Occurrence of major anti-retinal autoantibodies associated with paraneoplastic autoimmune retinopathy.

Autoantibodies (AAbs) against retinal antigens can be found in patients with cancer and unexplained vision loss unrelated to the cancer metastasis. Cancer-associated retinopathy (CAR) is a rare paraneoplastic visual syndrome mediated by AAbs. Our goal was to determine whether CAR patients with different malignancies have a specific AAb or repertoire of AAbs that could serve as biomarkers for retinal disease. We found AAbs against 12 confirmed retinal antigens, with α-enolase being the most frequently recognized. The significant finding of the study was a high incidence of anti-aldolase AAbs in colon-CAR, anti-CAII in prostate-CAR, and anti-arrestin in skin melanoma patients thus these AAbs could serve as biomarkers in the context of clinical presentation and could support the diagnosis of CAR. However, a lack of AAb restriction to any one antigenic protein or to one retinal cellular location makes screening for a CAR biomarker challenging.

Antigen-specificity of antiretinal antibodies in patients with noninfectious uveitis.

OBJECTIVE: Antiretinal antibodies (ARAs) have previously been described in noninfectious uveitis. However, the antigen specificity of these ARAs has not been investigated. The purpose of this study was to identify antigen-specific ARAs in noninfectious uveitis. METHODS: A total of 18 patients with noninfectious uveitis were enrolled. Surface plasmon resonance was used to measure binding responses of patient and control sera against several uveitogenic proteins: recoverin, S-antigen, interphotoreceptor retinoid binding (IRBP), retinal-pigment-epithelium-specific 65-kDa protein (RPE65), tyrosinase-related protein 1 (TRYP1), and tyrosinase-related protein 2 (TRYP2). RESULTS: The frequency of ARA positivity against S-antigen, IRBP, RPE65, TYRP1, and TYRP2 in patients with uveitis did not differ significantly from that of normal controls. However, ARA positivity for recoverin was more frequently observed in patients with uveitis (p = 0.002). A total of 10 patients in the uveitis cohort had birdshot chorioretinopathy, and all 10 were positive for anti-recoverin ARAs. CONCLUSIONS: Patients with noninfectious uveitis have increased frequency of ARA positivity against recoverin. This ARA deserves further investigations as a potential biomarker and pathogenic agent in noninfectious uveitis, especially in birdshot chorioretinopathy.

Carcinoma-associated retinopathy in a young teenager with immature teratoma of the ovary.

A 14-year-old African American girl presented with diminished vision in both eyes 1 week after undergoing an oophorectomy for a right ovarian mass. Systemic metastatic work-up was negative. Visual acuity was 20/40 in the right eye and 20/50 in the left eye. Slit-lamp biomicroscopy was unremarkable in both eyes. Fundus examination showed diffuse patchy areas of retinal pigment epithelial atrophy in the macula and peripheral retina bilaterally. Color vision had decreased in each eye. Electroretinography revealed nondetectable rod and cone responses. Both pattern and flash visual evoked potential (VEP) testing showed delayed latency in both eyes. She was treated with pulse intravenous methylprednisolone for 3 days along with intravenous immunoglobulins and rituximab, followed by systemic prednisolone and biweekly intravenous immunoglobulins and rituximab for 3 months. Antiretinal autoantibodies against 48-kDa (arrestin) and 64-kDa and 94-kDa proteins were positive, suggestive of carcinoma-associated retinopathy. After 3 months, visual acuity was 20/40 in each eye with improvement in color vision and VEP findings.

Autoimmune responses against photoreceptor antigens during retinal degeneration and their role in macrophage recruitment into retinas of RCS rats.

