Experimental autoimmune uveitis: Molecular mimicry and oral tolerance

Immuno log ic R e s e a w G h Immunol Res 1996; 15:323-346

V.K. Singh a K. Nagaraju b

a Department of Immunology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India;

b Connective Tissue Disease Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Md., USA

Experimental Autoimmune Uveitis: Molecular Mimicry and Oral Tolerance

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Key Words Uveitis, experimental

autoimmune Molecular mimicry Oral tolerance Autoantigens

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Abstract Intraocular inflammatory disease or uveitis, which affects the uveal tract and the retina of the eyes in human, is the major cause of visual impairment. Experimental autoimmune uveitis (EAU) is a T-cell-mediated autoimmune disease directed against retinal proteins and has been studied in several mammalian species including subhuman primates as a model for human posterior uveitis. Autoimmune responses provoked by molecular mimicry occur when the nonself and host determinants are similar enough to cross-react yet different enough to break immunological tolerance, and is one of the proposed mechanisms for induction of autoimmune diseases. Thera- peutic immunomodulatory strategies have been used to induce antigen-specific peripheral immune tolerance in animal models of T-cell-mediated autoimmune diseases by oral administration of autoantigens. Oral tolerance leads to unique mechanisms of tissue and disease-specific immunosuppres- sion, which would circumvent the immunotherapeutic problem of multiple target tissue autoreactivity. Several groups have investigated the effects of delivering autoantigens across gastric mucosal surfaces. This review briefly discusses molecular mimicry and the mechanism of induction of oral tolerance with respect to immunopathogenesis of T-cell-mediated autoimmune diseases in general and EAU in particular.

KARGER E-Mail karger@karger,ch Fax+41 61 306 12 34 http://www.karger.cb

�9 1996 S. Karger AG, Basel Dr. V.K. Singh Building 6, Room 310, MSC 2740 LRCMB, NE1, NIH Bethesda, MD 20892 (USA)

Introduction

The ocular disease developing in susceptible strains of animals immunized with S antigen or other retinal or uveal antigens has been termed experimental autoimmune uveitis (or uveoretinitis; EAU) [1]. The S antigen prepa- rations used in most studies were obtained from bovine or guinea pig retinas and both were found to be similarly potent. The capacity of S antigen to induce EAU depends on the inclusion of adjuvants in the immunization; no disease is induced in animals injected with S antigen without proper adjuvant [2]. Com- plete Freund's adjuvant (CFA), which is the adjuvant used by most investigators for induction of EAU, is a mixture of mineral oil with mycobacteria. However, later studies have shown that the induction of EAU is further enhanced by a simultaneous injection of the animals with Bordetella pertussis bacteria or a purified preparation of a single component of these bacteria, called pertussigen or pertussis toxin [3]. The clinical as well as histopathological features of EAU in animals closely resemble certain uveitic conditions in humans and are considered a model for these diseases [ 1, 4, 5]. The possible role of S antigen in the etiopathogenesis of human uveitis is further supported by the fact that many patients with intermediate or posterior uveitis have immune responses to the human and bovine sequences [6-10].

Molecular mimicry is a process in which the presence of epitopes (either linear or con- formational) is shared between a foreign and a host protein. When the shared determinants are identical, the host shows tolerance to the foreign epitope by recognizing it as self. Ho- mologous but nonidentical determinants dif- fering in one or more amino acids may be foreign enough to elicit an immune response. The immune response initiated against these foreign epitopes may, however, react with the

closely homologous 'self" host protein. Hu- moral and cell-mediated immune responses generated against this antigen might cross- react with a self epitope, thereby causing cellular injury leading to disease. Once the foreign agent initiates these processes, it need not to be present during the autoimmune destruc- tion that follows [11, 12]. Molecular mimicry has been suggested as one of the mechanisms for induction of autoimmune diseases.

South American Indians have been reported to have ingested the leaves from poi- son ivy plants in order to suppress a systemic delayed hypersensitivity response that is induced following contact with this plant [13]. Almost 50 years ago it was first noticed that experimental drug allergies could be suppressed by prior feeding of the sensitizing agent [14]. In the recent past it was demonstrated that the oral administration of specific antigen could attenuate disease activity in animal models of various autoimmune diseases such as multiple sclerosis, uveitis, rheumatoid arthritis, thyroiditis, myasthenia gravis, and insulin-dependent diabetes mellitus (IDDM) [13, 15-17]. Initial human trials in multiple sclerosis, rheumatoid arthritis, and uveitis have shown promising results [18-20]. The exact cellular and molecular mechanisms involved in the generation of antigen-specific unresponsiveness in adults are not well under- stood. Therefore, it is important to under- stand the anatomy and physiology of the gut- associated lymphoid tissue, and the immunopathogenesis of T-cell-mediated autoimmune diseases.

Generally, autoimmune diseases characteristically exhibit intercalating exacerbations and remissions or complete recovery indicat- ing that disease-associated symptoms may resolve through the activity of the immune system. This suggests that autoimmune diseases may be corrected by using natural immunological agents that participate in the selection

324 Immunol Res 1996;15:323-346 Singh/Nagaraju

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Uveitopathogenic Autoantigens in Retina

S antigen, also known as arrestin, is a 48-kDa protein and is the most potent retinal antigen which initiates autoimmune inflammatory reactions in the eye. It is specifically localized in visual tissues and is remarkably conserved through the phylogenetic development of vision organs, lmmunohistochemical and electron-microscopic studies showed that S antigen localizes mainly in the rod outer segments, on both sides of the disc membranes in the photoreceptor cell layer of the retina [21, 22]. The unique relationship between S antigen and the vision system is supported by the presence of this antigen in the pineal gland [23]. Pineal gland has a photoreceptor function in lower ver- tebrates and retains some anatomical features of the retina even in mammals. The mammalian pineal gland is believed to have lost its photoreceptor function as it evolved from a median eye into a secretory organ. In the course of evolution, major photoreceptor proteins like rhodopsin and ct-transducin might have been elim- inated or reduced to negligible amounts. In contrast, other photoreceptor cell proteins such as interphotoreceptor retinoid binding protein (IRBP) and rhodopsin kinase were localized to the pineal gland [4]. It is of interest to note that in accord with its content of S antigen and IRBP, the pineal gland is often affected in the inflammatory processes which develop in animals immunized with S antigen or IRBP and this condition is termed experimental autoimmune pinealitis (EAP) [24].

S antigen is one of the major proteins of the retina and pineal gland and is estimated to comprise approximately 0.5 and 0.2% of the total soluble protein of the retina and pineal gland, respectively. S antigen has been purified to homogeneity and reported to have an

inhibitory role in the activated phototransduction cascade [25-27]. Although the exact mechanism of the inhibition is not known, it is postulated that S antigen binds to photoexcited phosphorylated rhodopsin. The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding ofarrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (G1) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin in the phototransduction pathway [27].

