Igal Gery

TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1.

T-helper type 17 cells (TH17) are implicated in rodent models of immune-mediated diseases. Here we report their involvement in human uveitis and scleritis, and validate our findings in experimental autoimmune uveoretinitis (EAU), a model of uveitis. TH17 cells were present in human peripheral blood mononuclear cells (PBMC), and were expanded by interleukin (IL)-2 and inhibited by interferon (IFN)-γ. Their numbers increased during active uveitis and scleritis and decreased following treatment. IL-17 was elevated in EAU and upregulated tumor necrosis factor (TNF)-α in retinal cells, suggesting a mechanism by which TH17 may contribute to ocular pathology. Furthermore, IL-27 was constitutively expressed in retinal ganglion and photoreceptor cells, was upregulated by IFN-γ and inhibited proliferation of TH17. These findings suggest that TH1 cells may mitigate uveitis by antagonizing the TH17 phenotype through the IFN-γ–mediated induction of IL-27 in target tissue. The finding that IL-2 promotes TH17 expansion provides explanations for the efficacy of IL-2R antibody therapy in uveitis, and suggests that antagonism of TH17 by IFN-γ and/or IL-27 could be used for the treatment of chronic inflammation.

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 Behçets 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.

The involvement of sequence variation and expression of CX3CR1 in the pathogenesis of age-related macular degeneration

This study examined the association between the sequence variation/expression of CX3CR1, a chemokine receptor, and age‐related macular degeneration (AMD). Peripheral blood from 85 AMD patients and 105 subjects without AMD (controls), as well as ocular tissue from 40 pathological sections with AMD and two normal eye sections, were screened for V249I and T280M, two single nucleotide polymorphisms (SNPs) in CX3CR1. An increased prevalence, with the highest odds ratio of 3.57, of the I249 and M280 carriers was found among the AMD cases as compared with the controls. When comparing CX3CR1 expression in the archived eye sections, CX3CR1 transcripts were not detectable in the maculae of AMD eyes bearing T/M280; however, transcripts were detected in the maculae of normal eyes bearing T/T280 or T/M280 as well as in the AMD maculae bearing T/T280. Furthermore, lower CX3CR1 protein expression was observed in the maculae of AMD eyes bearing T/M280 compared with the controls bearing T/T280. The I249 and M280 alleles result in a lowered number of receptor binding sites and a decreased ligand affinity. Our data suggest that a decrease, caused by sequence variation and/or lower CX3CR1 expression, in CX3CR1‐induced cellular activities could contribute to AMD development.

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.

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 Responses of Patients with Uveitis to Retinal Antigens and Their Fragments

Of two patient populations totaling 82 patients, one in the United States and the other in Japan, we studied the cellular immune responses against S-antigen and interphotoreceptor retinoid binding protein as well as to fragments of each antigen. Behçets disease, birdshot retinochoroidopathy, pars planitis, ocular sarcoid, sympathetic ophthalmia, and the Vogt-Koyanagi-Harada syndrome were diagnosed in these patients. The response profile of both antigens paralleled each other. This profile was more commonly seen in patients suffering from diseases affecting the retina. Responders reacting to both antigens or to several fragments of an antigen were present. This pattern of response was seen in 26 of the patients tested. Patients with uveitis appeared able to recognize several autoantigens. This might be a consequence of the breakdown of the blood-retinal barrier and may help perpetuate the inflammatory process. Several patients were capable of responding to more than one epitope of the same antigen, which indicates that there are major differences between the experimental model and human autoimmune diseases in the response to autoantigens. Both of these findings may to help develop new immunotherapeutic strategies in the treatment of uveitis.