Autoimmunity may contribute to retinal degeneration. The studies examined the evolution of autoimmune responses against retina in naïve dystrophic RCS rats over the course of retinal degeneration. We showed that anti-retinal autoantibodies and T cells are generated in response to the availability of antigenic material released from dying photoreceptor cells during retinal degeneration but with distinctive activation trends. Passive transfer of anti-retinal antibodies enhanced disease progression by disrupting the BRB, upregulating MCP-1, attracting blood macrophages into retina, and augmenting apoptotic photoreceptor cell death. Our findings directly link anti-retinal autoantibodies to activated macrophage entry and their possible role in neurodegeneration.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.

Anti-retinal autoantibodies in experimental ocular and systemic toxoplasmosis.

BACKGROUND: Patients with ocular toxoplasmosis (OT) develop autoreactivity to several retinal antigens, including retinal S-antigen. By establishing an experimental rabbit model of systemic and of primary and secondary ocular toxoplasmosis, we wished to investigate the onset and development of humoral response to retinal S-antigen. METHODS: Of twelve infection-naïve rabbits, six were left untreated, and the other six were infected subcutaneously with 5,000 tachyzoites of the highly virulent, non-cyst-forming BK-strain of Toxoplasma gondii. Three months later, the left eye of each animal was infected transvitreally with 5,000 tachyzoites of the same strain. The right eye of each rabbit served as an uninfected control. Blood and aqueous humor were collected prior to infection, and up to 90 days thereafter. Using the ELISA technique, all samples were analyzed in parallel for total IgG, and antibodies against toxoplasmic, bovine retinal S-antigen and peptide 35 from human S-antigen. RESULTS: In infection-naïve rabbits Toxoplasma-specific antibodies were detected 10 to 15 days after systemic and ocular infection. Serum antibodies against retinal S-antigen and peptide 35 were not detected in response to systemic Toxoplasma infection. After ocular challenge, aqueous-humour levels of antibodies against retinal S-antigen and peptide 35 in the infected eye began to rise 10 to 15 days later in infection-naïve, but not in infection-immunized animals. During the early post-infection period, the concentrations of anti-retinal antibodies in the infected eye correlated with the severity of inflammatory tissue destruction, but returned to baseline later even though the inflammatory response persisted. In the uninfected partner eye, concentrations of anti-retinal and toxoplasmic antibodies did not correlate with each other. CONCLUSION: Our data afford no evidence of similarities between toxoplasmic and retinal antigens, nor of infection-induced humoral autoimmunity. They indicate rather that retinal autoantigens are liberated in the context of inflammatory tissue destruction due to ocular toxoplasmosis.

An arrestin-dependent multi-kinase signaling complex mediates MIP-1beta/CCL4 signaling and chemotaxis of primary human macrophages.

MIP-1beta/CCL4 is a principal regulator of macrophage migration and signals through CCR5. Several protein kinases are linked to CCR5 in macrophages including the src kinase Lyn, PI3K, focal adhesion related kinase Pyk2, and members of the MAPK family, but whether and how these kinases regulate macrophage chemotaxis are not known. To define the role of these signaling molecules, we examined the functions and interactions of endogenous proteins in primary human macrophages. Using siRNA gene silencing and pharmacologic inhibition, we show that chemotaxis in response to CCR5 stimulation by MIP-1beta requires activation of Pyk2, PI3K p85, and Lyn, as well as MAPK ERK. MIP-1beta activation of CCR5 triggered translocation of Pyk2 and PI3K p85 from the cytoplasm to colocalize with Lyn at the plasma membrane with formation of a multimolecular complex. We show further that arrestins were recruited into the complex, and arrestin down-regulation impaired complex formation and macrophage chemotaxis toward MIP-1beta. Together, these results identify a novel mechanism of chemokine receptor regulation of chemotaxis and suggest that arrestins may serve as scaffolding proteins linking CCR5 to multiple downstream signaling molecules in a biologically important primary human cell type.

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

PURPOSE: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU). METHODS: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence. RESULTS: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma. CONCLUSIONS: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.

S-antigen specific T helper type 1 response is present in Behcet's disease.

