Ocular immune privilege: a review

ELS EVI E R

Clinical Eye and Vision Care 12 (2000) 97-106

www.elsevier.com/locate/clineyeviscare

Special report

Ocular immune privilege: a review Steven B. Koevary” Department of Biological Sciences, Ocular Research Centev,New England College of Optometry, 424 Beacon Street, Boston, MA 02115, USA Accepted 28 January 2000

Abstract

The definition of the term ‘immune privilege’ has evolved over the last century. Current usage refers to a state within a particular organ or tissue in which elements of normal immunity are absent. The fact that this deficiency is thought to be generally beneficial has compelled others to go a step further and venture that immune privilege acts to minimize expression of immunopathology. The purpose of this article is to review which parts of the eye hold immune privileged status, what mechanisms contribute to it, and what clinical benefits may have driven the development of these unique immune environments. The article ends with an examination of recent studies which have sought to use components of ocular immune privilege to prevent systemic autoimmune disease. 0 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ocular immune privilege; Blood/ocular barrier; ACAID; Collagen-induced arthritis; Transforming growth factor beta

1. Introduction

The definition of the expression ‘immune privilege’ has evolved considerably over the years. Originally, the term referred to sites in the body that were capable of hosting donor histoincompatible tissues without rejection [l-31. These included such diverse regions as the placenta, testis, brain, hair follicle, hamster cheek pouch, liver, peritoneal omental pouch, mature cartilage, ovary, thyroid, and the retina, iris, ciliary body, anterior chamber, cornea, lens, and vitreous cavity of the eye 13-51. The factors which facilitated this survival were immune isolation of the graft and/or the inhibition of the effector immune mechanisms capable of destroying the graft. More recently, immune privilege has come to refer in more general terms to a location in which elements of the normal immune response to a host of antigens are absent.

* Tel.: + 1-617-236-6230;fax: + 1-617-369-0174. E-mail address: koevarysene-optometry.edu (S.B. Koevary).

The fact that this deficiency is, as we will see, thought to be generally beneficial to the tissue/organ has compelled others to go a step further and define immune privilege as ‘the processes that work to minimize expression of immunopathology in an organ or tissue’ [6]. Perhaps the best characterized immune privileged sites are located in the eye. The purpose of this article is to review which parts of the eye hold privileged status, what mechanisms contribute to it, and what clinical benefits may have driven the development of these unique immune environments. The article ends with an examination of recent studies which have sought to use components of ocular immune privilege to prevent systemic autoimmune disease. 1.1. Factors contributing to ocular immuneprivilege 1.1.1. Immunological isolation An attractive hypothesis to explain immune privilege is that it is due to isolation of antigens within the site from cells of the immune system. This lack of

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communication exists as a result of its absence of either a vasculature or a draining lymphatic system or to the presence of a vascular barrier.

1.1.2. Absence of a vascular supply Examples of ocular privileged sites which lack a blood supply are the lens and the central cornea; the nutritional requirements of these tissues are met through diffusion from neighboring tissues. Thus, the lens receives some oxygen from the aqueous humor which is used primarily by its epithelium and cortex. Most of the carbohydrate usage by the lens is through anaerobic metabolism. In the cornea, oxygen in the ambient air readily diffuses into the epithelium and most of the stroma, while the corneal endothelium probably receives its oxygen from the aqueous humor 171. Carbon dioxide produced in all levels of the cornea appears to diffuse outward. Vascularization of a previously non-vascularized privileged site, such as the central cornea, negates its privileged status and, in the case of the cornea, correlates highly with its rejection following transplantation 181. 1.1.3. Presence of a vascular barrier In vascularized immunologically privileged sites such as the brain, iris, ciliary body, and retina, the entry of immunological effectors is blocked by specialized endothelial intercellular junctions. In the iris, ciliary body, and retina, these specializations are referred to as the blood-ocular barrier. This barrier limits the intraocular movement of immune cells such as granulocytes, lymphocytes, and monocytes, and factors such as certain plasma proteins including complement components and a-2-macroglobulin7a protease inhibitor [9]. Immune reactions are therefore prevented by exclusion of the cells and factors that would promulgate the response. Interestingly, as articulated by Streilein [lo], since some of these factors can act to both promote and suppress immunogenic inflammation, the eye consequently lacks the ability to regulate ocular inflammation on two levels. The specific elements that contribute to the blood-ocular barrier (Fig. 1) differ in different re-

