonged oral feeding of IFNt (Soos et al, submitted). Here we have also demonstrated that IFNT-induced suppressor cells produce IL-10 and TGFp to synergistically inhibit MBP-specific T cell proliferation. Thus, the findings here serve as the basis for understanding how type I IFNs exert therapeutic effects in autoimmune neuropathies.

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CHAPTER 4 CONCLUSION IFNx, a recently discovered TEN, is a type I IFN that has pregnancy recognition hormone activity in ruminants. It possesses similar activities observed for the other type I IFNs, IFNa and DFNp, but in contrast, IFNx lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in animal studies. Considering the positive therapeutic value of the related IFNp for treatment of MS, IFNt has been examined for its ability to prevent the development of EAE, the animal model for MS. IFNx has been previously shown to prevent the development and superantigen-induced exacerbation of EAE in the absence of toxicity (Soos et ai, 1995a). These studies of IFNx protection against EAE involved initiation of IFN treatment before MBP immunization or before disease development. IFNx protected against both acute and chronic, relapsing EAE in mice, however, administration of IFN did not block sensitization, since cessation of treatment resulted in development of EAE (Soos et ai, 1997). In order to have potential for treatment of MS in humans, the IFNx must be effective in treatment of active EAE. In this study we show that both oral administration and ip injection of IFNx induced remission in SJL/J mice that had ongoing chronic active EAE disease and protected mice against secondary relapses. IFNx treatment reversed lymphocyte infiltration and microglial activation in the CNS. IFNx inhibition of antibody production against MBP may be a contributing mechanism by which IFNx inhibits further relapses of EAE. proliferation of effector B cells and T cells of EAE mice are inhibited by IFNx in both chronic and acute forms of EAE. Inhibition of MBP-specific T cell clones and reduced B cell responses could contribute to the reversal of disease and histopathological changes shown. Furthermore, IFNx can prevent EAE by induction of suppressor cells. Injection of these 58

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59 suppressor cells into mice delayed the onset of EAE. The suppressor cells were found to produce the inhibitory cytokine IL-10 and TGFp, which acted synergistically to inhibit MBP activation of T cells from EAE mice. This CD4 T suppressor cell is most likely the Th2 type based on the detection of TGFp and IL-10 in suppressor cell supernatants. Further, since this suppressor cell is induced by IFNx and probably also by other type I EFNs in the absence of MBP, it is most likely to be antigen-nonspecific in its effect. In fact, preliminary data suggest that suppressor cell supernatant inhibits mitogen stimulation of mouse spleen cells, and superantigen induced effects were similarly suppressed by CD4 T suppressor cells (Figure 15) and their supernatant (Figure 16) via IL-10 and TGFp. There was no evidence that non-CD4 T cells, including CD8 cells, possessed suppressor cell activity. This observation is in contrast to some other studies on suppressor cells (Nouri et al, 1991; Mukasa et al, 1994; Blank et al, 1995; Castedo et al, 1993). Other studies have also shown that the immune response is suppressed by antigen-specific CD4 Th2 cells (Karpus and Swanborg, 1991; Nabozny etal, 1991; Martinotti et al, 1995; Smith etal, 1991). Additional potential mechanisms for IFNt prevention of EAE could include altered cell migration into the CNS, as well as downregulation of MHC I and II molecules and certain co-stimulatory/adhesion molecules. We have previously shown that type I IFNs inhibit the progression of Daudi B cells through the Gl phase of the cell cycle (Subramaniam et al, 1998). Therefore, the inhibition of the cell cycle of cells in Gl may be a mechanism by which IFN-t could inhibit MBP-specific antibody production directly. Thus, other lines of investigation remain to be explored to completely understand the mechanism by which EFNt can prevent EAE. Others have seen similar inhibition of clinical disease and reversal of histopathological changes with IFNp used as the treatment for EAE mice (Yu et al, 1996). Furthermore, treatment of MS patients with EFNfJ in vivo as well as treatment of T cells

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O 3 C3 inj s w 8 "5 3 «fi • c x: o u c g E 8 a5 ^ c3 Z Si O S CL,