Cleavage of the S antigen molecule by enzymatic (trypsin, chymotrypsin, Staphylococcus aureus V8 pro- tease) or chemical (cyanogen bromide) agents has been used to obtain information about the peptide components involved in the immunogenic and uveitogenic activities of this molecule [28]. The complete amino acid sequence of human, bovine, rat and mouse retinal S antigen as well as rat pineal gland S antigen has been determined [29-37]. Comparison of the amino acid sequences indicates a high degree of sequence homology among these species [37]. The number of amino acid residues in S antigen are 403 (rat as well as mouse), 404 (bovine) or 405 (human). Based on amino acid sequence data, molecular weights of bovine S antigen and human S antigen are determined to be 45,275 Da and 45,045 Da, respectively [28, 37]. Analysis of nucleotide sequences of S antigen cDNA also showed a high degree of sequence homology among the mouse, rat, bovine and human. S antigen genes are highly homologous and have 16 exons interrupted by 15 introns spanning approximately 50 kbp in length [37]. The introns are significantly larger than the exons and the average size of the introns is approximately 3 kbp in length. The genes comprise 97% introns and 3% exons. Each splice junction was in good agreement with the GT/AG rule. Comparison of the amino acid sequences of rat pineal gland and retinal S antigen indicated a significant degree of homology. Analysis of nucleotide sequences of the above two cDNAs isolated from retinal and pineal gland libraries showed high sequence similarity [29, 36]. These results suggest that the S antigen in the retina and pineal gland has a high degree of similarity at the transcript as well as polypeptide level. The S antigen gene is assigned to chromosome 2q24-37 in the human and to the centromeric portion of chromosome 1 near the IDH-1 locus in the mouse [38, 39]. Secondary structure prediction and circular dichroic spectroscopy show that S antigen has predominantly a ~sheet conformation [28].

Although S antigen was previously believed to be a highly specialized single protein, it is now apparent

Experimental Autoimmune Uveitis Immunol Res 1996; 15:323-346 325

that S antigens are a family of multiple similar proteins [37]. Several arrestins have been sequenced from different organs of various species and few of them are human, bovine, rat, mouse retinal arrestin (S antigen), rat pineal S antigen, 13-adrenergic arrestins from human (13-1 and 13-2), bovine (13-1 and 13-2), rat (13-1 and 13-2), human thyroid, lateral eyes of Limulus polyphe- mus (horseshoe crab), compound eyes of Drosophila rnelanogaster I and II, Drosophila miranda, locust antennae (Locusta migratoria), corn earworm antennae (Heliothis virescens) and human cone arrestin, cone arrestin of Xenopus laevis [40-44]. All of the above arrestin sequences have shown significant sequence homology among them [37, 45]. Furthermore, additional arrestins have been identified by immuno- detection [46]. The existence of different subtypes of arrestins as well as the possibility of modifying the proteins via binding of certain ligands or via phosphorylation may allow arrestin to play different roles in cellular signalling.

Another major retinal antigen capable of inducing EAU and EAP is IRBP. It is a 140-kDa evolutionarily conserved glycoprotein with a 4-fold partially homologous repeat structure with 30-40% amino acid sequence homology between repeats. It has been isolated and well characterized. IRBP is thought to be involved in the transport of vitamin A derivatives between the retinal pigment epithelium and the photoreceptors. It has been sequenced from cDNA and gene [47, 48] and found to be equally potent as a uveitopathogenic molecule as S antigen, producing EAU at very low doses. EAU induced by IRBP resembled that induced by S antigen also in its basic clinical and histological ocular changes, and by the involvement of the pineal gland [4]. Animal experiment studies have shown that there are several uveitopathogenic epitopes in bovine IRBP, and peptide 1169-1191 has been shown to be immunodominant [49, 50]. Recently, Silver et al. [51] have reported an additional uveitopathogenic epitope ( 161- 180) in human IRBP for B 10.RIII mice. Furthermore, rhodopsin or opsin, A antigen, transducin and cGMP phosphodiesterase have also been reported to induce EAU [41.

EAU as a Model for Uveit is

S antigen is a highly uveitopathogenic protein which induces EAU and EAP in susceptible animal strains [1-3, 26]. EAU has been reproducibly induced in a variety of animals,

including primates [5], rabbits [52], guinea pigs [26], rats [53], and recently in mice [54, 55]. The clinical and histopathological ap- pearance of uveitis occurring in these models closely resembles certain uveitic conditions in man [1, 4, 5, 28]. Usually EAU has been found to be associated with EAP [24]. These auto immune inf lammatory condit ions have been studied extensively as an animal model for human uveitis [ I, 5].

The clinical symptoms observed in rats with EAU are hyperemia of the conjunctiva, pericorneal and iris vasodilation and accumulation of inflammatory exudate and cells in the anterior chamber and vitreous. Within 2 - 4 days after onset of the disease, the ocular inflammation reaches its peak such as pro- truding eyes due to severe periocular inflammation, corneal edema, intense cell infiltration in the anterior chamber and vitreous, and posterior synechiae of the iris. The clinical changes disappear rapidly in the rat and the eyes become clinically quiet. The histopathological changes in rats with EAU following immunizat ion with various doses of S antigen or IRBP demonstrate a high degree of confbr- mity. Initial histological changes are exudate formation and accumulation of inf lammatory cells in the photoreceptor cell layer. The retina is most often detached with accumulation of serum underneath. Other components of the eye rapidly become involved as well, with inflammatory cells infiltrating the vitreous, iris, ciliary body and anterior and posterior chambers. The choroid becomes affected 2-4 days after the disease onset. The infiltrate consists mainly of a mixture of polymorpho- nuclear and mononuclear leukocytes (lymphocytes and histiocytes). The inf lammatory cells disappear from the anterior chamber followed by cells from the posterior chamber. During this inflammatory process the photoreceptor cell layer is partially or completely lost [4].

326 |mmunol Res 1996;l 5:323-346 Singh/Nagaraju

Changes in primates with EAU are of particular interest because of their possible relationship to those in certain human uveitic conditions. The histological changes consisted of vasculitis and chorioretinitis with remark- able involvement of plasma cells and poly- morphonuclear cells, periphlebitis, focal loss of photoreceptor cells as well as areas of sub- retinal inflammation and occasional bulging of the retina into the vitreous cavity. Some of these changes resemble those observed in human disease such as birdshot retinochoroi- dopathy [5].

