Current Eye Research

Volume I I supplement 1992, 33-40

Experimental autoimmune uveoretinitis: a model system for immunointervention: a review J.V.Forrester, Janet Liversidge, H.S.Dua, A.Dick, Fiona Harper and P.G.McMenamin'

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Department of Ophthalmology, University of Aberdeen, Medical School, Foresterhill, Aberdeen AB9 2ZD, UK and 'Department of Anatomy and Human Biology, University of Western Australia, Nedlands, Perth, Western Australia 6009 ABSTRACT Experimental autoimmune uveoretinitis (EAU) is a useful model of human posterior uveitis and as such, permits the analysis of strategies for immuno-intervention. Modulation of the autoimmune response may be attempted at the stages of induction of EAU, during homing of autoreactive lymphocytes to the target organ, the retina, or during the effector stage of the disease. This paper presents a brief overview of current immuno-therapeuticmodalities and assesses the usefulness for extrapolation to human disease. INTRODUCTION Human uveitis is classified as anterior or posterior on the basis that both conditions are patho-genetically and clinically quite distinct. Posterior uveitis presents in a wide variety of clinical syndromes but many of these have overlapping if not identical features such as focal choroidoretinal inflammatory lesions and retinal vasculitis (reviewed in ref. 1). Pathologically typical lesions are found to contain many different cell types particularly monocyte/macrophages and CD4+ T cells (Fig 1a.b). However much of the histopathology is difficult to interpret since it derives from eyes with end-stage disease. Experimental autoimmune uveoretinitis (EAU) provides a useful model for human posterior uveoretinitis since many of the clinical manifestations can be mimicked closely. EAU is induced by immunization of animals with retinal antigens in various adjuvants and its value lies in its precise end-points, its titratability with dose of antigen and its susceptibility to immunomodulation. All of the antigens described so far are located at the photoreceptor/retinal pigment epithelial interface and this may reflect the organ specificity of these proteins and the fact that there is a considerable turnover of these proteins during photoreceptor renewal. In addition there is considerable information available on the nature of the antigens and their uveitogenic epitopes(2-4). In this paper we review some of the approaches to

immunointervention in EAU and discuss the possibilities for similar approaches to the treatment of human posterior uveitis. STRATEGIES FOR IMMUNOINTERVENTION EAU is induced by intradermal injection of retinal antigen in adjuvant at a distant site and some 9-11 days later an inflammatory response is detected in the eye (5). Induction of EAU by this technique is assumed to occur through antigen capture by antigen presenting cells (APC) at the site of injection in the skin (presumably Langerhans dendritic cells) which then migrate to the draining lymph node where they present the processed antigen to autoreactive T cells (Fig. 2). These cells then undergo blast transformation in the lymph node and enter the circulation. At specific sites in the blood-retinal barrier they are trapped and migrate into the parenchyma of the target organ, accompanied by many other non-specific cells. Specific homing of cells may involve only a few antigen-specific cells (6). Damage to the tissues is then induced by effector cells with release of further specific antigen. It is likely that local organresident cells plus other migratory bone-marrow derived cells become involved in continued presentation of this released antigen to ever-increasing numbers of activated T cells entering the inflamed tissue, as has been shown to occur in experimental autoimmune encephalomyelitis (EAE) (7). The main criticism of this model as a paradigm for human uveitis is that it requires exogenous introduction of the presumed (auto)antigen while most forms of human uveitis, apart from sympathetic ophthalmia, are endogenous. However, for many years it has been recognised that endogenous uveitis may be associated with exogenous foreign antigen exposure some weeks or months prior to the onset of the disease. The concept of molecular mimicry may provide an explanation of the mechanism of uveitis induction whereby cross-reactive epitopes

Received on January 17. 1992; accepted on May 8. 1992

0 Oxford University Press

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Current Eye Research

Figure 1: (a)Dalen-Fuch's nodule stained for iron (macrophages) in case of human sympathetic opthalmia.(H&E). @)Massively thickened choroid with granuloma formation in case of sympathetic ophthalmia. (c)Frozen-section of human choroid from same eye as l b stained for ICAM-1 (red stain, APAAP). Note marked staining of retinal pigment epitheluim. (d)EAU in Lewis rat, 9 days after immunisation with S antigen 34

showing ICAM- 1 staining of choroidal vasculature.(peroxidase stain) (e)EAU in Lewis rat, 11 days after immunisation with IRBP, show dual fluorescent staining of cells in subretinal exudate: green, LFA-1(CD11b)(ED7); red,macrophage maker (EDI). (f)EAU in Lewis rat, mild disease; note infil@ationof retina with ED3 positive macrophages (peroxidase stain).