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63

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64 in vitro resulted in an inhibition of T cell activation (Noronha et al, 1993; Rudick et al, 1993). But, it was previously shown that the type I IFNs murine IFNcc and murine IFNp induced toxic side effects manifested as flu-like symptoms, fever, nausea and malaise when used as a therapeutic in humans (Degre, 1974; Fent and Zbinden, 1987). Although we have not focused on the lack of toxicity of IFNx for the mice in these studies, we have previously shown such lack of toxicity in tissue culture and in mice by monitoring for weight loss and bone marrow suppression (Soos et al, 1995a; Soos et al, 1997). The study here involved the use of ovine EFNx. There has not been a successful expression of a human EFNx with similar properties like those of ovine EFNx. Therefore, current studies are focused on "humanizing" the ovine IFNx by construction and expression of an ovinelFNx/human IFNaD chimeric. The chimeric is made up of residues 1-27 of the ovine IFNx and residues 28-166 of the human IFNaD, and differs from human IFNaD by 15 residues. The chimeric was constructed based on studies showing that the N-terminus of type I IFNs played a central role as to their toxicity or lack thereof (Pontzer et al, 1994; Subramaniam et al, 1995). These recent studies show that the IFNx/IFNaD chimeric lacks the toxicity associated with IFNaD with human PBMC and mouse splenocytes (Mujtaba et al. 1999). Preliminary data show that the IFNx/IFNaD chimeric also inhibits MBP stimulation of MBP-sensitized spleen cell (Table 2). The IFNx/IFNaD chimeric suppressed proliferation more effectively than IFNx but not as effectively as IFNaD. Viabilities were determined and showed that the EFNaD was the most toxic as compared to IFNx and the IFNx/IFNaD chimeric. Thus, the IFNx/IFNaD chimeric may be a better therapeutic for use in human diseases. Overall, the finding reported here indicates that IFNx is an effective treatment for ongoing active EAE, and this amelioration of disease is mediated by suppressor cells and their synergistically acting suppressor factors such as EL10 and TGF(3.

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65 Table 2. IFN inhibition of MBP-sensitized spleen cells EFN MBP-induced Proliferation Cell Viability (U/mD (% Inhibition) (%) IFNt (7000 U/ml) 21 ± 6.6 91 IFNt ( 1 5000 U/ml) 42 ± 2.0 89 IFNt (3000 U/ml) 57 ± 6.1 85 IFNx/IFNaD chimeric (7000 U/ml) 57 ± 2.4 86 IFNT/IFNaD chimeric ( 1 5000 U/ml) 58 ± 1.1 81 IFNx/IFNaD chimeric (30000 U/ml) 73 ± 1.3 81 IFNaD (7000 U/ml) 60 ± 2.4 84 IFNaD (15000 U/ml) 75 ± 2.3 83 IFNaD (30000 U/ml) 76 ± 4.6 76 CPM and cell viability values for media-treated spleen cell cultures were 2710 ± 77 and 93%, respectively.

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BIOGRAPHICAL SKETCH Mustafa Ghulam Mujtaba was born in Kabul, Afghanistan, on September 18, 1973, to Ghulam and Uzra Mujtaba. During the war with the communist Soviet Union, the Mujtaba family left Afghanistan for the neighboring country, Pakistan, in 1983 and then to Cape Coral, Florida in June 1984. Mustafa completed his middle school in Cape Coral. The Mujtaba family then moved to Lake City, Florida in 1988 after his father had a job transfer. He graduated from Columbia High School in 1992. Mustafa attended college at the University of Florida majoring in Microbiology and Cell Science. In 1995, he received his Bachelor of Science degree in Microbiology and was accepted by the same department as a graduate student. He was kindly taken into the laboratory of Dr. Howard Johnson. After completion of his doctoral program, Mustafa plans to pursue research in a similar area as a postdoctoral fellow. 78

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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,,Chair Graduate Research Professor of Microbiology and Cell Science 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. Edward MJ Professor of Cell Science icrobiclogy and 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. Julie fviaupin Assistant Professor 6i Microbiology and Cell Science 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. Jaf^K. Associal Yamamo associate Professor of Veterinary Medicine

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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. t Wolfgang J. Streit Professor of Neuroscience This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August 1999 Dean, College of Agriculture Dean, Graduate School


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