Histopathology of pineal glands of rats with EAU shows focal cell infiltration, mainly at the peripheral or subcapsular areas of the gland. In severe cases, however, infiltrating cells are found in central areas as well. Unlike the massive infiltration with polymorphonu- clear leukocytes in the eyes, infiltration in the pineal glands of the same rats consists virtual- ly only of mononuclear leukocytes [4].

Although humoral responses are shown to play a role in EAU [56], the cell-mediated responses play the dominant role in the pathogenesis of EAU; since the disease does not develop in nude rats, the disease develops following the adoptive transfer of lymph node cells from animals previously immunized with S antigen, the majority of cells participat- ing in the inflammatory response can be identified as T cells, and the disease can be inhibit- ed by cyclosporin A [53, 57, 58]. Homozygous nude rats failed to develop EAU on challenge with S antigen, while heterozygous rats re- sponded very well. In addition, EAU could be induced in the nonresponder nude rats by the adoptive transfer of in vitro stimulated lymphocytes from responder rats [53].

The knowledge of S antigen amino acid sequences allowed investigators to identify the uveitopathogenic sites by the use of small synthetic peptides. Many investigators have focused on the identification of epitopes re-

sponsible for the immunogenicity and immu- nopathogenicity of S antigen. Three sites of bovine or human S antigen were identified as being immunopathogenic in Lewis rats: peptide 303-320 (peptide M) and 286-297 (peptide N) which are nondominant, and 343-362 (peptide G) which has immunodominant characteristics [59-62]. Later, de Smet et al. [63] mapped the immunogenic and immunopathogenic determinants of human S antigen in the Lewis rat by using overlapping synthetic peptides. They identified most pathogenic sequences as 180-200, 340-360 and 350-370 peptides. Ten peptide sequences induced visi- ble inflammation in the eye. A total of 23 peptides gave an in vitro proliferative response following immunization in animals. These studies have shown that there are several uveitopathogenic determinants within S antigen, sugesting the same may be true in uveitis patients. The existence of multiple determinants would place considerable restrictions on the feasibility of immunotherapy in patients. Immunotherapy directed against a single determinant is unlikely to be effective in humans. Only approaches like oral tolerance that can induce nonspecific suppression, even if limited to a specific inflammatory site, are likely to be effective.

Molecular Mimicry and EAU

The phenomenon by which the host re- sponds to exogenous antigens that cross-react with self by sharing of linear or conformation- al epitopes common to microbial antigens and host structures is known as molecular mimicry and it may provide a mechanism for triggering autoimmune diseases and tolerance [11, 64]. Earlier investigations have focused on cross-reaction between B cell epitopes and the subsequent generation of autoantibodies. However, mimicry at the B cell level cannot

Experimental Autoimmune Uveitis Irnrnunol Res 1996;15:323-346 327

provide the pathogenic basis for T-cell-mediated organ-specific autoimmune diseases where CD4+ T cells either produce inflammatory cytokines or provide help to B ceils. Cross-reaction between self and nonselfT cell peptides has also been documented in various systems [ 12].

The question of how autoimmune diseases such as endogenous posterior uveitis are initiated remains to be fully answered. Circum- stantial evidence suggests a link between autoimmune diseases and triggering events such as a recent viral or bacterial infection. This has led to the hypothesis that circulating autoreactive T cells, present in all healthy indi- viduals, recognize epitopes on processed foreign antigen, which possesses extensive amino acid sequence homology with host autoantigens.

According to the above hypothesis of molecular mimicry, invading microorganisms are rapidly attacked by innate immune mechanisms involving acute inflammatory cells such as neutrophils and monocytes. The partially degraded foreign antigens are then taken up by dendritic cells and transported to lymph nodes where the immunodominant antigenic peptides are presented to T cells, most of which induce a full range of immunological sequelae including cytotoxic T cell responses, B cell responses and memory T and B cell responses. Due to their sequence homology with autoantigenic peptides, certain foreign peptides may also activate autoreactive T cells. These activated T cells then enter the circulation, but do not necessarily home to their target tissue unless the endothelium has also been activated by cytokines such as interleukin-1 (IL-1) and interferon-,/which induce specific adhesion molecules on their surface. This may occur if the response to the original attack by microorganisms is sufficiently severe and sustained to the point where a threshold concentration of cytokines and/or

endotoxin is released into the circulation [65].

Several examples of possible molecular mimicry between retinal antigens and proteins from microorganisms exist. For in- stance, studies have demonstrated that the onchocerca worm can induce a keratitis associated with anticomeal autoantibodies when injected into the subconjunctival space. The onchocerca parasite is responsible for en- demic river blindness, but usually does so by involving the posterior segment. Recently, an onchocercal antigen (Ov39) has been found to cross-react with a 44-kDa antigen located in retinal pigment epithelium, the neural retina and the optic nerve. The retinal antigen has not yet been fully characterized, but it does not demonstrate identity with S antigen. However, when Lewis rats were immunized with Ov39, they developed a low grade anteri- or and posterior uveitis with some similarities to EAU and experimental autoimmune anterior uveitis, but without evidence of autoimmunity to retinal S antigen [65]. It is probable that the autoimmune response is to the 44- kDa antigen. Ov39-induced uveitis, therefore, represents the appropriate example of common linkage between immune responses to foreign and self-antigens in the eye.

The knowledge of the uveitopathogenic sites of S antigen enabled us to look for similar sequences in foreign antigens. We compared amino acid sequences of various uveitopathogenic peptides of S antigens with a variety of proteins in the National Biomedical Research Foundation data base for sequence homology. We found 4- to 6-amino acid sequence homology between one of the uveitopathogenic peptides of S antigen (peptide M) and various proteins, such as DNA polymerase of hepatitis B virus, gag-pol polyprotein ofAKV murine leukemia virus, Moloney murine leukemia virus, Moloney murine sarcoma virus, gag-pol polyprotein of baboon

328 immunol Res 1996;15:323-346 Singh/Nagaraju

Table 1. Sequence homology between human retinal S antigen fragment (peptide M) and other proteins

No. Source ofpeptide Amino acid sequence Ref. No.

