concanavalin-a and Uveitis

The purpose of this study was to investigate the effect of intraperitoneal ghrelin on vitreous levels of interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-α) and to compare its effects with those of intraperitoneal infliximab in an experimental uveitis model.. Twenty-four male rats were assigned to four groups of six rats in each. All the rats, except for those in group 1 (controls), were injected intravitreally with concanavalin A to induce experimental uveitis. Rats in group 2 (sham) were not given any treatment after uveitis was induced. Rats in group 3 were given intraperitoneal infliximab 0.5 mg/100 mL on days 0, 1, 3, 5, and 7 following induction of uveitis on day 14 of the study. Rats in group 4 were given intraperitoneal ghrelin 10 ng/kg/day for 7 days following induction of uveitis. On day 21 of the study, enucleated globes were subjected to histopathologic examination. Vitreous levels of IL-1, IL-6, and TNF-α were measured by enzyme-linked immunosorbent assay.. Vitreous levels of IL-1, IL-6, and TNF-α were significantly increased in the sham group relative to the control group (P < 0.05), but showed a significant decrease in the group treated with infliximab (P < 0.05). Cytokine levels also decreased in the ghrelin-treated group, but the decrease was not statistically significant (P > 0.05).. Ghrelin failed to decrease the IL-1, IL-6, and TNF-α levels that play a critical role in the pathogenesis of uveitis.

Topics: Animals; Concanavalin A; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Ghrelin; Injections, Intraperitoneal; Interleukin-1; Interleukin-6; Male; Rats; Rats, Inbred Lew; Time Factors; Tumor Necrosis Factor-alpha; Uveitis; Vitreous Body

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.

Topics: Animals; Autoantigens; Concanavalin A; Disease Models, Animal; Horse Diseases; Horses; Retina; Synaptotagmin I; Uveitis

Inducible costimulator (ICOS) is an important costimulatory molecule involved in T-cell activation. In this study, the role of ICOS in the pathogenesis of uveitis in Behçet's disease (BD) was investigated.. Peripheral blood mononuclear cells (PBMCs) were obtained from BD patients with uveitis in the active or remission phase and in healthy subjects. Total RNA was isolated from PMBCs, and mRNA expression was analyzed on an oligonucleotide microarray. ICOS expression on CD4(+) T cells was determined by flow cytometry, and the functional costimulatory effect of ICOS/B7RP-1 interaction was assessed on stimulation with concanavalin A (conA) or IRBP in the presence or absence of anti-ICOS mAb.. As the result of microarray analysis, ICOS in PBMCs showed the greatest difference in expression in BD patients with uveitis compared with healthy control subjects. ICOS expression on CD4(+) T cells in BD patients with uveitis was significantly higher than that in healthy individuals, both before and after conA stimulation. Among the BD patients, ICOS expression on CD4(+) T cells was significantly higher in those with active uveitis than in those with remitted uveitis. Blockade of ICOS/B7-related protein-1 (B7RP-1) interaction by anti-ICOS mAb significantly decreased IFN-γ, IL-17, and TNF-α production by PBMCs when stimulated with conA or IRBP in BD with active uveitis.. High ICOS expression in BD patients with uveitis contributed to the upregulation of IFN-γ, IL-17, and TNF-α production, suggesting that abnormal ICOS costimulation may play an immunopathologic role in the pathogenesis of uveitis in BD.

Topics: Adult; Antibodies, Blocking; Antigens, Differentiation, T-Lymphocyte; B7-1 Antigen; Behcet Syndrome; Biomarkers; CD4-Positive T-Lymphocytes; Concanavalin A; Cytokines; Eye Proteins; Female; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation; Humans; Inducible T-Cell Co-Stimulator Ligand; Inducible T-Cell Co-Stimulator Protein; Lymphocyte Activation; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Retinol-Binding Proteins; RNA, Messenger; Th1 Cells; Uveitis

To investigate the role of nitric oxide (NO), produced by the inducible form of NO synthase (NOS-2) in the development of experimental autoimmune uveoretinitis (EAU), we immunized C57BL/6x129Sv (H-2(b)) mice carrying a targeted disruption of the gene encoding NOS-2 (NOS-2[-/-]), and wild-type (WT) C57BL/6x129Sv controls with interphotoreceptor retinoid binding protein (IRBP). NOS-2[-/-] mice developed a clinical EAU with delayed onset and decreased severity compared to WT controls. The ocular tissues from WT mice contained activated F4/80 macrophages with NOS-2 expression and retinal destruction whereas less intense EAU was detected in NOS-2[-/-] mice. The expression of NOS-2 mRNA was detected in the retina at the peak of EAU in WT. Analysis of cytokine production in the spleen from NOS-2[-/-] mice by RT-PCR showed high levels of IL-10 mRNA. Our results demonstrate that NO is clearly involved in EAU and may be important for the regulation of immune responses through the regulation of IL-10.