PURPOSE: To investigate the frequency and phenotypic and functional characteristics of S-antigen (S-Ag) specific T cells in patients with Behcet's disease (BD). METHODS: Blood was taken from 23 active BD patients, 12 inactive BD patients, and 14 healthy controls. The clinical features of the patients were summarized. T cell response against 40 mixed S-Ag peptides was identified by interferon gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT). CD69 and CD45RO were used to characterize the phenotype of S-Ag specific T cells. The functional property of S-Ag specific T cells was investigated by measuring the production of cytokines. RESULTS: Response to the mixed S-Ag peptides was found in 56.5% and 25% of active and inactive BD patients, respectively. The responsiveness to S-Ag peptides was unrelated to the clinical features of the patients. About 65.8% of IFN-gamma(+) CD4(+) T cells in active BD patients expressed CD69 and CD45RO concomitantly. S-Ag peptides significantly induced a production of IFN-gamma and tumor necrosis factor (TNF)-alpha but not interleukin (IL)-2, IL-4, and IL-17 by peripheral blood mononuclear cells (PBMCs) in active BD patients with a response to S-Ag. CONCLUSIONS: S-Ag specific T cells are present in certain active BD patients, and most of them are activated memory CD4(+) T cells. These T cells may be involved in the pathogenesis of BD via producing Th1-dominant cytokines.

Cancer-associated retinopathy in patients with breast carcinoma.

INTRODUCTION: Cancer-associated retinopathy (CAR) is a paraneoplastic neurological syndrome resulting in progressive loss of vision and clinical signs of retinal degeneration. It is associated with various types of cancer and is also considered to be an autoimmune disorder that involves cross-reaction between autoantibodies and retinal proteins. The aim of this study was to establish whether immunoreactivity to retinal antigens (RAs) observed in patients with breast cancer is accompanied by any visual impairments. MATERIALS AND METHODS: Sera of 295 patients with diagnosed breast cancer were screened for the presence of anti-RAs antibodies using immunoblotting. Cellular immunoreactivity to RAs present in retinal extracts and to purified recoverin and arrestin was determined by means of a lymphocyte proliferation assay. Six patients with high-titer antibodies to RAs then underwent ophthalmic and neurological examinations. RESULTS: Four serum samples contained high-titer antibodies to a 46-kDa protein, most probably retinal alpha-enolase, three had antibodies to a 48-kDa protein identified as retinal arrestin, while 56-, 43-, 41-, and 34-kDa antigens were recognized only by one serum sample each. Moreover, weak cellular response to all the RAs tested was observed in one patient and another patient responded only to retinal extract. Two of the examined patients displayed symptoms of CAR. CONCLUSIONS: Immunoreactivity to RAs in patients with breast cancer may also be present in cases without clinical signs of CAR.

Eales' disease: oxidant stress and weak antioxidant defence.

Eales' disease (ED) is an idiopathic retinal periphlebitis characterized by capillary non-perfusion and neovascularization. In addition to the existing system, a new staging system has been proposed by Saxena et al. Immunological, molecular biological and biochemical studies have indicated the role of human leucocyte antigen, retinal S antigen autoimmunity, Mycobacterium tuberculosis genome, free radical damage and possibly hyperhomocysteinemia in its etiopathogenesis, which appears multifactorial. Oxidant stress has been shown by increase in the levels of thiobarbituric acid reactive substances (lipid oxidation) in the vitreous, erythrocytes, platelets and monocytes. A decrease in vitamins E and C both in active and healed vasculitis, superoxide dismutase, glutathione and glutathione peroxidase showed a weakened antioxidant defence. Epiretinal membrane from patients of ED who underwent surgery showed, by immunolocalization, presence of carboxy methyl lysine, an advanced glycation end product formed by glycoxidation and is involved in angiogenesis. OH. free radical accumulation in monocytes has been directly shown by electron spin resonance spectrometry. Free radical damage to DNA and of protein was shown by the accumulation of 8 hydroxy 2 deoxyguanosine (in leucocytes) and nitrotyrosine (in monocytes), respectively. Nitrosative stress was shown by increased expression of inducible nitric oxide synthase in monocytes in which levels of iron and copper were increased while those of zinc decreased. A novel 88 kDa protein was found in serum and vitreous in inflammatory condition and had antioxidant function. Platelet fluidity was also affected. Oral, methotrexate in low dosage (12.5 mg/week for 12 weeks) as well as oral vitamin E (400 IU) and C (500 mg) daily for 8 weeks are reported to have beneficial effects.

