PURPOSE: The purpose of this study was to clarify the interactions between macrophages (MPs) and RPE cells in coculture systems to investigate the functional plasticity of RPE cells. METHODS: Adherent peritoneal cells or murine MP cell line Raw 264.7 was cocultured with primary RPE cells taken from C57BL/6 mice, with or without lipopolysaccharide (LPS) or TNF-α stimulation. The cytokine levels of the culture supernatants (CSs) were then analyzed with the Bio-Plex murine 23-Plex Panel Assay Kit (Bio-Rad Laboratories). Monocyte chemoattractant protein-1 (MCP-1), IL-6, VEGF, and TNF-α in CS were further quantified by ELISA. The expression profiles, in cocultures, of complement-associated genes, TNF-α, and angiogenesis-associated genes were analyzed by quantitative real-time PCR. RESULTS: The production of MCP-1, IL-6, and VEGF was synergistically elevated when primary MPs or RAW264.7 cells and RPE cells were cocultured compared with those derived from sole cultures of MPs and RPE cells. The synergistic effect was confirmed without direct cell contact and was more prominent in the presence of LPS or TNF-α. TNF-α production by MPs was suppressed by RPE cells. Coculture of RPE cells with RAW264.7 cells increased the gene expression of C3, CFB, and VEGF genes, whereas it reduced those of complement regulatory factors CFH, CD59, clusterin, and TNF-α and antiangiogenic pigment epithelium-derived factor (PEDF). CONCLUSIONS: Our findings indicate the presence of ingenious interactions between MPs and RPE cells that forces the inflammation and complement activation in the vicinity of RPE cells under pathologic conditions.
PURPOSE: The purpose of this study was to clarify the interactions between macrophages (MPs) and RPE cells in coculture systems to investigate the functional plasticity of RPE cells. METHODS: Adherent peritoneal cells or murine MP cell line Raw 264.7 was cocultured with primary RPE cells taken from C57BL/6 mice, with or without lipopolysaccharide (LPS) or TNF-α stimulation. The cytokine levels of the culture supernatants (CSs) were then analyzed with the Bio-Plex murine 23-Plex Panel Assay Kit (Bio-Rad Laboratories). Monocyte chemoattractant protein-1 (MCP-1), IL-6, VEGF, and TNF-α in CS were further quantified by ELISA. The expression profiles, in cocultures, of complement-associated genes, TNF-α, and angiogenesis-associated genes were analyzed by quantitative real-time PCR. RESULTS: The production of MCP-1, IL-6, and VEGF was synergistically elevated when primary MPs or RAW264.7 cells and RPE cells were cocultured compared with those derived from sole cultures of MPs and RPE cells. The synergistic effect was confirmed without direct cell contact and was more prominent in the presence of LPS or TNF-α. TNF-α production by MPs was suppressed by RPE cells. Coculture of RPE cells with RAW264.7 cells increased the gene expression of C3, CFB, and VEGF genes, whereas it reduced those of complement regulatory factors CFH, CD59, clusterin, and TNF-α and antiangiogenic pigment epithelium-derived factor (PEDF). CONCLUSIONS: Our findings indicate the presence of ingenious interactions between MPs and RPE cells that forces the inflammation and complement activation in the vicinity of RPE cells under pathologic conditions.