Topics: Animals; Autoimmune Diseases; Cell Division; Concanavalin A; Eye Proteins; Female; Gene Expression Regulation, Enzymologic; Immunization; Immunoglobulin G; Interferon-gamma; Interleukin-10; Lymphocytes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Retina; Retinitis; Retinol-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Spleen; Tumor Necrosis Factor-alpha; Uveitis

MX-68 is a novel antifolate which is chemically designed not to undergo intracellular polyglutamation thus preventing the development of adverse effects. The present study was carried out to examine both the in vitro and in vivo effects of MX-68 on experimental autoimmune uveitis (EAU) and to compare its effect on collagen-induced arthritis (CIA) in rats. EAU was induced by injecting Lewis rats with retinal S-antigen in complete Freund's adjuvant. Either MX-68 or methotrexate (MTX), which forms several polyglutamates intracellularly, was orally administered five days a week for three weeks beginning on the day of immunization. In vivo, both MX-68 and MTX significantly delayed the onset of EAU and inhibited the antibody response to S-antigen in a dose-dependent manner. High dose MX-68 (2.5 mg kg-1 day-1) completely abrogated the induction of EAU. No adverse effects were observed in either MX-68- or MTX-treated rats. However, the cessation of MX-68 administration after a period of three weeks resulted in the induction of EAU. In contrast, both MX-68 and MTX suppressed the severity of CIA without affecting the onset of the disease and inhibited anti-collagen antibody production in a dose-dependent fashion. Discontinuation of the drugs did not result in the recurrence of CIA. In vitro, both MX-68 and MTX significantly suppressed the proliferation of S-antigen- and Con A-stimulated lymph node cells obtained from immunized rats in a dose-dependent fashion. These data suggest that MX-68 may be useful for the treatment of autoimmune diseases including EAU and that the pathophysiology of EAU could be different from that of CIA.

Topics: 2-Aminoadipic Acid; Animals; Arrestin; Arthritis, Experimental; Autoimmune Diseases; Cell Division; Cells, Cultured; Collagen; Concanavalin A; Dose-Response Relationship, Drug; Female; Folic Acid Antagonists; Immunization; Immunosuppressive Agents; Lymphocytes; Methotrexate; Rats; Rats, Inbred Strains; Uveitis

The in vitro and in vivo effects of a new immunomodulating agent, bucillamine, on experimental autoimmune uveitis (EAU) was studied in the rat. The capacity of S-antigen-sensitized lymphocytes to proliferate in response to the antigen or to produce antigen-specific antibodies was significantly suppressed by bucillamine in culture in a dose-dependent manner. The inhibitory effect of bucillamine was significantly enhanced by adding cyclosporin A (CYA) in the culture. The in vivo effects of bucillamine alone or in combination with CYA were further examined in Lewis rats immunized with S-antigen. All untreated rats developed severe EAU 17 days after S-antigen immunization, while rats treated with either bucillamine (200 mg kg-1 day-1) or CYA (2 mg kg-1 day-1) demonstrated milder symptoms of EAU. A combination therapy with bucillamine (20 or 200 mg kg-1 day-1) and CYA (2 mg kg-1 day-1) exhibited much more significant suppression of EAU induction. Although the in vivo treatment with bucillamine or CYA had no effects on the T-cell populations of spleen cells, the combination therapy significantly decreased the CD4+ T-cell population. As for the immune responses to S-antigen in drug-treated rats, bucillamine suppressed the lymphocyte proliferation to S-antigen, which was further suppressed by combination therapy with CYA. The serum antibody levels specific to S-antigen were not affected by tested dose of bucillamine.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibody Formation; Antigens; Arrestin; Autoimmune Diseases; Cell Division; Cells, Cultured; Concanavalin A; Cyclophosphamide; Cysteine; Drug Synergism; Eye Proteins; Immune Tolerance; Male; Rats; Rats, Inbred Lew; Spleen; Uveitis

During the later stages of soluble-antigen (sAg)-induced experimental autoimmune uveoretinitis (EAU), an increase in the relative number of CD8+ lymphocytes has been observed at the site of inflammation in the retina. It has been suggested that these late-appearing CD8+ cells might down-regulate this acute disease process. To determine the role of the CD8+ cells in EAU, Lewis rats were depleted of CD8+ cells prior to and during disease and the enucleated eyes examined histologically. The spleen cells from CD8-depleted rats were also examined for their ability to respond to concanavalin A (Con A) and to allogeneic targets as determined by mixed lymphocyte reaction (MLR) and cytotoxicity assays. The results suggest that depleting CD8+ cells had no effect on the course of disease and that CD8+ cells do not play a crucial role in the immunoregulation of EAU.