1 Retinal S antigen (peptide M) 2 Yeast histone H3 3 E. coli elongation factor 4 E. coli hypothetical protein 5 Potato inhibitor IIa 6 Hepatitis B virus protein 7 Baboon endogenous virus 8 Moloney murine leukemia virus 9 Moloney murine sarcoma virus

10 AKV routine leukemia virus

D T N L A S S T I I K E l 59 D T N L A A I H A K R V 1 68-70 A R H L A A S I A F K E 68 L A N L A S S T Q L C K ~ 67 D T N I A S Y K S U C E 66 L T N L L S S N L S W L l 66 P T N L A K V R T I T Q I 66 P T N L A K V K G I T Q ~ 66 P T N L A K V K G I T Q 1 66 P T N L A K V K G I T Q ~ 66

Underlinings indicate identical amino acid residues with peptide M. Peptides have been found to consistently induce EAU in Lewis rats.

endogenous virus, Escherichia coli hypothetical protein E-116, yeast histone H3 and pota- to proteinase inhibitor IIa (table 1) [12]. These amino acid sequences were used to syn- thesize oligopeptides of 12-20 amino acids by conventional solid phase chemistries on resin using an automated peptide synthesizer and purified by high-performance liquid chroma- tography [61]. Lewis rats were immunized intradermally with various peptides emulsi- fied in CFA followed by intravenous injection with B. pertussis. Interestingly, our results showed induction of EAU with peptides derived from DNA polymerase of hepatitis B virus, gag-pol polyprotein ofAKV murine leukemia virus, gag-pol polyprotein of baboon endogenous virus [66], E. coli hypothetical protein E-116 [67] and yeast histone H3 [68]. In addition, native yeast histone purified from Saccharomyces cerevisiae was found to be capable of inducing EAU in Lewis rats [68]. Yeast histone H3 peptide also induced EAU in monkeys [69]. Furthermore, rats developing EAU as a result of immunization with microbial peptides showed an associated

EAP characterized by a lymphocytic infiltration of the subcapsular area of the pineal glands [68]. The histology of the diseased retina and pineal glands from animals immunized with peptide M or various microbial peptides was indistinguishable [66-68]. Lymph node cells from rats immunized with either peptide M or one of the peptides men- tioned above showed a significant degree of cross-reaction, i.e. lymph node cells from animals immunized with peptide M showed significant in vitro T-cell-proliferative response in the presence of one of the above microbial peptides or vice versa [70]. Lymph node cells from animals immunized either with peptide M or yeast histone H3 peptide and stimulated in vitro with either peptide M or yeast histone H3 peptide induced EAU when injected in- traperitoneally in naive syngeneic rats. The histopathotogy showed severe inflammation in the uveal tract and complete loss of photoreceptor cells which was indistinguishable from EAU induced with peptide M or native S antigen [70].

Experimental Autoimmune Uveitis lmmunol Res 1996;15:323-346 329

Table 2. Some important examples of molecular mimicry

No. Diseases Homologous antigens Ref. No.

1 EAU 2 EAE 3 AS 4 Myocarditis 5 Rheumatoid arthritis 6 Ovarian autoimmunity 7 Celiac disease 8 IDDM

S antigen and various microbial proteins 66-70 MBP and HBVP 73 HLA-B27 and proteins from Klebsiella, Yersinia, Salmonella, Shigella 75, 76 Heart tissue myosin and coxsackievirus capsid protein 77, 78 Human heat shock protein and mycobacterial heat shock protein 79, 80 Ovarian and nonovarian peptides 82 Wheat gluten EIB protein of adenovirus 12 83 Islet antigens and CVB protein 84, 85

HLA = H u m a n leukocyte antigen.

The likelihood of any particular autoimmune disease and an infectious disease having a common antigenic ontogeny depends on many factors such as the degree of epitope homology between the antigens, the immuno- dominance of the common antigenic sequence, the degree of MHC restriction and the avidity of class II binding, the dose of antigenic peptide released for presentation, and the number of potentially primed autoreactive T cell clones. The chances are increased after infection with superantigens. Superanti- gens are proteins that activate the T cell receptor (TCR) not via the conventional MHC- peptide mechanism, but by complexing the MHC class II to the TCR directly via the TCR 13 chain outside the peptide binding site. Superantigens have been implicated as causa- tive agents in a variety of autoimmune diseases [71]. Infection with certain superantigens may lead to the activation of up to 10% of the T cell population, which significantly en- hances the chances that one or more of these clones may be responsive to presentation of a self peptide. In particular, it has recently been demonstrated that the superantigen Staphylo- coccus M peptide contains sequences homologous to certain peptides of retinal S antigen that can induce EAU. In addition, these pep-

tides preferentially associate with certain V13 T cell receptor chains which appear to be used at greater frequency in patients with posterior uveitis [72].

Molecular Mimicry in Other Autoimmune Diseases

There are various examples of molecular mimicry in different systems (table 2) and some of them are discussed below. Fujinami and Oldstone [73] have reported 6-amino acid sequence homology between hepatitis B virus polymerase (HBVP) and the encephalitogenic site of myelin basic protein (MBP) which causes experimental allergic encephalomyelitis (EAE), the animal model for multiple sclerosis. Rabbits injected with synthetic peptide showed antibody responses that cross-reacted with MBP. The peripheral blood mononuclear cells proliferated significantly when in- cubated with either MBP or HBVP. In addition, these rabbits showed central nervous system lesions reminiscent of EAE induced by the immunization with MBP.

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disorder particularly affecting the axial skeleton. A link between AS


and the major histocompatibility antigen HLA-B27 has been well established. Over 95% of patients with AS are HLA-B27-positive. There are seven subtypes of HLA-B27 ranging from B'2701 to B'2707. It has been suggested that B'2705 is the most common HLA-B27 subtype in the Caucasian population, whilst it is proposed that B'2703 is the most common in black populations, where AS is rare. The observation has led to the sugges- tion that B'2703 is not associated with AS. The cause of AS remains controversial, although an environmental agent in the form of the gram-negative bacterium, Klebsiella pneumoniae, present in the gut, has been suggested as a possible etiological agent to which antibodies have been shown. Schwimmbeck et al. [74] identified an amino acid homology, QTDRED found in residues 72-77 ofB'2705 and residues 188-193 of K. pneumoniae ni- trogenase enzyme. Others have shown homology between HLA-B27 and Yersinia pseudo- tuberculosis adhesin, outer surface of protein of Yersinia enterocolitica and Salmonella ty- phimztrium and Shigella flexneri [75]. The validity of this homology, as a cross-reactive antigen, has, however, been criticized, in both biochemical and immunochemical terms. Re- cently, Fielder et al. [76] have shown novel homology between two sequences of K. pneumoniae pulD secretion protein (DRDE) with HLA-B27 (DRED) and pulA (pullulanase) enzyme (Gly-X-Pro) with type I, III and IV collagen, respectively. Elevated levels of antibody in AS patients were detected against 16 mer synthetic peptides of HLA-B27 and pulD, pulA and type I and IV collagen.

