SYNERGISTIC EFFECTS OF GAMMA-INTERFERON ON INFLAMMATORY MEDIATORS THAT INDUCE INTERLEUKIN-6 GENE-EXPRESSION AND SECRETION BY HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS

The retinal pigment epithelial (RPE) cell is a potent regulatory cell within the retina. It helps to maintain normal retinal activity, and f ollowing gamma interferon (IFN-gamma) exposure, it may express major h istocompatibility complex class II molecules and function as an antige n-presenting cell. Since interleukin-1 (IL-1) and IL-6 are potent cyto kines observed in ocular inflammatory processes, we initiated studies to evaluate conditions which enable RPE cells to produce these cytokin es. Cultures of human RPE cells from two eye donors were established a nd characterized, and enzyme immunoassays were employed to screen for IL-1 and IL-6 production. Treatment of RPE cells with lipopolysacchari de (LPS) or recombinant tumor necrosis factor alpha, IL-1, or IFN-gamm a resulted in a significant level of secretion of IL-6, In contrast, t reatment with recombinant epidermal growth factor, basic fibroblast gr owth factor, platelet-derived growth factor, or transforming growth fa ctor beta 1 did not result in IL-6 production. IFN-gamma in combinatio n with suboptimal levels of IL-1, tumor necrosis factor alpha, or LPS can dramatically augment the secretion of IL-6 by RPE cells. Thus, the se inflammatory mediators can act alone or synergistically with IFN-ga mma to activate RPE cells and dramatically increase the expression and secretion of IL-6. In contrast, IL-1 was not detected following stimu lation with any of the above-mentioned cytokines or LPS. Characterizat ion of IL-6 protein production by RPE cells revealed that 98% of the p rotein is promptly secreted by the cell, its induction is dependent up on the time and concentration of the stimulant, and the continuous pre sence of the stimulant is required for IL-6 production. Moreover, West ern blot (immunoblot) analysis of secreted proteins revealed that IL-6 was produced in multiple molecular forms. Characterization of gene tr anscription by Northern (RNA) blot analysis revealed that mRNA for IL- 6 was detected shortly after IL-1 treatment. However, within hours of IL-1 withdrawal, IL-6 mRNA levels returned to control levels. These da ta demonstrate that LPS and cytokine activation of RPE cells results i n the activation of IL-6 gene transcription and IL-6 protein secretion . Moreover, these studies indicate that cytokine activation of RPE cel ls may be one of the critical factors in ocular inflammation and that it is an important consideration in RPE cell transplantation studies.