Effect of denaturization on the immunogenicity and uveitogenicity of retinal S-antigen.

Previous studies have shown that alteration of pH or temperature of retina extract can affect its complement fixing reactivity with anti-S-antigen serum. To examine the effect of pH or heat on the immunogenicity and uveitogenicity of purified bovine S-antigen, guinea-pigs were injected with pH- or heat-treated S-antigen and evaluated for clinical and histopathological signs of uveoretinitis, histopathology of pineal gland, serum and intraocular S-antigen antibody reactivity, and S-antigen skin test reactivity. Guinea-pigs that received pH 7 or pH 10 treated S-antigen responded as did those that received untreated S-antigen. Guinea-pigs injected with pH 4 or heat-treated S-antigen exhibited lower incidence, later onset and less severe uveitis than those that received untreated S-antigen. Systemic responses of skin test reactivity to S-antigen were not different from those of the control group; pineal gland involvement and serum anti-S antibody reactivity were slightly reduced. Skin test responses of animals receiving treated antigen were less to the treated (injected) antigen than to untreated S-antigen. In addition, antibody responses of guinea-pigs receiving pH 4 or heat-treated antigen were less to the treated (injected) antigen than to untreated S-antigen. These results suggest that the sites on the S-antigen molecule responsible for various aspects of pathogenicity and immunogenicity do not have the same sensitivity to physical/chemical treatment and may reside on different parts of the molecule. Furthermore, the reactive sites especially for antibody and skin test reactivity, may be continuous sites.

Retinal autoimmunity: two decades of research.

A brief review of our current knowledge of retinal autoimmunity and the pathogenesis of experimental autoimmune uveitis developed over the past two decades is presented. Attention is focused upon the discovery of retinal S-antigen, its isolation and characterization, manifestations of the disease produced, mechanisms of experimental autoimmune uveitis, analysis of the activities of the peptide fragments of the antigen, and potential relationships of the model to human ocular disease.

Rabbit ocular and pineal autoimmune response to retina antigens.

Different forms of experimental autoimmune uveitis (EAU) can be produced by varying protocols to present different autoantigens to several species of experimental animal. We have studied the clinical, histological and serological responses of rabbits to footpad injection of various fractions of retina extract. Rabbits injected with retina extract or S antigen developed posterior uveitis. However, rabbits injected with retina extract, also developed an anterior uveitis and pinealitis not seen in rabbits receiving S antigen. The Serological response of rabbits to retina extract was different than that to purified S antigen. Antisera of rabbits receiving retina extract reacted with rabbit retina and pineal gland as well as with guinea pig retina but not with guinea pig pineal gland. In contrast anti-S antigen sera reacted with rabbit retina and guinea pig retina and pineal gland but not with rabbit pineal gland. Gel filtration chromatography of the ammonium sulfate supernate of retina extract was used to differentiate the antigens with which these two sera reacted. An analysis of these experiments gives preliminary evidence of an autoantigen(s) of rabbit retina and pineal gland that is not S antigen. The existence of multiple autoantigens common to retina and pineal gland in various species is significant in that it further underscores the relationship of these tissues. Furthermore, it is not unrealistic to expect more than one autoantigen of retina or uvea to be involved in autoimmune uveitis.

Treatment of intraocular inflammatory disease with cyclosporin A.

Eight patients with bilateral sight-threatening posterior uveitis of non-infectious aetiology that had not responded to corticosteroid or cytotoxic therapy were given cyclosporin A. Seven of the eight responded with improvement in visual acuity and disappearance of ocular inflammatory activity. The seven included two with Behçet's disease, who also had improvement in non-ocular symptoms. Peripheral blood lymphocytes from the seven cyclosporin A responsive patients, but not those from the non-responder, gave positive in-vitro blastogenic responses to the retinal S-antigen, a highly uveitogenic organ-specific material. In four patients OKT4/OKT8 ratios fell after the start of therapy, as the result of an increase in the OKT8 fraction. In the other four patients the fall was preceded by an increase in the OKT4/OKT8 ratio. Cyclosporin A did not influence natural killer cell activity. Side-effects ascribed to cyclosporin A were tolerated or disappeared with continued therapy, sometimes at a lower dosage. No neoplasms were observed. Cyclosporin A seems to be effective treatment for selected patients with severe bilateral uveitis of non-infectious aetiology.