gions of the eye. In the iris, vessels are enclosed in a thick, fibrous, acellular connective tissue. In the ciliary body, the cells of the two epithelial layers are joined together by junctional complexes which serve a dual purpose. First, they transmit the movement of the ciliary muscle to the inner layer and second, they serve as a barrier between the blood and the intraocular environment. The retinal pigment epithelial cells are interconnected by junctional complexes consisting of apical gap junctions, tight junctions, and a zonula adherens. The retinal outer plexiform and outer nuclear layers, and the fovea, are vessel-free zones, and the endothelial cells of all other retinal vessels are linked by tight junctions. Finally, both the pigment epithelial cells and retinal endothelial cells have a paucity of endocytic vesicles and tightly regulate their ionic and metabolic gradients [ll]. The above morphological evidence of the existence of a blood/ocular barrier notwithstanding, work by Prendergast et al. [12] has called its physiological significance into question. These authors used fluorescent labeling to track the distribution of activated T cell blasts in the retina. Their results showed that labeled cells freely entered and exited the retina but only remained there if they were activated and were responding to retinal antigens as in uveoretinitis. In this case, there was a progressive inflammatory disruption of the barrier ultimately resulting in more pronounced leukocyte infiltration and edema. If their study can be confirmed, it could open debate about the immunological role of all tight junction-mediated barriers. 1.1.4. Role of lymphatics For many years after its initial characterization, immune privilege was thought to be due to the absence of a lymphatic drainage in the privileged site. Lymphatic vessels channel antigens and antigen presenting cells (APCs) bearing antigens to draining lymph nodes which trap them in the cortex. Free antigen then binds to appropriate APCs and lymphocytes in the cortex, and antigen-bearing APCs present their antigen to T cells, thereby initiating an immune response. It was theorized that if antigens placed into

Thick, fibrous, acellular connective tissue surrounding iris vasculature Ciliary body epithelial cells linked by junctional complexes Retinal pigment epithelial cells linked by junctional complexes Retinal endothelial cells linked by tight junctions Reduced endocytic vesicles in pigment epithelium and retinal endothelium Fig. 1. Elements of the blood/ocular barrier.

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privileged sites did not have access to draining lymph nodes, they would be effectively concealed from the immune system. While certain immune privileged sites, such as the hamster cheek pouch, do in fact lack a lymphatic drainage 1131, others (e.g. testes) have indeed been shown to possess such vessels or to otherwise have access to draining nodes. Still others, like the brain and the eye, were shown to drain antigens both directly into the vasculature (a means that does not promote immune activation but rather promotes immune tolerance) and also into regional nodes such the cervical and submandibular nodes. In the eye, the bulk of the aqueous humor carrying antigens from the privileged intraocular environment drains directly into the vasculature by way of the canal of Schlemm. Drainage of intraocular fluid into lymph nodes has been shown to occur, however. Lymphatic drainage of the intraocular environment occurs by the uveoscleral pathway [14]. It has been estimated that approximately 10% of the aqueous humor drains by this mechanism into lymphatic vessels in the head and neck [lo]. There is also suggestive evidence that antigens placed into the posterior chamber may drain into, and activate cells within, the submandibular lymph nodes 1151. What physiological role these drainage pathways play in normal ocular immunity is uncertain. 1.1.5. Role of APCs and MHC (major histocompatibili& complex) antigen expression APCs, which are characterized by their expression of class I1 MHC molecules, play a critical role in the initiation and propagation of an immune response. These cells pick up antigens which enter tissues and migrate to a draining lymph node where they present them to T cells. This process leads to T cell activation and to the generation and activation of effector cells and to the release of effector cytokines. APCs can also present antigen to, and activate, lymphocytes within a tissue itself. It follows that if a tissue or organ lacks APCs, it might be far less capable of initiating a T cell response. Thus, the localized absence or reduction in number of APCs could play a role in mediating immune privilege. For many years, there have been conflicting reports concerning the presence of APCs in the iris and ciliary body. Resolution of this controversy began in the late 1980s with the introduction of better methods for the preparation of these tissues. Using these methods it was reported that the iris and ciliary body of rodents contained a significant number of cells which expressed class I1 molecules and which expressed a macrophage marker [16]. Ultrastructurally, these cells were shown to resemble classical APCs