Treatment of experimental autoimmune uveoretinitis with atorvastatin and lovastatin.

Statins, which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are approved for cholesterol reduction and are commonly used to treat atherosclerosis and coronary artery disease. Statins may also be potent immunomodulatory agents and may be beneficial in the treatment of autoimmune diseases. In this study, we investigated therapeutic effects of atorvastatin and lovastatin on experimental autoimmune uveoretinitis (EAU). EAU was induced in Lewis rats using bovine S-antigen (S-Ag) peptide. Atorvastatin was suspended in 0.5% aqueous methylcellulose and was administered orally at a dose of 10 mg/kg and at a low-dose of 1 mg/kg. Lovastatin was dissolved in DMSO:PBS (1:1) and was administered by intraperitoneal (i.p.) injection at a dose of 2 mg/kg. Both statin treatments were initiated after the clinical onset once daily for 14 days. The rats were examined every other day for clinical signs of EAU. The histological scores and delayed-type hypersensitivity (DTH) were evaluated on day 28 post-immunization. Morphologic and immunohistochemical examinations were performed with light and confocal microscopy, respectively. Lymphocyte proliferation was measured by [(3)H]thymidine incorporation into antigen-stimulated T cells from inguinal lymph nodes. After 72 h, supernatants were collected and assayed for IFN-gamma by ELISA. Clinical and histological scores of EAU were decreased in both the atorvastatin (10 mg/kg)- and lovastatin (2 mg/kg)-treated groups. The invasion of T cells and macrophages, and Müller cell proliferation, were inhibited in both atorvastatin- and lovastatin-treated groups. DTH was significantly inhibited in both groups, compared with vehicle-treated groups (controls). Lymphocyte proliferation assay demonstrated decreased proliferation in the presence of 25 microg/ml S-Ag peptide in both groups, compared with controls. In the supernatants of lymph node cells stimulated with S-Ag peptide (5 microg/ml), 77 or 87% inhibition of IFN-gamma production was observed in rats treated with atorvastatin or lovastatin, respectively, compared with controls. The current results indicate that atorvastatin administrated orally following the clinical onset has therapeutic effect in EAU as well as lovastatin administrated intraperitoneally. Statins may be useful for treating intraocular inflammation.

Photoreceptor mitochondrial oxidative stress in early experimental autoimmune uveoretinitis.

AIMS: In early S-antigen induced experimental uveitis (EAU), photoreceptor mitochondrial proteins are nitrated prior to macrophage infiltration of the retina, suggesting that oxidative stress is an initial event in the development of EAU. We attempted to detect the oxidative stress and localise it in the EAU retina. METHODS: Lewis rats were immunised with S-antigen in complete Freund's adjuvant (CFA). Animals were injected with CFA alone and non-immunised animals served as controls. Immunised and non-immunised animals were killed on day 5 and subsequent days. Isolated retinas were processed for inducible nitric oxide synthase (iNOS), tumour necrosis factor (TNF)alpha, interferon (IFN)gamma, interleukin (IL)Ialpha and CD28 expression by real time polymerase chain reaction. In addition, iNOS was colocalised with cytochrome c oxidase on day 5 of EAU. Oxidative stress was detected by 2', 7'-dichlorodihydrofluorescein diacetate and localised by a mitochondrial specific marker. Leucocyte and T cell infiltration in the retina/choroid was evaluated by immunohistochemistry. RESULTS: The iNOS, TNFalpha, IFNgamma, IL1alpha and CD28 transcripts were significantly upregulated on day 5 in EAU, and iNOS was colocalised with cytochrome c oxidase in the photoreceptor mitochondria. Oxidative stress was seen primarily in the photoreceptor mitochondria. Occasional T cells were present in the retina at this stage. CONCLUSIONS: During early EAU, mitochondrial oxidative stress is selectively noted in the photoreceptor inner segments. The oxidative stress appears to result from iNOS upregulation in the photoreceptor mitochondria and cytokine generation in the retina by a few antigen specific infiltrating T cells.