Clinical studies show that a higher proportion of patients with chronic myocarditis or dilated cardiomyopathies contain serum IgM antibodies to coxsackievirus group B (CVB) than patients with heart disease of other infectious etiologies or immunological causes. Mo- lecular mimicry has been proposed to be a

mechanism that may explain some cases of CVB-induced chronic myocarditis in humans and in some strains of mice that model the human disease [77]. The hypothesis of shared epitope between CVB capsid proteins in and on normal cells is supported by the fact that a neutralizing monoclonal antibody (mAb) against CVB4 was shown to bind to viral capsid polypeptide VP 1 and to heart tissue of several animals. This mAb was directed against an epitope in heart tissue-specific myosin and not skeletal myosin. A neutralizing mAb against CVB3 bound to epitopes on normal mouse myocytes, HeLa cells and HEp2 cells. A nonneutralizing mAb that was generated against murine heart myocytes, and perhaps against the highly myocarditic CVB3 receptor was found to recognize CVB3. This mAb can partially protect BALB/c mice against CVB3- induced murine myocarditis and can also recognize an epitope on the adhesin nucleotide translocator protein of murine myocytes. None of these mAbs to CVB4 and CVB3 was reported to induce cytopathic changes in cells in vitro or pathological alterations in heart tissues of mice inoculated with a mAb [78].

In patients with rheumatoid arthritis, peptides from a mycobacteria165-kDa heat shock protein have been shown to cross-react with peptides from proteoglyca_q link proteins and human heat shock protein 60 [79, 80]. More recently, viral peptides were demonstrated to activate MBP-specific T cell clones isolated from multiple sclerosis patients [81]. Garza and Tung [82] have demonstrated that close to 50% of nonovarian peptides that share some critical residues of the ovarian peptides can cause ovarian autoimmunity.

Celiac disease is characterized by small intestinal mucosal injury and malabsorption. The disease is activated by dietary exposure to wheat gluten. Patients with celiac diseases show higher titer of antibodies against A-gliadin and the E1B protein of adenovirus sub-

Experimental Autoimmune Uveitis Immunol Res 1996:15:323-346 331

type 12, but not normal controls, suggesting that a cross-reactive immune response against the wheat protein A-gliadin initially induced by adenovirus 12 intestinal infection plays a role in the pathogenesis of celiac disease [83]. Recently, similarity between one peptide of the CVB and islet autoantigen, carboxypepti- dase H, autoantibodies to which have been claimed to be characteristic of IDDM, has been reported [84]. This is in addition to another homology between islet autoantigen, glutamic acid decarboxylase and the CVB which was reported earlier [85].

Oral Tolerance in Autoimmune Diseases

Gttt-Associated Lymphoid Tissue and Handling of Ingested Antigens by the Gut The lymphoid system of mammals is com-

posed of primary lymphoid organs, the bone marrow and the thymus, and secondary lymphoid organs such as diffuse lymphoid tissue, lymphoid follicles, Peyer's patches, tonsil, lymph nodes and spleen. The bone marrow and the thymus are responsible for the maturation of stem cells into B and T lymphocytes, respectively. The secondary lymphoid organs are the sites where the antigen-dependent and antigen-independent interactions of mature lymphocytes take place leading to an effective immune response. The secondary lymphoid organs are conveniently situated near the vast mucosal surface, lining the digestive, respiratory and urinogenital system. Gut-associated lymphoid tissue (GALT) consists of aggre- gated lymphoid follicles in the small intestines, the appendix vermiformis, colonic patches and solitary lymphoid nodules [86, 87]. GALT has two functions: to protect the host from pathogens entering through the gut, and preventing the host immune system from mounting an immune response to ingested

antigens. The wall of the intestine consists of seven layers which from the inner towards the outer surface are: epithelium, lamina propria, lamina muscularis, mucosae, submucosa, the layer of circular muscles and the layer of lon- gitudinal muscles. Of these the first three form the intestinal villi. The submucosa contains diffuse lymphoid tissue with dense areas of solitary lymphoid follicles especially in the ileum. This portion of the small intestine has clusters of lymphoid follicles called Peyer's patches. Each patch is seen on the inner surface as a group of bare dome-shaped structures in the microvilli. The epithelium of the dome overlying each follicle is comprised of specialized cells with many microfolds called M cells. These M cells take up particulate antigens by endocytosis and process and present antigens either directly to T cells or alternatively transfer them to antigen-presenting cells (APCs) such as dendritic cells or Lan- gerhans cells. In Peyer's patches the B cells are committed to immunoglobulin classA and subsequently populate the lamina propria [88]. The origin of the lymphocytes present in the lamina propria and in the intraepithelial portion of the intestine is not known. Charac- terization of lymphoid populations in these regions shows that these are predominantly T cells with et/13 TCR and are of the CD8 phe- noptype, CD45 RO antigen suggesting they are MHC class I-restricted memory T cells. These ceils are specific for a limited number of antigens [89]. A small proportion of intraepithelial lymphocytes (IEL) have V/~5 TCR.

Traditionally, GALT is considered as a secondary lymphoid tissue. The following observations made during the past few years suggest that GALT may have functions similar to the thymus. (1)Murine IEL can develop via an extrathymic pathway [90-96] giving rise to two distinct subsets of T cells, one with a/J3 TCR repertoire suggesting thymus selection and phenotypically with CD8 et/13 heterodi-

332 Immunol Res 1996:15:323-346 Singh/Nagaraju

mer, and another population appears to have developed extrathymically with a phenotype of CD8 a/a homodimer [90, 97]. (2)Yrans- genic mouse models expressing transgene for a T/8 TCR specific for MHC class I molecule suggest the presence of anergic autoreactive T/ 8 IEL in the absence of these clones in the peripheral circulation [98]. Similar results were obtained using mice transgenic for an a/ 13 TCR specific for male H-Y antigen [97]. (3) The presence of RAG 1 and RAG2 mRNA in the adult and fetal intestines. (4) Studies on athymic radiation chimeras showed generation of CD4+, CD8 a/J3+, CD4+/CD8 a/a++ mucosal T cell subsets in the gut upon recon- stitution with T-cell-depleted bone marrow or fetal liver [99].This evidence indicates that GALT may have some functions similar to those of primary lymphoid organs, such as thymus. It may be speculated that GALT might be involved in inducing clonal anergy and clonal deletion in a similar manner like the thymus.

