Francois G. Roberge

Standardizatlon of Vitreal inflammatory Activity in Intermediate and Posterior Uveitis

Standardization of observations is recognized as fundamental to clinical research. The methodology for the evaluation of anterior segment inflammatory disease has become well accepted, while the ocular inflammatory standardization of the posterior segment has not been so well described or accepted. A system for the evaluation of vitreal inflammatory activity in patients with intermediate and posterior uveitis is presented. A series of photographs representing various degrees of fundus vitreal haze is depicted. The observer examines the eye with an indirect ophthalmoscope, then chooses the photograph which most closely simulates what is being seen. This technique is rapid and its reproducibility is helpful in standardizing clinical observations.

Anti-retinal Auto-antibodies in Vogt-Koyanagi-Harada Syndrome, Behcet's Disease, and Sympathetic Ophthalmia

Sera of patients diagnosed as having the active Vogt-Koyanagi-Harada (VKH) syndrome, Behcets syndrome or sympathetic ophthalmia as well as normal controls were evaluated by ELISA and by staining of normal human retinal tissue using the avidin-biotin-peroxidase complex (ABC) technique for anti-retinal antibodies. No anti-retinal S-antigen antibodies were detected by ELISA. However, autoimmune antibodies were found against the outer segments of photoreceptors and Müller cells in patients with the VKH syndrome, with lower titers in some patients with Behcets syndrome, and in a few patients with sympathetic ophthalmia. These results suggested anti-retinal antibodies were present and that retinal autoimmunity may play a role in pathogenesis in varieties of posterior uveitis. In addition, the indirect immunoperoxidase staining technique may facilitate the diagnosis of VKH in uncertain cases.

Long-term culture of Muller cells from adult rats in the presence of activated lymphocytes/monocytes products

A method for the culture of retinal Muller cells from adult rats is described. Whereas isolated Muller elements did not grow in a medium composed of MEM + 10% FBS, the addition of supernatant from mitogen activated spleen cells induced a strong proliferative response. The factor(s) responsible for that growth were not species specific, as mouse spleen cells conditioned medium was also an effective stimulant. The highly significant degree of DNA synthesis induced by the supernatant from an S-antigen specific T-helper cell line (reacting to the retinal antigen presented on APC) indicates that the growth promoting activity is not restricted to mitogen driven spleen cells. It is suggested that products of inflammatory cells secreted in the course of intraocular immune processes may contribute to the formation of epiretinal membranes. These products also induced the expression of Ia surface antigens by Muller cells, supporting the hypothesis of a possible involvement in local immune reactions.

Treatment of autoimmune uveoretinitis in the rat with rapamycin, an inhibitor of lymphocyte growth factor signal transduction

Rapamycin (RAPA) is a macrolide antibiotic with unique immunosuppressive properties. RAPA inhibits T-cell function by interfering with IL-2 and IL-4 signal transduction. It does not prevent IL-2 production or IL-2R expression. The efficacy of RAPA in the treatment of autoimmune diseases was evaluated using the experimental autoimmune uveoretinitis (EAU) model. EAU was actively induced in Lewis rats by immunization with S-antigen in Hunters adjuvant. RAPA and control vehicle were administered by continuous intravenous infusion over a 14 day period by miniosmotic pump. RAPA treatment initiated on the day of immunization or 7 days later was found to efficiently inhibit EAU induction. The minimal effective dose was 0.1 mg/kg/d. EAU inhibition was correlated with reduced number of cells in the immunization site draining lymph nodes, as well as with a shift and lowering of the peak of the lymphocyte proliferative response curve. The anti-S-antigen antibody response was delayed by 3 days under RAPA treatment and the serum levels lowered in a dose dependent manner. An initial body weight loss was observed during the first week of drug administration, but there was a normal weight gain afterward.

Protective role of nitric oxide in ocular toxoplasmosis.

AIMS: To evaluate the role of nitric oxide (NO) in ocular involvement during systemic toxoplasmosis. METHODS: C57B1/6 mice were infected with Toxoplasma gondii strain ME49. The synthesis of NO was inhibited by an intraperitoneal injection of aminoguanidine every 8 hours, starting on the day of infection. Control infected mice received phosphate buffered saline vehicle alone. After 14 days, the ocular lesions were evaluated by histopathological examination. The expression of NO synthase induced in the spleen by toxoplasma infection was evaluated by immunostaining. The production of NO by the spleen cells of infected mice was measured by the colorimetric assay of Griess in the supernatant of cultures stimulated with toxoplasma antigen or concanavalin A. RESULTS: The inhibition of NO production in T gondii infected mice resulted in a marked increase in the symptoms of ocular inflammation. We observed a strong induction of NO synthase expression in the spleen of infected animals. In culture, the spleen cells from these mice produced high levels of NO in response to T gondii antigens. This elevation of NO synthesis was suppressed in the presence of aminoguanidine. CONCLUSION: This study indicates that NO plays a crucial role in the protection against T gondii infection as reflected by the severity of the ocular involvement.