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[17] and to reside between the two epithelial cell layers of the ciliary epithelium and beneath the single layer of the epithelium of the iris [MI. Similarly, confocal microscopy revealed a regular array of these cells in the human iris [19]. Other intraocular regions which were shown to possess APCs were the trabecular meshwork (but not in the direct outflow pathway) and along the uveoscleral pathway 1201, as well as near the ora serrata 1181. In humans, ramified retinal microglial cells were shown to have features in common with APCs including the constitutive expression of class I1 [21]. Muller cells, while not constitutive expressors of class I1 molecules, can be induced to express them [22]. Macrophages were also found throughout intraocular tissues. While the choroid lies outside the bloodocular barrier, it is important to note that it also contains APCs, specifically in the connective tissues surrounding the choriocapillaris and choroid, which play a role in ocular inflammation. It has been clearly demonstrated that the central cornea is devoid of APCs [23]. However, they are present in the corneal limbus and in response to infectious agents [24], chemical irritants [25], or localized damage [26], were shown to migrate into the central cornea. The presence of these APCs, called Langerhans' cells, in the central cornea was shown not only to facilitate inflammation, but also interfere with unique immunosuppressive features of the anterior chamber [27], as described below. While cells with the appearance of classical APCs are present in the immune privileged regions of the eye, as described above, they fail to activate immune cells. A great deal of data attribute this fact to the influence of factors, present in the aqueous humor, on these cells. Not only do these factors, discussed below, inhibit the T cell-activating ability of APCs in the eye, but they also directly immunosuppress lymphocytes and as such contribute on two levels to ocular immune privilege. 1.2. Suppression of effector immune mechanisms 1.2.1. Immunosuppressive factors in the eye It was first demonstrated in the early 1970s that the aqueous humor suppresses lymphocyte proliferation [28]. More recently, it was shown to inhibit mixed lymphocyte reactions and cytokine production in response to alloantigens, soluble antigens, and IL-2dependent T cell proliferation [29]. A notable exception to the immunosuppressive nature of the aqueous humor was the demonstration that it did not inhibit the killing activity of fully differentiated CD8' cytotoxic T cells. The absence of certain plasma proteins, such as many of the complement proteins and cortisol-

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Transforming growth factor beta (TGF-p) Vasoactive intestinal polypeptide (VIP) Alpha-melanocyte stimulating hormone (a-MSH) Calcitonin gene-related peptide (CGRP) Cortisol NK cell inhibitory factor (10 kD)

Factor(s) that promotes apoptosis of inflammatory cells

Fig. 2. Immunosuppressive factors in the aqueous humor.