Diet consists of a complex mixture of sub- stances which are potentially antigenic. The mucosal surface of the gut is covered for the most part by a monolayer of epithelial cells. A minority population of epithelial cells is highly specialized (the M cells) for presenting antigens to the cells of the immune system, whereas a major population of diverse epithelial and glandular cells selectively exports polymeric immunoglobulins to mucosal surfaces. The M cells are important because of their unique position over the organized lymphoid tissues that serve as inductive sites for mucosal immune responses and their ability to transport antigens with great efficiency. The immunological consequences of antigen uptake and transport are determined in part by the nature and capacity of the intracellular pathways through which antigens are directed. Macro- molecules cross intestinal epithelial cells in three ways: shuttle through absorptive cells on

specific receptors and molecules that bind to a receptor pass or they can pass through M cells [100] or antigen fragments cross epithelial cells for presentation by MHC molecules at the basolateral surface [I01]. Enterocytes present peptides originated in the cytosol through MHC class I to CD8+ intraepithelial lymphocytes [102]. These CD8+ cells prolifer- ate and secrete various cytokines which influence the immune response in a variety of ways. MHC class II antigens are present on the basolateral surface as well as in intracellular vesicles and on apical membranes of intestinal cells [ 103]. These pickup peptides are taken up by adsorptive or fluid phase endocytosis into common endosomes and present to CD4+ lymphocytes in the lamina propria. Since CD4+ lymphocytes are not generally found within epithelial cells, it is not known how fre- quently MHC class II-presented peptides in- teract with CD4+ lymphocytes in the lamina propria [104]. These epithelial MHC molecules might be involved in the development of thymus-independent IEL (TCR a/13 or T/8), suggesting that MHC class II+ enterocytes and lamina propria lymphocytes cooperate to modulate the lymphocyte repertoire in the gut. Primed T and B cells migrate through lymph to the peripheral blood circulation and extrav- agate mainly in the intestinal lamina propria. Intestinal B cells differentiate into IgA-secret- ing plasma cells in the lamina propria under the influence of macrophages, APCs and CD4+ T ceils. Most CD8+ T cells migrate into the villus epithelium, perhaps to mediate oral tolerance to food antigens.

Factors Determining the Induction and Maintenance of Oral Tolerance Various factors such as nature of the anti-

gen, concentration of the antigen, genetic makeup and the age of the animal determine the induction and maintenance of oral tolerance.

Experimental Autoimrnune Uveitis lmmunol Res 1996;15:323-346 333

Live organisms such as viruses and bacteria induce active immune response rather than tolerance by the oral route, whereas the same organisms if killed or inactivated induce oral tolerance [ 105]. How the gut immune system can distinguish between immunogenic and tolerogenic stimuli is currently not known. It might be due to different modes of antigen processing and presentation involved in handling live and killed antigens by the gut immune system.

Testing a given antigen systemically over a wide range of concentrations reveals that low antigen doses often induce tolerance, intermediate doses induce immunity and high doses induce tolerance. Unlike systemic responses, oral tolerance can be induced using a wide range of antigen doses from few micrograms to milligrams [106, 107], these differences might be due to tolerogenic epitope density in a particular antigen. Friedman and Weiner [108] showed that induction of anergy or active suppression following oral tolerance is determined by antigen dosage, single high dose of antigen-induced anergy and multiple low doses of antigen-induced tolerance by active suppression through transforming growth factor-J3 (TGF-[3) and IL-4. It is difficult to induce tolerance in animals previously primed via cross-reacting intestinal flora, therefore, multiple feeds of relatively large doses of antigen are required to induce oral tolerance in such cases [ 107]. How exactly the antigen doses elicit anergy or active suppression through cytokines is not known. It is speculated that it might be due to differential requirements for number of epitopes, costimulatory signals and cytokines.

Genetic influence on the ease of tolerance induction is exemplified by the mouse strains BALB/B and BALB/c. The former is relatively much more difficult to tolerize than the congenic BALB/c strain [109-111]. Since these mice differ only at the H-2 locus, it is

reasonable to assume the MHC genes at least partly might have role in regulating oral tolerance to protein antigens. The immunological basis of this MHC-linked effect remains to be investigated. It is easier to induce systemic immune tolerance in animals with an immature lymphoid system than in mature animals. The mice fed ovalbumin (OVA) during early postnatal life, i.e. first day or second, do not develop the tolerance of systemic immunity found in adults fed the same dose of antigen. In fact, OVA-fed infant mice develop a systemic immune response when challenged parenterally as adults. This reflects the inabil- ity of the immature immune system to re- spond appropriately to gut-derived tolerogen. The adult pattern of susceptibility to tolerance develops within the first week of postnatal life [ 112, 113]. Maturation into adulthood from 8 to 24 weeks of age significantly in- fluences the induction of oral tolerance in different strains of mice. Animals from strains which are susceptible to the induction of oral tolerance to OVA at 8 weeks of age became refractory at 2 weeks of age [I 14].

Factors such as cytokine milieu, adjuvants, nutritional status of the animal [ 115], intestinal flora [106, 116], permeability of the ingested antigen [117], chemical modification of the antigen by methylation and acetoacety- lation are also known to influence the tolerance induction.

Mechanisms and Modulation of Oral Tolerance

Antigens such as foreign S antigen, type II collagen or MBP pass from the lumen of the gut across multifold cells (M cells) or APCs lying under Peyer's patches. These cells then activate a local population of T cells which secrete TGF-13 and IL-4 [ 118]. Following activation, few of these cells wander out through

334 immunol Res 1996;15:323-346 Singh/Nagaraju

the lymphatics and blood stream and then through tissues until they again find their recall antigens. In a patient with uveitis, rheumatoid arthritis or multiple sclerosis these cells recognize via cross-reaction of the ex- posed selfS antigen, collagen II or MBP in the inflamed eye, joint or brain. The specialized T cells are then stimulated by their recall antigen to secrete TGF-13 and IL-4. These inhibitory cytokines suppress the activity of neigh- boring disease inducing Thl cells. The latter cells presumably recognize one or more autoantigens, the nature of which is unknown. Any organ-specific antigen could lure the suppressive T cells to the target organ which need not be a known autoantigen (S antigen, type II collagen or MBP).

Apart from the secretion of inhibitory cytokines and nonspecific suppression, there are basically three nonmutually exclusive hypotheses that have been proposed to explain the induction of adult unresponsiveness to antigen, any or all of which may potentially oper- ate in models of oral tolerance. These are anergy, deletion, and antigen-specific suppression, and indeed the stability and revers- ibility of peripheral tolerance in a given situation may depend on which of these mechanisms is operating [119].

Under certain conditions the contact of antigen with T cells in vitro would render them anergic [120, 121]. This was suggested to be caused by the lack ofa costimulatory signal required for T cell activation. Specific T cells from tolerant animals are shown to be unresponsive even to stimulation directly through their antigen receptor via VI3-specific antibodies [122]. Such data are further supported by analysis in which antigen-specific responses cannot be elicited in cells from tolerant animals in culture despite the presence of cells expressing VI3 chains that have been associated with a response to that antigen [123]. In some models, anergy is associat-

ed with a downregulation of cell surface proteins critical to T cell activation such as TCR and/or accessory cell surface molecules like CD8 [124]. Melamed and Friedman [125] showed that a single feeding of 20 mg OVA by gastric intubation induced a state of anergy in OVA-specific T cells. These ceils are characterized by lack of proliferative responses to OVA, absence of IL-2 and IL-2R expression and this nonresponsive state was reversed by preculture of tolerized cells in IL-2-containing medium. Subsequently, it was shown that induction of anergy depends on antigen dosage and frequency of feeding [126]. Since anergy has been suggested to be a postthymic-peripheral mechanism for tolerance, it could be val- id as a mechanism for oral tolerance to dietary antigens [ 127].

