Interferon-beta and adhesion molecules (E-selectin and s-intracellular adhesion molecule-1) are detected in sera from patients with retinal vasculitis and are induced in retinal vascular endothelial cells by Toll-like receptor 3 signalling.

Retinal vasculitis is a major component of ocular inflammation that plays a role in retinal tissue damage in patients with idiopathic uveitis and Behçet's disease. Here we show that type 1 interferons (IFN alpha/beta) were not detected in sera from normal individuals but were identified in up to 46% of the sera from retinal vasculitis patients. The predominant form of IFN observed was IFN-beta, which was detected in 39% of Behçet's disease patients and 47% of idiopathic uveitis patients. Seven patients whose sera contained IFN-beta were monitored prospectively. IFN-beta was shown to be present for 6-12 months in all seven of the sera samples tested. Furthermore, the adhesion molecule profile identified in this study was strikingly different when Behçet's and uveitis patient sera were compared to sera from normal controls. Sera from Behçet's disease patients contained significantly elevated levels of the soluble adhesion molecules, sE-selectin and s-intracellular adhesion molecule-1 (sICAM-1), whereas sera from patients with idiopathic uveitis contained significantly increased sE-selectin. In vitro studies evaluating the cell source of these cytokines revealed that polyriboinosinic polyribocytidylic acid (poly I:C) activated retinal vascular endothelial cells produce sE-selectin, sICAM-1 and IFN-beta. Production of these molecules was inhibited by pretreatment with anti-Toll-like receptor 3 (TLR-3) antibody. In conclusion, IFN-beta, sE-selectin and sICAM-1 are elevated in patients with retinal vasculitis and are induced in retinal vascular endothelial cells in vitro by activating the innate immune system through TLR-3. Further analysis of innate immune signalling may prove to be a novel target for future studies on pathogenic mechanisms and therapeutic approaches in retinal vasculitis.

Cyclooxygenase-2 gene expression and regulation in human retinal pigment epithelial cells.

PURPOSE: Cyclooxygenases (COX) orchestrate a variety of homeostatic processes and participate in various pathophysiological conditions. The retinal pigment epithelium (RPE) cell performs a variety of regulatory functions within the retina. The conditions under which COX-1 and COX-2 are expressed and upregulated in human RPE (HRPE) cells were determined. METHODS: COX gene expression was examined using RT-PCR analysis of untreated HRPE cultures or cultures exposed to bacterial lipopolysaccharide or various cytokines. COX proteins were detected by immunohistochemistry and Western blot analysis. Prostaglandin (PG) production was analyzed by EIA. RESULTS: Examination of untreated RPE cells revealed the presence of COX-2 mRNA and the absence of COX-1 mRNA. Moreover, cytokine stimulation more readily enhanced COX-2 gene expression than COX-1 gene expression. IL-1 beta, the most potent inducer of COX-2, also resulted in detection of COX-2 protein by immunocytochemical staining and Western blot analysis. There was a direct relationship between both the appearance and amount of COX-2 mRNA and protein synthesis and the degree of PG synthesis by RPE cells. Furthermore, COX inhibitors significantly decreased PG production. Pretreatment of RPE cells with a NF-kappa B inhibitor, PDTC, resulted in dose-dependent decrease in IL-1 beta-induced COX-2 gene expression and PG production. CONCLUSIONS: COX-2 was the predominant isoform of cyclooxygenase in untreated HRPE cells. When HRPE cells were treated with proinflammatory cytokines, COX-2 gene expression and synthesis of PGs were enhanced. NF-kappa B mediated the induction of COX-2 gene expression in HRPE cells. These studies indicate that RPE cells may participate in normal and pathologic retinal conditions through the induction of COX-2.

Macrophage uptake and retention of radiolabeled glycopeptidolipid antigens associated with the superficial L1 layer of Mycobacterium intracellulare serovar 20.

Glycopeptidolipid (GPL) antigens which are associated with the superficial L1 layer of Mycobacterium intracellulare serovar 20 were labeled with radioisotopes by means of internal labeling techniques and used in macrophage uptake and retention studies. The use of tritiated alanine and phenylalanine allowed the incorporation of label into the GPL invariant fatty acyl peptide core, which is common to all members of the Mycobacterium avium-M. intracellulare complex. Radiolabeled GPL antigens were then purified by a one-step column chromatographic procedure and subsequently used to determine the maximum uptake and retention in peritoneal macrophages isolated from C57BL/6 and CBA/J mice. Maximum uptake for peritoneal macrophages from both strains of mice occurred at a concentration between 200 and 250 micrograms of antigen per ml of medium when 3.4 X 10(5) cells were pulsed. Timed experiments demonstrated that approximately 20% of the antigens remained associated with the macrophages up to 4 days after a pulse of 200 micrograms of GPL, and examination of chloroform-extractable components from both macrophages and spent medium revealed that 98% or more of the radioactivity corresponded to intact GPL components. The ability of the GPL antigens to become associated with macrophages is demonstrated by these results, which strongly suggest that these potentially important mycobacterial antigens are inert to degradation by those cells.