Interleukin 10 inhibits inflammatory cells infiltration in endotoxin-induced uveitis

Abstract• Background: Endotoxin-induced uveitis (EIU) is a model for acute anterior uveitis associated with a variety of pro-inflammatory cytokines and nitric oxide production. Interleukin 10 (IL-10) down-regulates these inflammatory mediators. We report a study of the effect of systemic administration of IL-10 on the inflammatory parameters of EIU. • Methods: Uveitis was induced in C3H/HeN mice by subcutaneous injection of 200 μg lipopolysaccharide (LPS) per mouse. Intraocular inflammation was assessed by leukocyte count and measurement of the protein concentration in the aqueous humor (AH). Mouse recombinant IL-10 at 1000 U or its vehicle alone were administered by three intravenous injections given 4.0 h and 0.5 h before and 8.0 h after LPS injection. • Results: The inflammatory cell infiltration in the eyes was significantly reduced in four of five experiments from 40% to 64% in the groups treated with IL-10 compared to the control groups (P<0.05). In contrast, the level of protein exudation in the anterior chamber (AC) was not significantly affected by IL-10 treatment. • Conclusion: IL-10 reduces the cellular infiltration in the ocular inflammation produced by endotoxin. This result suggests potential usefulness for IL-10 in the treatment of severe anterior uveitis with a strong cellular component.

Glial cells as suppressor cells: Characterization of the inhibitory function

Rat retinal glial cells (Müller cells) profoundly suppress antigen-driven activation, as well as the subsequent interleukin-2 (IL-2)-dependent expansion of autoimmune and conventional-immune syngeneic T-helper lymphocytes, through a contact-dependent mechanism. Characterization of this inhibitory function showed that some activation parameters of autoimmune T-helper lymphocytes specific to the retinal soluble antigen, responding to antigen presented on syngeneic antigen-presenting cells, were differentially affected by coculture with Müller cells. In contrast to endogenous IL-2 production, IL-2-receptor generation and proliferation, the production of interleukin-3 and of interferon-gamma were not inhibited. Inhibition of IL-2-supported proliferation of cells which had already generated the IL-2 receptor was markedly potentiated in the presence of the specific antigen, in a dose-dependent manner. The extent of inhibition was proportional to the number of Müller cells in the culture. The suppression appeared not to involve a major histocompatibility complex (MHC) Class II-related interaction and could act across allogeneic and even xenogeneic barriers. Inhibition affected normal lymphocytes, including primed T cells responding to antigen or to IL-2, unprimed spleen cells responding to the T-cell mitogen concanavalin A (Con-A) and, to a lesser extent, unprimed spleen cells responding to the B-cell mitogen bacterial lipopolysaccharide (LPS). Several permanently transformed cell lines of mouse, rat and human origin were not affected. These results may suggest participation of organ-resident cells in regulation of locally occurring immune processes.

Treatment of uveitis with recombinant human interleukin-13

AIM To evaluate the anti-inflammatory cytokine interleukin-13 (IL-13) for the treatment of uveitis. METHODS Uveitis was induced in monkeys by immunisation with human retinal S-antigen. Starting at the onset of disease, the animals were treated with IL-13 at 25 μg/kg, or vehicle control, injected subcutaneously once a day for 28 days. Intraocular inflammation was scored by indirect ophthalmoscopy for a period of 56 days. Circulating leucocyte levels were monitored. RESULTS Uveitis started unilaterally in all but one animal. IL-13 inhibited inflammation both in the eyes in which the disease was present when the treatment was initiated (p=0.0001), and in the contralateral initially negative eyes (p=0.0001). After cessation of therapy, there was a progressive increase of inflammation in the IL-13 treated group. However, the beneficial effect of IL-13 extended into the 4 week follow up period. IL-13 produced an increase in circulating polymorphonuclear neutrophils and a decrease in lymphocytes. CONCLUSION Administration of IL-13 appears to be a promising modality of treatment for severe uveitis.

Treatment of corneal allograft rejection with the cytotoxin IL-2-PE40

IL-2-PE40 is a recombinant chimeric protein composed of IL-2, fused to a modified pseudomonas exotoxin. This molecule is extremely toxic to activated T cells expressing high-affinity IL-2R. We used this new molecule for selective immunosuppression to treat corneal allograft rejection in the rat, using Fisher and Lewis rats, a strain combination differing only in medial and minor histocompatibility antigens. The effect of IL-2-PE40 on the immunologic response was studied using both a heterotopic corneal graft model and orthotopic grafts. At the dose of 0.31 micrograms/g given intraperitoneally every 12 hr, IL-2-PE40 produced a significant reduction of both total lymph node cells and cytotoxic-T-cell (CTL) activity in draining lymph nodes (DLN) of heterotopically grafted animals. IL-2-PE40 treatment also significantly reduced the clinical rejection score and cumulative rejection rate (CRR) in orthotopic grafts and appears to be a very effective immunosuppressive agent.

Inhibition of T lymphocyte proliferation by retinal glial Müller cells: Reversal of inhibition by glucocorticoids

Study of interactions between retinal glial Müller cells and T lymphocytes have revealed a wide array of reciprocal influences on the functions of these cells. In the present study we show that these interactions can be further modified by corticosteroid hormones. The primary effect of Müller cells on T lymphocytes is an inhibition of the T-cell proliferative response, and it is exerted via a membrane-bound factor. In this report we show that glucocorticoids can reverse the inhibition by suppressing the expression of the Müller cell inhibitory factor. This effect was independent of the action of glucocorticoids on arachidonic acid metabolism.