IL-4 Potentiates IL-1ß- and TNF-a-stimulated IL-8 and MCP-1 protein production in human retinal pigment epithelial cells

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Abstract
Purpose. Human retinal pigment epithelial (HRPE) cells are involved in ocular inflammation by secretion of chemokines such as IL-8 and MCP-1. It has been shown in this and other laboratories that interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-a) are potent inducers of HRPE IL-8 and MCP-1 secretion. The induced IL-8 and MCP-1 expression is often modulated by other proinflammatory factors in a synergistic manner. Modulation of IL-8 and MCP-1 production by interleukin-4 (IL-4), a important mediator in Th2-mediated immunity, and granulocyte/macrophage-colony-stimulating factor (GM-CSF), one of the cytokines secreted by HRPE has been reported in non-ocular cells. The aim of the present investigation was to study effects of these two cytokines alone or in combination with IL-1ß or TNF-a on HRPE IL-8 and MCP-1 generation. Methods. The primary culture of HRPE cells was stimulated with various doses of IL-4, GM-CSF, IL1-ß and TNF-a alone or in combination for 8 or 24 hr. The supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) for IL-8 and MCP-1. The mRNAs were isolated from the corresponding cells for Northern blot analysis. Results. IL-1ß and TNF-a induced dose-dependent increases in HRPE IL-8 and MCP-1 secretion with maximal stimulation observed at 2–5 ng/ml. IL-4 alone (100 ng/ml) resulted in a slight increase of MCP-1 and IL-8 secretion. When IL-4 was co-administrated with IL-1ß or TNF-a, two to three-fold increases in IL-8 and MCP-1 were observed over the maximal levels induced by IL-1ß or TNF-a alone. Northern blot analyses revealed that IL-4 did not alter the steady-state MCP-1 mRNA stimulated by IL-1ß and TNF-a, or alter the IL-8 mRNA stimulated by TNF-a, although the IL-1ß-induced IL-8 mRNA was slightly enhanced by higher concentrations of IL-4 (100 ng/ml). Conclusion. The synergistic action by IL-4 occurs predominately at the post-transcriptional level. In contrast to IL-4, GM-CSF alone or in combination with IL-1ß or TNF-a did not generate additional secretion of HRPE IL-8 and MCP-1. HRPE IL-8 and MCP-1 gene expression and protein production are stimulated by IL-1ß or TNF-a through pathways differentially modulated by IL-4 and GM-CSF.