Topics: Animals; Autoimmune Diseases; CD4-Positive T-Lymphocytes; CD8 Antigens; Concanavalin A; Eye; Female; Immunoenzyme Techniques; Lymphocyte Culture Test, Mixed; Rats; Rats, Inbred Lew; Retinitis; Spleen; T-Lymphocytes; Uveitis

We have evaluated the effect of human Igs for intravenous use (IVIg) on the onset and development of experimental autoimmune uveoretinitis (EAU), a T cell-dependent autoimmune disease induced in rats by a single immunization with retinal S-antigen (S-Ag). Five consecutive daily infusions of IVIg, starting on the same day as S-Ag immunization, protected (Lewis x Brown-Norway) F1 rats against EAU. The prevention of EAU was IVIg-specific, i.e. mediated by pooled human IgG from multiple donors, since neither infusions of BSA nor infusions of pooled Ig from only two healthy individuals were effective. Treatment with IVIg decreased lymphocyte proliferative and antibody responses to S-Ag and the proliferative response to concanavalin A. Lack of proliferation was not dependent upon generation of suppressor cells. Lymph node (LN) cells from IVIg-treated and S-Ag-immunized animals neither proliferated nor secreted IL-2 in response to S-Ag but proliferated when co-cultured with LN cells from rats immunized with S-Ag. Our findings are compatible with an induction of a state of functional inactivation/anergy of T lymphocytes by infusions of IVIg. This functional inactivation may be due to the presence in IVIg of antibodies that bind both in vivo and in vitro to rat lymphocytes. Results from the present study suggest a novel mechanism by which IVIg may be beneficial in human autoimmune diseases.

Topics: Animals; Antigens; Arrestin; Autoimmune Diseases; Concanavalin A; Eye Proteins; Female; Flow Cytometry; Humans; Immunoglobulins, Intravenous; Interleukin-2; Lymph Nodes; Lymphocyte Activation; Male; Rats; Rats, Inbred BN; Rats, Inbred Lew; Retinitis; Tuberculin; Uveitis

The authors previously reported that FK506 effectively suppressed the induction of experimental autoimmune uveoretinitis (EAU) in rats with much lower doses than cyclosporine A. This study was aimed at analyzing the immune status of the FK506-treated and EAU-suppressed rats and examining the hypothesis whether the agent could induce antigen-specific suppressor T (Ts) cells. It was found that spleens from S-antigen-immunized and FK506-treated rats contained a population of Ts cells inhibiting the proliferative responses of S-antigen-sensitized lymphocytes to S-antigen, yet these cells did not affect the proliferative responses of interphotoreceptor retinoid-binding protein (IRBP)-sensitized lymphocytes to IRBP. The helper T (Th) cells did not exhibit such suppressor activities. Furthermore, transfer of Ts cells from S-antigen-immunized and FK506-treated rats to naive syngenic rats induced partial inhibition of EAU induction or delay of EAU onset after immunizing the recipient rats with S-antigen. Lymphocytes from the EAU-suppressed recipients showed low proliferative response to S-antigen and low levels of antibody to S-antigen. These data thus indicate that FK506 treatment after S-antigen immunization induces an activation of Ts cells specific to S-antigen and that the Ts cells might contribute, at least in part, to the uniquely prolonged and intensive immunosuppression by FK506.

Topics: Animals; Anti-Bacterial Agents; Antigens; Arrestin; Autoimmune Diseases; Concanavalin A; Epitopes; Eye Proteins; Immunization; Immunosuppressive Agents; Immunotherapy, Adoptive; Lymphocyte Activation; Male; Rats; Rats, Inbred Lew; Retinitis; Retinol-Binding Proteins; Spleen; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Tacrolimus; Uveitis