Identification of MHC class I H-2 Kb/Db-restricted immunogenic peptides derived from retinal proteins.

PURPOSE: To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. METHODS: Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. RESULTS: All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.

[Catalytic autoantibodies--a new molecular instrument in cardiology and ophthalmology].

AIM: To develop a conceptual model of using catalytic autoantibodies as diagnostic and monitoring tools in organ-specific autoimmune disorders. MATERIAL AND METHODS: A total of 99 patients (56 males and 43 females aged 21-52 years) with autoimmune myocarditis (AM) and 198 patients (77 males and 121 females aged 8-79 years) with autoimmune uveitis (A U) participated in the study. AM patients were examined for anticardiomyosin and anti-DNA autoantibodies (ACM, ADNAab), AU patients - for autoantibodies to S-antigen, IRBP, redopsin, phosphocine, autoDNA. RESULTS: AM patients had double level of DNA-binding autoantibodies. In 1/3 of them there was hydrolysing DNA and cytotoxic activity. In AU patients maximal titers were in Behcet's disease, sympathic ophthalmia, generalized uveitis and viral uveitis. CONCLUSION: Autoantibodies with different specificity and function including DNA-abzymes can be additional diagnostic and prognostic markers.

Thymic expression of peripheral tissue antigens in humans: a remarkable variability among individuals.

The majority of maturing T lymphocytes that recognize self-antigens is eliminated in the thymus upon exposure to their target antigens. This physiological process of negative selection requires that tissue-specific antigens be expressed by thymic cells, a phenomenon that has been well studied in experimental animals. Here, we have examined the expression in human thymi of four retinal antigens, that are capable of inducing autoimmune ocular disease retinal S-antigen (S-Ag), recoverin, RPE65 and inter-photoreceptor retinoid-binding protein (IRBP)], as well as four melanocyte-specific antigens, two of which are used as targets for melanoma immunotherapy [gp100, melanoma antigen recognized by T cells 1, tyrosinase-related protein (TRP)-1 and TRP-2]. Using reverse transcription (RT)-PCR, we found that all thymic samples from the 18 donors expressed mRNA transcripts of most or all the eight tested tissue antigens. Yet, the expression of the transcripts varied remarkably among the individual thymic samples. In addition, S-Ag, RPE65 and IRBP were detected by immunostaining in rare cells in sections of human thymi by antibodies against these proteins. Quantitative real-time RT-PCR analysis revealed that the retinal antigen transcripts in the human thymus are present at trace levels, that are lower by approximately five orders of magnitude than those in the retina. Our observations thus support the notions that thymic expression is a common feature for all tissue-specific antigens and that the levels of expression play a role in determining the susceptibility to autoimmunity against these molecules.

The uveitogenic potential of retinal S-antigen in horses.

PURPOSE: To investigate the uveitogenic potential of retinal S-antigen (S-Ag) in horses. METHODS: Horses were immunized subcutaneously with S-Ag or BSA as control antigen, emulsified in complete Freund's adjuvant. Simultaneously, Bordetella pertussis was given intravenously. Antigen specific T- and B-cell responses were analyzed in a 3-day interval. Disease development was judged clinically and histopathologically. Two identical booster immunizations were given every 4 weeks to test induction of recurrences. RESULTS: T- and B-cell responses specific for S-Ag were observed in all immunized horses but were absent in control animals. However, uveitis developed in only one of five animals. Reimmunization with S-Ag did not lead to a uveitic relapse in this horse. All other horses of the S-Ag- and BSA-treated groups neither showed any signs of uveitis, nor had inflammatory infiltrates of the inner eye. CONCLUSIONS: In contrast to interphotoreceptor retinoid-binding protein (IRBP), S-Ag is a weak autoantigen in horses. Even though S-Ag immunization leads to the activation of autoreactive T- and B-cells, infiltration of the inner eye and induction of uveitis are controlled in most horses.