binding globulin, certainly contributes to the immunosuppressiveness of the aqueous humor; interestingly, many of these proteins are increased in inflamed eyes as a compensatory mechanism. Aqueous humor contains low levels of IgG while other immunoglobulin isotypes are absent. However, it is becoming clear that it is not the absence of immune activators but the presence of immunosuppressive factors (Fig. 2) which contributes to the immunoinhibitory nature of the intraocular environment. These include vasoactive intestinal polypeptide (VIP), a-melanocyte stimulating hormone (a-MSH), calcitonin gene-related protein (CGRP), cortisol, a factor (or factors) that promotes the apoptotic death of inflammatory cells, and transforming growth factor beta (TGF-P). While all of these factors unquestionably play a role in suppressing immune reactions in the anterior chamber, many of the inhibitory effects of the aqueous humor can be attributed to TGF-P. TGF-P inhibits epithelial cell growth while promoting the growth of fibroblasts and stimulating the production of connective tissue matrix molecules. Its immune effects include inhibition of: T lymphocyte and thymocyte proliferation [30,31]; IL-2 receptor expression [32]; B cell proliferation and immunoglobulin production [33]; the development of cytotoxic T lymphocytes 134,351; the generation of lymphokine activated killer (LAK) cells; the generation and activity of NK cells [36]; and class I1 expression on tumor cells [37]. TGF-P is not always immunosuppressive, however. For example, it increases the secretion of IgA and does not block the activity of cytotoxic T cells. These effects have implications for ocular immunity. TGF-P is present in tears where it likely plays a role in suppressing ocular surface inflammation and promotes normal growth and differentiation of ocular surface epithelia and wound healing [38]. TGF-P was shown to be present in immunosuppressive concentrations in the normal aqueous humor

in several species including humans [39-41]. The source of aqueous TGF-P was established to be the ciliary epithelial cells [42,43].TGF-P is also produced by a host of other intraocular cells and tissues in the human and primate eye (Fig. 3) including retinal pigment epithelial cells 1441, the superficial limbal epithelial cells, conjunctival stroma, ciliary processes, and stroma adjacent to the pigment epithelium [45], Muller cells, ganglion cells, photoreceptors, hyalocytes, and cells associated with choroidal and retinal vessels [46]. 1.2.2. Fus ligund-induced apoptosis Fas and Fas ligand (FasL), which are present on lymphocytes, are thought to play a role in immune regulation. As the concentration of the antigen wanes, the probability increases that Fas on one lymphocyte will bind to FasL on another. When this occurs, the Fas-containing cell dies by an apoptotic mechanism. This was suggested as a means of downregulating the immune response after the elimination of antigen. FasL mRNA was shown to be expressed in the eye [47]. Specifically, immunohistochemical analysis localized FasL on the corneal epithelium and endothelium, iris and ciliary body, and throughout the retina 147,481. Intraocular FasL induced apoptosis in immune cells infiltrating into the anterior chamber in normal mice but not in gld mice, which lack functional FasL [47]. Thus, the interaction of lymphocytes expressing Fas with FasL in the eye appears to be another mechanism which contributes to ocular immune privilege. Interestingly, it has recently been reported that FasL may also damage the cornea under certain circumstances by stimulating neutrophil degradation [49].

Ciliary epithelium and process Retinal pigment epithelial cells Corneal limbal epithelial cells Conjunctival stroma Stroma adjacent to pigment epithelium in the pars plana Muller cells Ganglion cells Photoreceptors Hyalocytes Cells associated with choroidal and retinal vessels Fig. 3. Location of TGF-p in the eye.

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1.2.3. Role of ACAID (anterior chamber-associated immune deviation) in ocular immunity Twenty years ago, it was observed that allogeneic lymphoid cells injected into the anterior chamber of the rat eye failed to elicit significant delayed type hypersensitivity (DTH) inflammation [50]. This finding itself was not unexpected in light of what is currently known about the immunosuppressive nature of the aqueous humor. What was interesting, however, was that recipient rats had an impaired ability to reject orthotopic skin allografts derived from the same donor as the lymphoid cells. Since its first description, this phenomenon has been elicited experimentally in animals using a wide variety of antigens, such as bovine serum albumen (BSA) [51], ovalbumin [52], retinal proteins [53], hapten-derivatized cells [54], virallyencoded antigens (herpes simplex virus, HSV) [55], alloantigens [56] (an intact B cell population was also shown to be necessary for the induction of ACAID in response to alloantigens), and tumor-specific antigens [57,58]. The stereotypic, altered systemic immune response to antigens placed in the anterior chamber has been termed Anterior Chamber-Associated Immune Deviation or ACAID [59]. The immune deviation which occurs in ACAID is characterized by impaired development and expression of antigen-specific DTH [60,61] and impaired production of complement-fixing antibodies (isotype IgG2a) [62]. Elements of the immune response which are intact include the ability to produce non-complement-fixing antibodies [62] and the cytotoxic T cell-mediated lysis of target cells (Fig. 4) 1601. In order to induce ACAID, an ACAID-inducing signal needs to reach the spleen, where it activates regulatory lymphocytes. This signal was shown to be associated with either the serum component [63] or monocytes [64] in the blood, depending on the nature of the antigen [65]. Monocytes represent a significant population of MHC class I1 positive cells that are present in the eye. While these cells appear to be classical APCs, they fail to stimulate allogeneic T cells