Peripheral clonal deletion is an intriguing possibility in oral tolerance. The existence of peripheral clonal deletion from studies showing the selective absence of VI3 6+ cells in the periphery of adult thymectomized, Mls+ mice in comparison to adult thymectomized Mls- mice has been suggested [128]. Peripheral downregulation of TCR and CD8 molecules can be a novel mechanism for the establish- ment of peripheral tolerance [124, 129, 130]. Whitacre et al. [131] showed the specific absence o f t cells expressing the VI3 8.2 TCR in SJL mice fed MBP. Since the immune response in these mice is dominated by V[3 8.2+ T cells, these results support the possibility that clonal deletion of antigen-specific T cells may occur during oral tolerance. But these studies do not exclude downregulation of TCR and accessory molecules like CD8 on T cells [124]. The observations showing that the oral tolerance may be specifically abrogated by treating with cyclophosphamide, 2'-deoxyguanosine or by transfer of TCR ~//5 IEL suggest that clonal deletion is a less likely possible mechanism in oral tolerance [ 111, 132-134]. A recent study by Chen et al. [135] shows that

Experimental Autoimmune Uveitis Immunol Res 1996;15:323-346 335

orally administered antigen can induce tolerance not only by active suppression and clonal anergy but by extrathymic deletion of antigen- reactive Thl and Th2 cells.

Generally the term suppression is used to describe a situation where potentially reactive T cells are present but held in check by other T cells. For several years the idea that suppressor cells were able to control immune responses and could be responsible for peripheral tolerance received widespread credence [ 136, 137]. In models of transplantation tolerance, it was shown that adoptive transfer of ceils from tolerant animals into naive animals prevented the rejection of a subsequent graft [138]. In all cases of suppression it is neces- sary to make a distinction between specific and nonspecific suppression, and in the case of specific suppression for idiotype and antigen-specific suppression.

Antigen-specific suppressors can be de- fined as cells which suppress effector responses through recognition of the same antigen as that recognized by the effector population. Therefore, they may not be the same cells as those recognized by the effector population. They may not recognize exactly the same epitope, but they must be able to recognize a component of the stimulating antigen so that they can interfere with the overall response to the antigen. Richman et al. [ 139] showed that oral tolerance could be transferred through splenic T cells to naive, syngeneic irradiated recipients. It was also shown that orally tolerized recipients suppressed responses of normal transferred spleen cells [140]. The suppression was abrogated by using cyclophosphamide, antisuppressor cell an- tiserum [111,127, 140-144]. Antigen-specific suppressor cells have been described in in vitro systems [ 145-147]. One of the models of suppression suggests involvement of three different types of cells acting in a cascade to produce suppression [148]; the first level of the

cascade involves T cells which are antigen- specific CD4+ suppressor inducers. It is also possible that anti-idiotypic suppression may be operating in oral tolerance. Orally administered antigens may selectively stimulate T cell clones which recognize pathogenic T cells, thereby controlling autoaggressive clones.

Natural suppressor cells which suppress an immune response in an antigen and MHC- independent manner were described in graft versus host disease [149, 150]; the induction of such cells in oral tolerance could not be ruled out. Lider et al. [151] showed that CD8+ cells isolated from orally tolerized animals could adoptively transfer the protection. These MBP-specific CD8+ T cells mediated suppression by releasing TGF-I3 and administration of anti-TGF-[3 abrogated the protection [152]. Yoshino et al. [153] showed that oral collagen II suppressed arthritis, and the effect was dose dependent occurring at low doses of collagen II, demonstrating the biological relevance of bystander suppression associated with oral tolerance.

It was shown that secretion of anti-inflammatory cytokines as well as protection from disease can be enhanced by feeding the bacterial adjuvant lipopolysaccharide together with the antigen [118, 154, 155]. Rizzo et al. [155] showed that three feedings of 0.2 mg IRBP every other day before immunization did not protect against EAU, whereas a similar regimen of five doses was protective. However, supplementation of the three-feeding regimen with IL-2 resulted in disease suppression similar to the five-feeding regimen. They also showed that this effect was due to increased production of anti-inflammatory cytokines like TGF-13, IL-4 and IL-10 induced by IL-2 administration and the tolerance induced in the five-feeding regimen was due to anergy. Using knockout models of IL-4 and IL-10, they demonstrated that these cytokines have a role in the induction of low dose tolerance,


Table 3. Oral tolerance in various autoimmune diseases

Features EAE EAU AA IDDM

Animal model Lewis rat Lewis rat Rat NOD mice Disease induction MBP in CFA S antigen in CFA Mycobacterium Spontaneous

tuberculosis Oral tolerance induction MBP S antigen Collagen type II Porcine

insulin Inflammatory infiltrate Decreased Decreased Decreased Decreased Severity/incidence Decreased Decreased Decreased Decreased Lymphoproliferation/DTH Decreased Decreased Decreased Decreased Adoptive transfer of tolerance Yes Yes Yes Yes Ref. No. 13, 126, 151 16, 17, 176, 177, 179 159, 173 15, 160

AA = Adjuvant arthritis; NOD mice = nonobese diabetic mice; DTH = delayed-type hypersensitivity.

because these mice were successfully tolerized with a high dose feeding regimen [156].

Relevance of Oral Tolerance in T-Cell-Mediated Autoimmune Diseases

Autoimmunity develops into a disease when the components of the immune system begin to damage the individual's own tissues. T lymphocytes are strongly implicated in the pathogenesis of certain autoimmune diseases like IDDM, uveoretinitis, multiple sclerosis and arthritis (table 3). In experimental models of these diseases it was shown that the pathogenic lymphocytes were able to transfer autoimmune disease [ 157-160]. Both cytotoxic and helper T lymphocytes have been shown to cause tissue damage. The cytotoxic T cells presumably attack the target tissue directly; the helper T cells act by providing help to cytotoxic T cells but also by releasing cytotoxic lymphokines and by contributing to a developing inflammatory response. A number of studies on effector T cells mediating organ- specific autoimmune diseases in spontaneous or induced models have documented that the

key effector cells are CD4+ T cells which mediate autoimmune disease as helper T cells for autoantibody forming B cells, as amplifi- ers for cytotoxic or as mediators of cell- mediated immune responses akin to delayed- type hypersensitivity [161-163]. These find- ings suggest the existence of two distinct populations ofCD4+ T cells in terms of maintaining self-tolerance and induction of autoimmune disease, one mediating autoimmune disease and the other inhibiting it. This further leads to the hypothesis that one aspect of self-tolerance may be maintained by the cellular interaction between these two CD4+ populations. The autoimmune inhibiting CD4+ T cells are dominant in the normal physiological state. The experimental evidence for this hypothesis was provided by studies using athymic nude mice reconstituted with particular T cell subpopulations CD4+ CD51o autoimmune-inducing clones, CD4+ CD5hi suppressor of disease-inducing clone.