Immune responses to retinal self-antigens in CD25(+)CD4(+) regulatory T-cell-depleted mice.

PURPOSE: Prior work has shown that autoimmune uveoretinitis develops spontaneously in CD25(+)CD4(+) regulatory T-cell-depleted mice (Tr-depleted mice). In this study, the generation of autoantibodies and autoreactive T-cells specific to retinal antigens was examined in Tr-depleted mice with uveoretinitis, and the pathogenic and immunogenic abilities of the autoreactive T cells were evaluated. METHODS: Tr-depletion was achieved in (C57BL/6 x A/J) F1 (B6A) mice by thymectomy on day 3 of life followed by intraperitoneal injection of an anti-CD25 mAb. At 6 months of age, autoantibodies to the retina were evaluated by indirect immunofluorescence, and total IgG2a levels in sera were assessed by ELISA. The pathogenic abilities of the splenic T cells were examined by adoptive transfer to syngeneic nu/nu mice, and the proliferation responses and the secretion of granulocyte-macrophage-colony-stimulating factor (GM-CSF), IFN-gamma, and IL-10 on stimulation by retinal self-antigens was also evaluated. RESULTS: Autoantibodies to the retinal photoreceptor cell layer were detected in Tr-depleted mice, and the titers correlated well with the grades of inflammatory lesions. The splenic CD4(+) T cells of Tr-depleted mice induced uveoretinitis in the recipients by adoptive transfer and exhibited proliferative responses and secretion of IFN-gamma, but not of IL-10, by in vitro stimulation with S-Ag and interphotoreceptor retinoid-binding protein (IRBP). Moreover, the total IgG2a level in serum was markedly and significantly augmented in Tr-depleted mice. CONCLUSIONS: The results suggest that in Tr-depleted mice in which uveoretinitis develops, S-Ag and IRBP-specific T cells are spontaneously sensitized and shifted to a Th1-phenotype. These sensitized T cells may account for the development of autoimmune uveoretinitis in Tr-depleted mice.

Human S-antigen: peptide determinant recognition in uveitis patients.

Uveitis is an inflammation of the uveal tract and is one of the major causes of visual impairment. Several lines of evidence suggest an important role for activated T lymphocytes in the perpetuation of posterior uveitis. In sequel to our preliminary observations with human S-antigen, we have further investigated the proliferative response of peripheral blood lymphocytes of posterior uveitis patients against 20 linear and 9 overlapping peptides of retinal S-antigen. The expression of surface markers CD4, CD8, CD29, CD45RA in peripheral blood was detected by flow cytometry. We have also assessed the pattern of cytokines present in peripheral blood mononuclear cells (PBMCs) using ribonuclease protection assay (RPA). Nineteen out of 32 patients' lymphocytes showed proliferative response to S-antigen, one or more of its 20 linear and nine overlapping synthetic peptides. Six patients showed significant lymphoproliferative response against various peptides. The maximum response was found to peptides from the 231-270 amino acid region of human S-antigen sequence. The percentage of CD29(+) (memory cells) and CD45RA(+) (naive cells) T-lymphocytes was higher in patients compared to healthy volunteers. There was a demonstrable difference in the percentage of CD4(+) and CD8(+) lymphocytes in the patients (P <== 0.05) as compared to controls. Higher message for interleukin (IL)-5, IL-10, IL-15, IL-9, IL-2, IL-13, and interferon (IFN)-gamma was observed in uveitis patients than in healthy individuals. In brief, our study suggests that a particular region of S-antigen plays an important role in idiopathic uveitis.