Monocyte chemotactic protein gene expression by cytokine-treated human retinal pigment epithelial cells

Abstract

Inflammation involving the retina and choroid is a common clinical problem, but the mechanisms that elicit and maintain ocular inflammation remain poorly understood. Interposed between the sensory retina and the systemic blood circulation within the choroid is the neural-derived retinal pigment epithelium (RPE), which forms part of the blood-retina barrier. The RPE is actively phagocytic and shares several features with mononuclear phagocytes of bone marrow origin, including the production of a neutrophil chemotactic factor, interleukin 8, after stimulation with interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha). Because monocyte-derived macrophages are present in retinal lesions of many common and blinding diseases, we monitored human RPE cells or monocyte chemotactic protein (MCP) mRNA expression and activity following cytokine stimulation. Cultured human RPE cells were left unstimulated or exposed to recombinant human IL-1 beta, TNF-alpha, or lipopolysaccharide. MCP mRNA expression in RPE cells and biologically active MCP in RPE cell supernatants were present 1 hour after stimulation and maintained for 24 hours. Conditioned media from RPE cells stimulated with 20 ng/ml of IL-1 beta or TNF-alpha for 24 hours contained biologically active monocyte chemotactic activity that rose rapidly from baseline levels over 4 hours and plateaued over the subsequent 20 hours. RPE chemotactic activity was dose dependent using concentrations of these cytokines ranging from 20 pg/ml to 20 ng/ml of 4-hour assays. Time- and concentration-dependent expression of RPE cell MCP mRNA was also found in the same cultures. Peak MCP mRNA expression occurred after 8 hours of stimulation with IL-1 beta or TNF-alpha. Maximal steady-state MCP mRNA expression occurred at 20 ng/ml for IL-1 beta. Immunohistochemical staining using specific anti-MCP antibodies resulted in distinctive RPE cell staining, confirming the presence of MCP in human RPE cells. These findings demonstrate that cytokine-stimulated RPE cells may evoke or augment mononuclear phagocyte-mediated ocular inflammation by synthesizing MCP.