Our understanding of the T-cell-mediated autoimmune diseases is derived to a large extent from the study of animal models of disease like EAE, EAU, IDDM and adjuvant arthritis. Considerable effort has been di-


rected towards defining the autoreactive T cell repertoire in many human diseases including multiple sclerosis [ 164, 165], autoimmune thyroiditis [166], myasthenia gravis [167], IDDM [168] and chronic active hepatitis [ 169]. However, interpretation of the data obtained has not been conclusive. Some authors provide evidence for a restricted T cell response [ 165-169], while others find significant diversity both with respect to autoantigenic determinants and in the range of TCR genes [167, 170]. A solution to this contradic- tion was first shown in a rodent model of EAE that relates to the MBP-specific repertoire at different time points. These data show that the expressed autoimmune repertoire is not fixed but evolves during the course of disease. The initial responses are restricted to a specific self-protein in the target organ, these responses subsequently spread to additional epitopes of the same autoantigen (intramo- lecular spread) or to different autoantigen in the target organ (intermolecular spread). Thus multiple autoantigens become targets of autoimmune responses [171, 172]. This indicates that therapeutic approaches for human diseases based on deleting and rendering anergic specific antigen-reactive clones using immunodominant peptides or anti-idiotypic strategies may not work. Generally oral tolerance leads to unique mechanisms of tissue- directed suppression that occur following oral administration of proteins which would circumvent the immunotherapeutic problem of multiple target tissue autoreactivity. Oral tolerance is tissue- and disease-specific, i.e. orally administered MBP does not affect adjuvant arthritis [173] or autoimmune uveitis [17]. Likewise, orally administered collagen or S antigen suppress these diseases, respectively, and do not affect EAE. Therefore, the induction of oral tolerance may be a suitable therapeutic intervention in T-cell-mediated autoimmune diseases.

Oral Tolerance in EAU

As discussed above, EAU is a T-cell-mediated autoimmune disease which can be induced by many well-characterized photoreceptor cell proteins including S antigen, IRBP [28] or their pathogenic peptides or sensitized cells [58-63]. It was shown that EAU can be prevented by oral administration of native bovine S antigen or its peptides prior to immunization [16, 17, 174, 175]. The exact mechanism by which the protection is con- ferred is not well known. Vrabec et al. [174] suggested that adoptive transfer of protection from EAU or EAE using splenocytes from orally tolerized animals should not be possible if anergy was the protective mechanism. In addition, it has been suggested that oral tolerance in EAU operates by at least two distinct mechanisms that depend on the feeding dose; low doses induce suppression, whereas high doses induce unresponsiveness [176]. Avail- able experimental evidence tilts more towards suppression as a mechanism of oral tolerance in EAU for the following reasons. (1)Oral administration of antigen induces suppressor T cells and splenic CD8+ T cells protect the animals from subsequent active induction of EAU. Furthermore, these cells also inhibit antigen-specific in vitro prolifcrative responses [17]. (2)The antigen-specific in vitro suppression was blocked by anti-CD8 antibodies demonstrating that suppression was CD8+ T-cell-mediated. Recently, Vistica et al. [177] suggested that CD8 T cells are not essential for the induction of low-dose oral tolerance. Additional studies showed that tolerance to retinal antigen could be induced via the upper respiratory tract with microgram doses of antigen preventing subsequent induction of EAU [I 78]. The authors also spec- ulate that the inhalational tolerance may be better than oral tolerance due to small amounts of antigen used to induce the toler-


ance. Recently, we have shown that feeding of E. coli expressing retinal S antigen to Lewis rats prior to uveitopathogenic challenge with S antigen resulted in significant suppression of EAU and of the cellular responses to S antigen [179].

Conclusion and Future Direction

Despite the fact that several examples of sequence homology between retinal S antigen and microbial peptides and the induction of EAU by the latter group of peptides have been clearly demonstrated, the specific role for microbial proteins in the pathogenesis of uveitis is obscure. Molecular mimicry has been one of the hypotheses, and no convincing evidence has been brought forward.

The evidence for molecular mimicry in autoimmune disease, however, remains mostly circumstantial at the present time. An alterna- tive theory is that autoimmune diseases are, in fact, primarily the result of infection and that of reactive autoantigens, which are nor- mally sequestered from autoreactive T cells, occurs because of bystander tissue damage releasing partially degraded antigens to be presented by local APCs to primed/activated T cells. This may, in part, explain the development of autoimmunity to corneal antigens in onchocerca infection.

In general, the course of autoimmune disease characteristically exhibits intercalating exacerbations and remissions, or complete recovery indicates that associated symptoms or the disease itself may resolve through the activity of the immune system, even after periods of profound dysfunction. This indicates that the autoimmune disease may be appropriately corrected by using natural immunological reagents that participate in selection of the lymphocyte repertoire under physiological conditions. Therefore, in the future

the autoimmune disease therapy should be based on utilization of natural components of the immune system, rather than immunosup- pressive drugs. This view points to the impor- tance of understanding the anatomy and physiology of the components of the immune system. The above hypothesis also suggests that one of the means of practical immune intervention in autoimmune states is through oral tolerance. In the future, research efforts should be directed to understanding the po- tential mechanisms operating in the induction and maintenance of oral tolerance. The best experimental evidence for peripheral suppression is through cytokines secreted by Tht and Th2 cells. Until now oral tolerance was investigated in rodent models of autoimmune disease; in the future it will be important to direct efforts toward understanding the phenomenon of oral tolerance through primate and human studies.

Acknowledgements

We would like to express our thanks to Dr. Timo- thy J. Schoen for helpful comments during preparation of the manuscript. Financial assistance from the In- dian Council of Medical Research, New Delhi, and Uttar Pradesh Council of Science and Technology, Lucknow, India, to V.K.S. is gratefully acknowledged. The laboratory infrastructure was provided by a JICA grant-in-aid to the SGPGt projects.


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Experimental autoimmune uveitis: Molecular mimicry and oral tolerance

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