S-antigen uveitis in primates. A new model for human disease.

Uveitis was induced rhesus monkeys immunized with purified retinal S-antigen. Focal retinal and subretinal infiltration, in addition to periphlebitic changes, were observed histologically. Immunized monkeys had similar cell-mediated in vitro immune responses to the S-antigen as do humans with posterior uveitis. Circulating immune complexes at the time of disease could not be detected. This primate model supports the notion that the S-antigen may play a role in human uveitis.

Cyclosporin a. Inhibition of experimental autoimmune uveitis in Lewis rats.

Cyclosporin A (CS-A), a selective inhibitor of T lymphocytes, is reported here to prevent S antigen (S-Ag) induced uveitis in Lewis rats. The S-Ag, found in all mammalian retinas, is uveitogenic under experimental conditions and patients with certain uveitic entities demonstrate cell mediated responses to this antigen. Daily treatment with CS-A (10 mg/kg) begun on the same day as S-Ag immunization totally inhibited the development of the uveitis in this experimental autoimmune model. Moreover a greater CS-A dose (40 mg/kg) efficiently prevented the disease process when therapy was started 7 d after S-Ag immunization. Anti-S-Ag antibody titers were observed to be similar in rats either protected or not protected with CS-A. Our data support strongly the need for T cell participation in this disease model. Since ocular inflammatory disease is an important cause of visual impairment, the data further suggest that CS-A may be useful in the treatment of patients with intractable uveitis.

Cellular immune responsiveness of uveitis patients to retinal S-antigen.

S-antigen, an organ-specific substance isolated from the retinal photoreceptor region, was shown to be a potent agent for the induction of experimental autoimmune uveitis. S-antigen and other ocular antigens were tested for their ability to induce blast transformation of lymphocytes from patients with a variety of ocular inflammatory diseases. Lymphocytes from 22% of patients tested manifested a positive memory response to the S-antigen prepared from bovine eyes. Responses to human S-antigen paralleled those found with the bovine. All of those with a positive response had active or inactive retinal lesions. Some posterior uveitis patients responded to crude retinal extracts but not to S-antigen, indicating the possible role of other retinal antigens still to be purified. Control subjects did not manifest a positive immune response to the S-antigen, nor did patients with anterior uveitis. Possibly, these responses play some role in the pathogenesis of the disease.

Experimental allergic uveitis: clinicopathologic features associated with varying doses of S antigen.

Considerable differences were observed in the experimental autoimmune disease elicited by retinal S antigen, depending on the immunizing dose. An inoculum of 50 microgram produced a massive panophthalmitis containing many polymorphonuclear leukocytes, eosinophils, and mononuclear cells. A less severe endophthalmitis was seen in animals receiving 25 microgram of antigen. Animals receiving between 5 and 10 microgram of antigen developed a disease characterized by a granulomatous uveitis. The inflammatory infiltrate consisted primarily of mononuclear and epithelioid elements and appeared virtually identical to that seen in sympathetic ophthalmia. One microgram of S antigen produced primarily a nongranulomatous posterior uveitis composed chiefly of mononuclear cells. The principal change in the character of the disease occurred at dose levels between 10 and 25 microgram. This change consisted of the disappearance of polymorphonuclear leukocytes and eosinophils at the lower dose levels. These histopathologic changes suggest that at higher dose levels an immune complex disease may be superimposed on or replace a presumably cell-mediated hypersensitivity response.

Pineal gland involvement in retina-induced experimental allergic uveitis.