A possible role for retinal pigment epithelial cells (RPE) as local antigen presenting cells in immune inflammatory eye disease was investigated by studying the in vitro response of human RPE cells to stimulation with purified IFN-gamma or Con A induced lymphokine. RPE cells cultured with a single dose of 50-1000 u/ml IFN-gamma for up to 8 days to allow maximal Class II gene transcription, expressed HLA DP, DR and DQ antigens in a dose-dependent manner with 80% or more of cells positive for each antigen at the higher concentration. After removal of a suboptimal IFN-gamma stimulus, HLA-DR antigen expression persisted for at least 15 days. HLA-DP and DQ antigens persisted only after maximal IFN-gamma stimulation. Lymphokine from Con A stimulated lymphocytes induced higher levels of DR and DQ expression (80%) over DP (15%) implying complex interactions with other mediators present in the lymphocyte culture supernatant. Since RPE cells phagocytose and recycle autoantigen-rich retinal rod outer segments and co-express HLA DR and DQ Class II antigens in response to IFN-gamma stimulation, an immunoregulatory role in conditions in which retinal autoimmunity is implicated, such as chronic idiopathic posterior uveitis and retinal vasculitis is postulated for these cells.

Topics: Autoimmune Diseases; Cells, Cultured; Concanavalin A; HLA-DP Antigens; HLA-DQ Antigens; HLA-DR Antigens; Humans; Interferon-gamma; Kinetics; Lymphokines; Pigment Epithelium of Eye; Uveitis

Topics: Adult; Antibodies, Monoclonal; Antigens, Surface; Concanavalin A; Female; Humans; Male; Middle Aged; T-Lymphocytes; Uveitis; Uveomeningoencephalitic Syndrome

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.

Topics: Adult; Animals; Antibodies, Monoclonal; Antigens, Surface; Cell Line; Complement System Proteins; Concanavalin A; Cytotoxicity, Immunologic; Epitopes; Female; HeLa Cells; Humans; Iritis; Killer Cells, Natural; Kinetics; Lymphocytes; Male; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Middle Aged; Rabbits; Syndrome; T-Lymphocytes, Cytotoxic; Uveitis

Lymphokines were prepared from rabbit lymph node cells specifically activated in vitro with the insoluble antigen ovalbumin or nonspecifically activated with the insoluble mitogen concanavalin A. Intravitreal injection of either lymphokine caused uveitis in the normal rabbit eye, more severe and more prolonged with the concanavalin A-activated supernatants than with the ovalbumin-activated supernatants. The most striking difference between the two lymphokine preparations was in their ability to induce a secondary intraocular antibody response in the trinitrophenyl bovine gamma globulin-primed recipient. Concanavalin A supernatants stimulated polyclonal B cell activation with appreciable anti-TNP responses in animals primed either systemically or locally. In contrast, ovalbumin supernatants stimulated an intraocular anti-TNP response only in those animals that had been primed within the eye. We speculate that two or more different lymphokines are active, some of which attract lymphocytes nonspecifically to the local site and others of which excite polyclonal B cell activation and plasma cell formation.

Topics: Animals; Antibody Formation; Antibody-Producing Cells; B-Lymphocytes; Concanavalin A; Female; Hemolytic Plaque Technique; Iris; Lymphocyte Activation; Lymphokines; Male; Rabbits; Uveitis

Lysosome proteins derived from peripheral blood granulocytes of patients with multiple sclerosis (MS), lupus erythematosus (LE), rheumatoid arthritis (RA) and recurrent uveitis cause the impairment of Con A-induced suppressor cell activity in two-step culture in vitro. This effect is independent of lysosome protease activity. Such a phenomenon, observed in vitro, may cause disturbances in suppressor cell function in the course of the above diseases.

Topics: Adult; Arthritis, Rheumatoid; Autoimmune Diseases; Concanavalin A; Female; Humans; Isoflurophate; Lupus Erythematosus, Systemic; Male; Middle Aged; Multiple Sclerosis; Neutrophils; Proteins; Recurrence; T-Lymphocytes, Regulatory; Uveitis

Suppressor-cell function was evaluated by two in-vitro assay systems in forty patients with intraocular inflammatory disease (uveitis) and in sixteen healthy age-matched controls. A concanavalin-A (Con A)-induced suppressor-cell assay showed that patients with posterior uveitic conditions had greater suppressor activity than did the anterior uveitic group or the control group (p < 0.005); but an assay for non-induced suppressor-cell activity showed that the posterior uveitic group had less suppressor activity (p < 0.001). These findings suggest (a) that at least two cell types may be involved in immunoregulation, and (b) that not all diseases of presumed autoimmune origin are the result of reduced suppressor activity, which has been shown in some "autoimmune" conditions.

Topics: Adult; Autoimmune Diseases; Choroid; Ciliary Body; Concanavalin A; Female; Humans; Iris; Male; T-Lymphocytes, Regulatory; Uveitis