Sumressed: DTH

Production of complement-fixing antibodies (IgG2A)

Intact: Cytotoxic T cell activity Production of non-complement-fixing antibodies

Fig. 4. Systemic immune alterations in ACAID.

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[27,66]. When harvested from eyes previously injected with BSA, they induced ACAID when injected into syngeneic recipients; if they were harvested from the peritoneal cavity 24 h after intraperitoneal (i.p.1 injection of BSA, they did not. However, when peritoneal exudate cells (PECs) from naive mice were pulsed with BSA overnight in vitro and then injected into the anterior chamber of normal mice, the recipients developed ACAID [51]. These data suggested that the local microenvironment of the anterior chamber influenced the action of classical APCs, conferring upon them ACAID-inducing properties. Specifically, it was shown that TGF-P in the aqueous humor was responsible for bestowing ACAID-inducing properties on these PECs 167-691. In fact, culture of PECs, or peripheral blood monocytes 1701, along with antigen and TGF-P was shown to be sufficient to convert them into ACAID-inducers. Exogenous TGF-P was shown to have two effects on PECs: (1) it enhanced the secretion of TGF-P and IL-10 by the PECs [71,72]; and (2) it reduced the production of IL-12 and expression of CD40 by the PECs [73]. These effects were speculated to play a role in the inability of the PECs to promote the development of T h l cells involved in the DTH response [73]. As mentioned above, TGF-P is present in abundance in the aqueous humor and has immunosuppressive effects. However, it does not inhibit the activity of cytotoxic T cells and its effects on antibody production are variable.

1.2.4. Nature of the final immune effector cells which suppress DTH The effector mechanism of ACAID seems to differ depending on whether the animal was primed or unprimed to the antigen 174,751. In unprimed animals, when PECs are cultured with antigen in the presence of TGF-P, the antigen is cleaved and peptide fragments are preferentially incorporated into class I molecules on these cells, a fact that probably accounts for the selective activation of CD8' regulatory (suppressor) and cytotoxic cells in the spleens of animals with ACAID [71]. Thus, DTH responses and the development of precursors of cytotoxic T cells are inhibited, while the activity of cytotoxic T cells is promoted. In primed animals or in animals in which T cells are injected along with antigen into the eye or the antigen (e.g. HSV) elicits inflammation that includes T cells [74], it appears that a soluble ACAID-inducing factor is involved. Specifically, the soluble factor appears to resemble the 01 chain of the T cell antigen receptor [76] that was either deposited or migrated into the eye [74]. It has been suggested that the binding of Fas' T cells to FasL in the eye may

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promote the deposition of these anterior chamber [74].

01

chains in the

1.2.5. Clinical sign@cance of ocular immune privilege The eye, like other tissues and organs, must possess the immunological resources to rid itself of foreign invaders and transformed cells. The immune response that most effectively eliminates these antigens is the DTH reaction. Although the DTH reaction can occur without adverse consequences in most organs, this is not true of the eye. The cytoarchitectural organization of the neural retina is unique and its integrity must be preserved for normal vision to take place. A DTH reaction in the eye, while effectively eliminating a pernicious antigen, would result in catastrophic consequences in the retina. Consequently, a distinctive immune milieu was thought to have evolved in the eye which permitted immune responsiveness to antigens while at the same time preserving the integrity of the retina and the visual axis. Specifically, the immune effectors which the eye uses to combat disease are cytotoxic T cells and non-complement-fixing antibodies. DTH and complement-fixing antibodies are inhibited by local factors in the eye, FasL on cells that line the anterior chamber, and systemically by ACAID. The systemic inhibition of these immune effectors ensures that no extraocular immune response is generated against antigens which can cross react with those in the eye; such a systemic response could become potent enough to enter the eye, overcome its immunosuppressive environment, and destroy the retina. Cytotoxic T cells and non-complement-fixing antibodies are not as competent as DTH in removing antigens. Thus, the eye makes, as Streilein stated, a ‘dangerous compromise’ with the immune system in order to preserve retinal integrity (Fig. 5 ) 1771.