A form of experimental allergic uveitis (EAU) can be elicited in guinea pigs by injection of an extract of homologous retina homogenate (S preparation). These guinea pigs exhibit clinical and histological ocular disease as well as cellular and humoral immune responses to S preparation. Recent studies have shown an immunofluorescent crossreactivity of the sera of these animals (anti-S) with retinal photoreceptors and pinealocytes. The experiments presented in this paper demonstrate pineal gland involvement during the course of S preparation-induced EAU. There is lymphocytic infiltration of the pineal gland, with corresponding loss of antigen reactivity and some disturbance in cell structure. Pineal gland infiltrate can be seen before observable retinal involvement. Similarity of the reactive substances in retinal photoreceptors and pinealocytes is demonstrated by a reaction of identity in immunodiffusion studies and similar mobilities in immunoelectrophoretic studies of S preparation and pineal gland extract with anti-S serum. The relationship of the retinal and pineal substances to each other, as well as their role in disease development in the animal model and possibly in human uveitides, are discussed.

Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors.

A radial diffusion assay for tissue collagenase (EC 3.4.24.3) has been devised which is simple, sensitive and capable of application to large numbers of samples. The assay employs an agarose matrix containing solubilized lathyritic rat skin collagen as substrate. Fibril formation is induced for 2 h at 37 degrees C subsequent to 41 h digestion at 28 degrees C. The procedure results in sharply defined zones of lysis which may be measured directly or after photography. The characteristics of the procedure are otherwise similar to those reported for other radial diffusion assays. The new method was used to examine the action of 10 compounds which were known or potential inhibitors of tadpole collagenase. The concentration of inhibitor required to produce 50% inhibition is reported for the following compounds: alpha2-macroglobulin, 142 microng/ml; N-acetylcysteine, greater than or equal to 100 mM; cysteine, 8.7 mM; EDTA, 0.46 mM; histidine, greater than or equal to 100 mM; 2,3-dimercaptopropanol, 0.5 mM and mercaptoacetic acid, 70 mM. The procedure also has potential for clinical determinations (e.g. tears, synovial fluid) since assay dishes may be prepared in advance and only 15 micronl of sample is required.

Pineal reactivity of anti-retina sera.

Specific immunofluorescence has been demonstrated in the guinea pig pineal gland by homologous sera from guinea pigs injected with an extract of retina homogenate. This fluorescence appears to be in the cytoplasm and is evenly distributed among the cells of the pineal gland. Specific immunofluorescence in the retina has been previously demonstrated by these sera.

The role of uveal and retinal antigens in experimental autoimmune ocular pathology.

Guinea pigs immunized with retina antigen, located by immunofluorescence in the photoreceptor cell layers, develop retina-specific immune responses and autoimmune uveoretinitis. Ocular pathology is characterized by cellular infiltration and destruction of the photoreceptor cells subsequent to inflammation of the iris, ciliary body and choroid. Guinea pigs immunized with homologous choroid antigen, localized by immunofluorenscence in the area of Bruch's membrane or pigment epithelium, developed choroiditis, subretinal exudation and loss of photoreceptor outer segments. The pathology is associated with immune responses directed primarily against antigens recovered with choroid, but which demonstrated partial cross reactions with outer segment antigens. Possible relationship of uveal and retinal antigens is discussed.

Use of immunofluorescent localization in the normal guinea pig eye to differentiate three autoantisera.

Three distinct immunofluorescent patterns were demonstrated in normal guinea pig eyes by autoantisera prepared to three different ocular tissue preparations. Anti-U serum (antiserum to ureal homogenate) exhibited specific fluorescence in the area of Bruch's membrane. Anti-P serum (antiserum to a suspension of the sedimentable portion of retina homogenate) exhibited specific fluorescence in the outer segments of the photoreceptor cells. Anti-S serum (antiserum to retinal homogenate extract) demonstrated specific fluorescence in the outer portion of the retina extending from the outer plexiform layer to the outer segments of the photoreceptor cells. The ability of anti-S and anti-P sera immunofluorescent activities to cross species lines was demonstrated.