Relationship between production and release of lymphocyte-activating factor (interleukin 1) by murine macrophages: I. Effects of various agents

Abstract The relationship between the production and release of lymphocyte-activating factor (LAF, or interleukin 1) by cultured murine peritoneal macrophages (Mφ) was investigated. Unstimulated Mφ produce high levels of intracellular LAF within a few hours after culturing, but release little of this activity into their culture medium. Addition of various agents was found to increase significantly the production and release of LAF, with three different patterns: (a) Both intracellular and extracellular LAF activities were increased in response to latex beads, (b) A marked increase of intracellular LAF, with just a minimal elevation of extracellular activity, was stimulated by LPS. (c) Sharp increases in LAF release, with small increments of the intracellular activity, were induced by silica and glucocerebroside (GL 1 ). Silica and GL 1 damaged the cultured Mφ, as indicated by the increased release of lactate dehydrogenase. It is significant, therefore, that silica and GL 1 increased both intracellular and extracellular LAF levels, suggesting that damage of Mφ may stimulate total LAF production. A combination of LPS with silica or GL 1 acted synergistically on Mφ to release very high levels of LAF, which far exceeded those released by the individual agents. The agents were also tested on Mφ which were precultured, to deplete their LAF content. Latex, LPS, or silica increased LAF production and release by precultured Mφ, but the levels were lower than those obtained with freshly cultured Mφ. The results of this study thus show that the level of LAF release does not necessarily reflect the level of total LAF production by cultured Mφ and suggest that injurious agents may promote LAF production.

Phenotype Switching by Inflammation-Inducing Polarized Th17 Cells, but not by Th1 Cells

Th1 and Th17 cells are characterized by their expression of IFN-γ or IL-17, respectively. The finding of Th cells producing both IL-17 and IFN-γ suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-γ was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-γ or both IFN-γ and IL-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-γ or IFN-γ/IL-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with 20–25% nonpolarized cells than in recipients of 35–40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-γ or IFN-γ/IL-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-γt was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-γ or IFN-γ/IL-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.

Inflammatory Mediators in Uveitis: Differential Induction of Cytokines and Chemokines in Th1- Versus Th2-Mediated Ocular Inflammation

Ocular inflammation leads to vision loss through the destruction and scarring of delicate tissues along the visual axis. To identify inflammatory mediators involved in this process, we used real time RT-PCR to quantify the expression of mRNA transcripts of 34 cytokines, 26 chemokines, and 14 chemokine receptors at certain time points during T cell-mediated ocular inflammation. We induced disease by adoptive transfer of Ag-specific Th1 or Th2 cells into recipients expressing the target Ag in their eyes. We also compared the mediator expression patterns seen in adoptive transfer-induced inflammation with that seen in mouse eyes developing experimental autoimmune uveoretinitis. In addition, we used laser capture microdissection to examine chemokine mRNA production by both retinal pigment epithelium cells and infiltrating leukocytes in inflamed eyes. Major findings included the following: 1) Three patterns of expression of the inflammation-related molecules were seen in recipients of adoptively transferred Th cells: preferential expression in Th1 recipients, or in Th2 recipients, or similar expression in both recipient groups. 2) In experimental autoimmune uveoretinitis, the inflammatory mediator expression pattern largely paralleled that seen in Th1-induced disease. 3) Both retinal pigment epithelium and infiltrating leukocytes expressed chemokine transcripts in distinct, but overlapping patterns in inflamed eyes. 4) Interestingly, trancripts of multiple cytokines, chemokines, and chemokine receptors were constitutively expressed in high levels in mouse eyes. Seven of these molecules have not been previously associated with the eye. These data underscore the multiplicity of mediators that participate in the pathogenesis of eye inflammation and point to upstream cytokines as potential therapeutic targets.

Immunocytochemical demonstration of retinal S-antigen in the pineal organ of four mammalian species

SummaryBy means of immunocytochemistry retinal S-antigen is selectively demonstrated in retinal photoreceptor cells of the rat and in pinealocytes of the hedgehog, rat, gerbil and cat. Brain areas surrounding the pineal organ are immunonegative. The immunoreactive material is evenly distributed in the perikarya of the cells. Occasionally, inner segments of retinal photoreceptors and processes of pinealocytes are also stained. The outer segments of retinal photoreceptors display a strong immunoreaction. In both pinealocytes and retinal photoreceptors the intensity of the immunoreaction varied considerably among individual cells.The immunocytochemical demonstration of retinal S-antigen in mammalian pinealocytes indicates that these cells still bear characteristics of photoreceptors. This finding is in accord with the concept that mammalian pinealocytes are derived from pineal photoreceptor cells of poikilothermic vertebrates.