Current Eye Research CD4 + T Cell

Skin

f

/

Lymph Node Spleen

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INDUCTION

HOMING

Eye EFFECTOR STAGE (+LOCAL INDUCTION)

Figure 2: Experimental model for induction of EAU. CFA, complete Freund's adiuvant

resting T cell

IL-2 _------_

W--"'. activated T cell 'blast'

APC

Figure 3: Schematic diagram of antigen processing and presentation via interaction with MHC antigen in the intracellular compartment. Ag,antigen; APC, antigen presenting cell

on foreign and autoantigens permit the escape from tolerance of autoreactive T cells. For these reasons, EAU and many other "autoimmune" experimental models are accepted as valuable in the study of autoimmune mechanisms(8). On this basis it can be Seen that immuno-intemention can be initiated at various levels including the process of antigen presentation, the stage of specific homing of autoreactiveT cells to the target organ or the activation of the effector cells in the tissues. ANTIGEN PRESENTATION Presentation of antigen by "professional"APC (dendritic cells, macrophages or B cells) involves the endocytosis of the antigen, its degradation in the endosomal pathway to produce peptides, peptide association with MHC Class II antigen in the proteasome and transport of the MHC Class I1 antigedpeptide complex to

the cell surface when interaction with the variable component of the T cell receptor occurs (Fig.3)(9). Effective antigen presentation also requires a second signal the precise n a m of which is unknown. However, several other molecules are involved including the "adhesion" molecules which not only assist in optimising the cell-cell contact but also have a role in second messenger responses (10). In addition, the CD4 antigen participates in reactions involving MHC Class II antigens (the CD8 antigen is required for MHC Class-I mediated antigen presentation) and as such represents an "accessory" molecule. Immunointervention at the stage of antigen presentation can therefore be directed at several points in this process. In EAU it has been shown that monoclonal antibodies directed against retinal S-antigen can inhibit induction of the disease either directly (1 1) or via an anti-idiotype mechanism( 12). Not all antibodies are effective and certain antibodies are effective only in some species(13). This has led to the identification of uveitogenic sites on the molecule which are quite distinct from lymphocyte proliferative sites and from other antibody binding sites(l4) and raises the possibility of utilising similar antibodies in human disease, perhaps by "humanising" the antibody. In other autoimmune diseases such as EAE, peptide blocking therapy has been successfully attempted (15). Although such approaches are possible in EAU the fact that there are several uveitogenic sites on the molecule poses significant problems. Many of these sites have been identified using synthetic overlapping peptides of the entire molecule and this may not be relevant to in vivQ peptide generation during antigen processing. In this regard, it has been shown that limited digestion of Santigen with trypsin or papain generates a series of peptides which are still functional in their interaction with an EAU inhibiting monoclonal anti-S antibody and in their induction of EAU. whereas limited digestion with Staphylococcus a m u s protease yields peptides which are non-functional in either test (14)(Fig.4). Thus the precise mode of proteolysis in vivQ will determine whether one peptide is likely to be more pathogenic than another even if there is considerable sequence overlap. Apart from antibodies directed against the antigen or peptides which may act by "blocking" antigen-MHC interactions, monoclonal antibody directed against other components of the antigen presentation machinery has been tested in the therapy of EAU. Thus antibodies against the MHC Class II antigen (16) have been shown to be effective in inhibiting EAU as have antibodies against the CD4 antigen (17). The immunological basis for the action of these antibodies is more easily understood

