SUMMARY

Experimental autoimmune uveoretinitis (EAU)'2 is an established model for human endogenous posterior uveitides, the study of which has led to a greater understanding of the underlying immunopathogenesis ofthis group of conditions.5 EAU is a CD4+ T-lymphocyte mediated disease which can be induced by at least two soluble retinal antigens (S-Ag) and interphotoreceptor retinol binding protein (IRBP), both of which have several uveitogenic epitopes.6-8 The S-Ag induced EAU model is of particular interest as patients demonstrate abnormal in vitro T-cell proliferative responses to S-Ag and its peptide fragments,9 although humoral antibody titres to S-Ag occur in both patients and normal subjects.'0 EAU has proved to be an extremely valuable model for preclinical trials of immunosuppressive therapy as has been shown with cyclosporin A" and with several antigen-specific and immunospecific targeted therapies such as anti-S Ag antibody therapy,'2-'4 anti-Ia antibodies,'5 and antigen coupled splenocyte transfer. 16 Recently, tolerance (immune unresponsiveness) to S-Ag induced EAU has been achieved in susceptible animals by oral administration of milligram quantities of S-Ag.'7 Oral feeding of specific immunopathogenic antigens suppresses successfully the inflammatory response in many other models of autoimmune diseases'8 and the mechanism of 'oral tolerance' is mediated by CD8+ T-lymphocytes. ' ANTIGENSSoluble bovine retinal extract (RE) was prepared by hypotonic lysis of the retina in the dark into 0-02% TRIS-HCI pH 8-0, followed by ultracentrifugation at 25 000 rpm for 15 hours at 4°C and was dialysed against phosphate buffered saline (PBS) over 24 hours. Bovine S-Ag was prepared by HPLC on a TSK-DEAE column as previously described'4 or by affinity column extraction. The S-Ag by both methods was homogeneous by silver staining on SDS polyacrylamide gel using Pharmacia Phast System, according to the manufacturer's instructions. The protein concentration of RE used in the experiments ranged from 5-7 mg/ml and S-Ag

The Fas and Fas ligand (Fas/FasL) pathways may play a central role in cytotoxicity or immunoregulation in liver transplantation. Here, in an attempt to examine the role of Fas/FasL on drug-free tolerance, we measured mRNA levels of Fas/FasL in livers by reverse transcriptase-polymerase chain reaction (RT-PCR), and also protein levels of Fas/FasL in livers by immunohistochemistry and in serum by dot blot assay. PVG recipients bearing DA livers showed serious rejection between post-operative (POD) days 7 and 14, but this rejection was naturally overcome without any immunosuppression. Fas gene and protein products were expressed on almost every cell in livers taken from naive rats, and at any time point in both syngeneic and allogeneic orthotopic liver transplantation (OLT) rats. In contrast, FasL mRNA in DA livers was detectable at POD 2, peaked at POD 14, and declined at POD 63 in allogeneic OLT (DA-PVG). Although the FasL gene was detectable in isografts at POD 14, its expression was much lower than in allografts. The time course and localization of FasL expression indicated that the expression of FasL gradually switched from infiltrating cells to hepatocytes when the rejection was naturally overcome and tolerance was induced in this OLT model. Soluble Fas could constitutively be detected at any time point in the serum of the tolerogenic OLT (DA-PVG) rats and was not diminished during the rejection phase. Soluble FasL peaked at POD 14 in allogeneic OLT, while sFasL was significantly lower in the serum of normal and syngeneic OLT rats. These findings suggest that the Fas and FasL pathways, including soluble forms, may contribute to the control of the immune response in this drug-free tolerance OLT model.

CD5+ B lymphocytes and TCR gamma-delta T lymphocytes, phenotypes implicated in the pathogenesis of autoimmune disease, were isolated from the vitreous in a case of acute sympathetic ophthalmitis. These cells were obtained using a method which allows the selective maintainance in vitro of in vivo activated T lymphocytes. Dual colour flow cytometry showed that after 3 days culture in IL-2 containing medium 61% of cells were CD5/CD19 + ve and 41% CD3/TCR gamma delta + ve. Of the total CD3 + ve population, 15% were gamma/delta negative. These cells formed a population which also responded in a proliferation assay to retinal antigens. Histologically the eye showed a marked mononuclear cell infiltration of the retina, ciliary body and choroid. Granulomatous lesions within the choroid contained lymphocytes, plasma cells and multinucleate giant cells. Immunocytochemistry showed lymphocyte populations to be predominantly CD2 + ve CD3 + ve T lymphocytes of the CD4 sub-set. Distribution of monocytes/macrophages throughout the lesions and restriction of B-lymphocytes to granulomata were all consistent with a DTH type reaction. Despite immunosuppressive therapy, the expression of activation antigens HLA-DR and ICAM-1 on infiltrating and resident ocular tissue cells was high, although IL-2 receptor (CD25) expression was virtually absent. Flow cytometric analysis of peripheral blood cells prior to treatment with Cyclosporin-A showed systemic activation of lymphocytes, with high levels of HLA-DR and CD25 expression and a raised CD4/CD8 ratio.

We studied the surface expression of activation markers IL2-R, HLA-DR and CD45-RO on peripheral T-lymphocytes in two groups of patients (n = 26) with idiopathic uveoretinitis, compared with controls. Thirteen patients were analysed by alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemistry, which demonstrated a significant rise in expression of HLA-DR and IL2-R surface markers. Flow cytometric analysis was performed on a further 13 patients, which confirmed a significant rise in IL2-R expression in uveitis patients. Within this group systemic activation was confined to patients with idiopathic retinal vasculitis. Dual flow cytometry confirmed a CD4+,IL2--R+ T--lymphocyte phenotype. A further 4 patients with retinal vasculitis who had been treated with cyclosporin A demonstrated a 32% reduction in IL2-R expression over a 3-month period. Analysis of CD45-RO and CD5+ cells was found to be uninformative in this study. We have demonstrated activated peripheral lymphocytes in patients predominantly with retinal vasculitis, the significance of which is discussed.