1.3. Adverse effects of a limited intraocular immune response 1.3.1. HSV infection While humoral and cytotoxic T cell responses, which are not inhibited in ACAID, generally suffice in providing the eye with protection, they are ineffectual in posterior chamber HSV-1 infection. In this case, the infected eye initially appears to clear the virus effectively through the action of cytotoxic T cells and anti-viral antibodies. However, these effectors are insufficient in preventing the spread of the virus through neurons to the contralateral eye, where acute retinal infection occurs leading to necrosis. 1.3.2. Tumor growth Ironically, the action of cytotoxic T cells and antibodies, which are preserved locally and systemically in ACAID, relatively effectively limit the systemic spread of intra-ocular tumors while being ineffective in eliminating the original ocular tumor, which continues to grow progressively. 1.4. Beneficial effects of ocular immuneprivilege 1.4.1. Stromal keratitis ACAID appears to have been an evolutionary adaptation acquired by higher vertebrates, being absent in amphibians and fish [78]. It therefore follows that its advantages outweigh its disadvantages. In addition to the preservation of retinal integrity, other benefits of ACAID have been identified. Stromal keratitis, evident during anterior chamber HSV-1 infection, is thought to represent a DTH reaction to viral antigens in the corneal stroma as opposed to a direct toxic effect of the virus. However, this condition de-

Adverse Effects: Allows spread of HSV-1 to contralateral eye, causing retinal necrosis Ineffective in eliminating ocular tumors Beneficial Effects: Prevents stromal keratitis in HSV-1 infection Facilitates corneal allograft acceptance Inhibits systemic metastases from intraocular tumors Prevents ocular autoimmunity

Fig. 5. Clinical significance of ACAID.

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velops relatively infrequently (- 20% of cases) in HSV infection due to the inhibition of DTH reactions in the intraocular environment [ 101. Furthermore, eradication of anterior chamber HSV infection occurs in spite of the absence of an anti-viral DTH response. 1.4.2. Corneal allograft acceptance The high rate of acceptance of corneal allografts is thought to owe its success, in part, to ACAID. Abolishing ACAID by corneal cauterization, neovascularization, or keratoplasty was variably associated with loss of corneal, and iris and ciliary body immunosuppressive factor production, movement of Langerhans' cells into the central cornea, and leakiness of corneal neovessels [26]. Furthermore, denervation of the central cornea was also associated with the abolition of ACAID. In such animals, explants of iris and ciliary body tissues lacked the normal capacity to suppress T cell activation in vitro 1261. It is likely that disruption of neural connections to the cornea adversely affects immunosuppressive factor release by tissues lining the anterior chamber [77]. Moreover, this latter finding suggests that neurally-derived factors contribute to ACAID, as mentioned earlier. This fits nicely with the demonstrated presence of neuropeptides such as VIP, CGRP, and a-MSH in the aqueous humor. 1.4.3. Ocular autoimmuniQ ACAID is thought to play an important role in preventing uveoretinal autoreactivity under normal and pathological conditions and following trauma, such as in sympathetic ophthalmia. It has been shown that ACAID induction to the retinal antigens S-Ag and interphotoreceptor retinoid-binding protein (IRBP), prevented experimental autoimmune uveoretinitis (EAU) in rodents 179,801. Perhaps more clinically relevant was the finding that IRBP-induced

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Fig. 6. ACAID induced to CII antigen suppressed CII-directed DTH, as measured by a reduced footpad thickness in response to CII immunization.