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Current Eye Research

Figure 4: Limited digestion of retinal S-antigen with (A)papain (B)staphylocococcusaureus protease, and accompanying immunoblots using monoclonal antibody S2.4.C5 (with permission from ref. 14). S-antigen was incubated with the

enzymes for varying periods of time and the peptides identified by SDS-PAGE prior to immunoblotting with monoclonal antibody to S-antigen (S2.4.C5)

but their specificity in targeting only those cells involved in the uveitis response may be questioned. This may not pose a problem in practice however, if only 'activated' T cells are inhibited and only over a short time span. Drug-based immunointewention may act at several levels in the immune response from antigen presentation to effector cells. Cyclosporin-A (18), FK506 and rapamycin have been shown to

be effective in EAU (19) and clinically for CyA and FK506 and the mode of action appears to be on the antigen-primed T cells by preventing the elaboration of IL-2 (20) although precisely how this occurs varies for each drug (21). These drugs, therefore, not only prevent EAU but can be effective some time after immunisation. Dissecting this mechanism should prove valuable to the development of the next generation of drugs.

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Eye Research

Figure 5: Elecron micrograph of rat retinal endothelial cells from eye with EAU 21 days after immunisation. . Note marked

HOMING Once primed, the antigen-specific autoreactive T cell must find its way to the target organ if autoimmune damage is to occur. Recent studies in EAE have shown that brain microvascular endothelial cells undergo morphological changes which mimic the appearance of high endothelial venules in lymph nodes, i.e. those cells specifically adapted to facilitate trans-endothelial migration of lymphocytes (22). A preliminary report documented similar findings in retinal microvascular endothelial cells in EAU and also demonstrated marked changes in the morphology of the retinal pigment epithelial cells, the second site of the blood-retinal barrier, during trans-epithelial migration of lymphocytesand other cells (23). More recently, we have quantified these changes in EAU using morphometric techniques (24) and have demonstrated that the endothelial cell undergoes phenotypic changes immediately prior to the onset of the disease (Fig.5). Thus these cells may be 'primed' or 'activated' in a tissue specific manner to interact with antigen-specific and nonspecific T-cells. 'Activated' endothelial cells also express specific markers or adhesion molecules, which are otherwise involved in antigenpresentation (for review see ref. 25). Those molecules include ICAM-1 and -2, V-CAM, ELAM-1 and -2 plus the integrin receptors, and are currently the focus of much interest. Retinal pigment epithelial cells constitutively express ICAM-1 and retinal endothelial cells can be induced to do so in vitrQ after induction with interferon-g (26). Furthermore, adhesion of CW+T lymphocytes to monolayers of these cells can be specifically inhibited in virQ by antibodies to ICAM-1 (26). ICAM-1 is present at high levels in human retinal pigment

HEV-like changes in endothelium(a)and adhesion of lymphocytes to the endothelium(b) (insert of a).

I

0RPE + IFN -d K Inhlbltlon ol adhmlon

20. 10

.

0.

FK-508 on RPE 6 lymphocytes

a, FK-5m

on lymphocytes only

FK-508 on RPE only

Figure 6: Adhesion of human CD4+T lymphocytes to monolayers of RPE cells in virro after pretreatment of cells with FK506.

epithelium in some forms of inaaocular inflammation (Fig. lc), and also appears within choroidal blood vessels immediately prior to the onset of EAU (Figld). Furthermore, many of the mononuclear cells in the subretinal inflammatory exudate in EAU express the CD1 l b marker (LFA-1) which is the ligand for ICAM-1 (Figle). Therefore, it is likely that adhesive interactions between 'activated' inflammatory cells and cells of the blood-retinal banier central to the mechanisms of homing of cells in EAU, and that inhibition of this adhesive response might block the disease. Indeed, 'anti-adhesive' therapy has already been shown to be effective in other inflammatory models (27). It is possible that these mechanisms are also affected by therapies directed at other stages in the immune response. For 37

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41 280ng/rnl 28nglml

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DAYS POST IMMUNISATION

Figure 7: Induction of EAU in animals tolerised with 10 daily insufflations of whole retinal antigen extract. Inhibition of EAU is still induced with doses of retinal extract (RE)

Experimental autoimmune uveoretinitis: a model system for immunointervention: a review.

Experimental autoimmune uveoretinitis (EAU) is a useful model of human posterior uveitis and as such, permits the analysis of strategies for immuno-in...
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