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uveoretinitis could be prevented by the in vitro ACAID-generating technique in which animals were injected with PECs previously cultured with IRBP and TGF-P. 1.4.4. Systemicautoimmunity The approach of using PECs to generate ACAIDinducing cells in vitro holds promise for treating inflammatory eye diseases such as uveoretinitis. Since ACAID inhibits systemic, as well as ocular, DTH, it follows that this technique could conceivably be used to prevent adverse DTH-like reactions elsewhere in the body. Specifically, it is feasible that a systemic autoimmune disease which utilizes T cells and macrophages might be preventable by such an approach. Indeed, we used such a strategy to reduce the incidence of spontaneous autoimmune Type I diabetes in a rat model [81]. We also have preliminary data (described below) supporting a role for ACAID in suppressing systemic autoimmunity rheumatoid arthritis in a rat model. Rheumatoid arthritis (RA) is an inflammatory joint disease with evidence suggesting that autoimmune processes associated with HLA class I1 haplotypes may be involved. Joint destruction is mediated by leukocytes and T cells, primarily through their release of cytokines. Collagen-induced arthritis (CIA), induced by the intradermal injection of native collagen I1 (CII) emulsified in incomplete Freund's adjuvant (IFA), is an animal model of RA which has been useful for the study of disease pathogenesis and of potential therapies. all Arthritis was induced in rats (DR BB/Wor animals were cared for in accordance with NIH and institutional guidelines) by injection with 400 pg of heterologous CII (10 mg/ml in 0.1 M acetic acid) emulsified in an equal volume of IFA intradermally at ~

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the base of the tail. The clinical severity of the disease was scored on a subjective scale. Histopathology was confirmed at sacrifice approximately 3-4 weeks after immunization. ACAID was induced in DR rats by injecting 50 pg of CII in 3 p l into the anterior chamber, 1 week prior to immunization with CII for arthritis induction. For DTH measurement, intraocularly-injected rats were injected subcutaneously with 200 p g of CII in complete Freund’s adjuvant (CFA) in the base of the tail, followed in 1 week by the injection of 200 p g of CII alone into the footpad. Footpad thickness was measured 24 h later. As another measure of anti-CII immunoreactivity, draining inguinal lymph node cells from rats with arthritis were assayed for their ability to proliferate in response to CII in a lymphocyte proliferation assay and were compared to similar cells in disease-free ACAIDinduced rats (values are means f S.E.M.). Our results showed that we were able to induce ACAID to CII as determined by the suppression of CII-directed DTH in the footpad swelling assay (see Fig. 6). Lymphocytes from rats in which ACAID was induced to CII also showed reduced proliferation to CII compared to control animals exhibiting arthritis (Fig. 7, values are means f S.E.M.). Rats intraocularly injected with CII showed a reduced incidence of CIA compared to control rats (2/9 or 22.2% vs. 8/10 or 80%, P = 0.02; Fig. 8). The mean time from immunization to disease onset was 16 days. Interestingly, one of the two animals that became arthritic in the experimental group reverted to normal after 11 days this did not occur in any of the control animals. All arthritic rats developed severe disease with full ankylosis, with inflammation confirmed histologically. These data support the notion that the induction of ACAID to antigens implicated in the development of systemic autoimmunity can prevent disease. Thus, not only are there inherent beneficial effects of ACAID

for the eye but the ACAID process can seemingly be of therapeutic use for the treatment of systemic disease. Acknowledgements

The work cited in this paper was funded by the American Diabetes Association and the Juvenile Diabetes Foundation International. References 111 Van Dooremaal JC. Die entwickelung der in grund versetzten 121 131 141 151

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161 171

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1111 1121 1131

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Fig. 8. Rats intraocularly injected with CII showed a significantly reduced incidence of CIA compared to control rats (2/9 or 22.2% vs. 8/10 or SO%, P = 0.02).

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