PURPOSE: The purpose of the study was to examine the effect of T-lymphocyte products on human retinal pigment epithelial (HRPE) cell interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and gene expression. METHODS: HRPE cells were stimulated for 2, 4, 8, or 24 hours with 20% conditioned media (CM) from T-lymphocytes stimulated with CD3 or CD28 monoclonal antibodies (mAbs) or phorbol myristic acid. In some experiments, CM from CD3 mAb-stimulated T-lymphocytes was preincubated with neutralizing anti-(alpha)-tumor necrosis factor (TNF), alpha-interferon-gamma (IFN-gamma), or alpha-interleukin-1 (IL-1) mAb (control) to determine the contributions of each of these cytokines to HRPE chemokine induction by stimulated T-lymphocyte CM. HRPE cells were stimulated for 8 and 24 hours with IL-1 beta (0.2 to 20.0 ng/ml) (positive control), TNF-alpha (0.2 to 20.0 ng/ml) (positive control), IFN-gamma (1 to 1000 U/ml), IFN-gamma + IL-1 beta, IFN-gamma + TNF-alpha. Interleukin-2 (IL-2; 100 ng/ml) alone or in combination with IL-1 beta, TNF-alpha, or IFN-gamma also was tested. Enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses were performed to determine secreted IL-8 and MCP-1 and their steady state mRNA expression, respectively. RESULTS: ELISA showed significant increases in HRPE IL-8 and MCP-1 secretion by CM from T-lymphocytes stimulated with CD3 or CD3 + CD28 mAb. Smaller, but significant, increases in IL-8 and MCP-1 resulted from CM phorbol myristic acid-stimulated T-lymphocytes. CM preincubated with neutralizing alpha-TNF or alpha-IFN-gamma mAb induced significantly less HRPE IL-8 and MCP-1, whereas preincubation of CM with neutralizing alpha-IL-1 mAb failed to inhibit CM-induced IL-8 or MCP-1. Northern blot analysis showed increased HRPE IL-8 and MCP-1 mRNA expression within 2 hours of stimulation and was maintained up to 24 hours. CM from T-lymphocytes stimulated with CD3 mAb or CD3 + CD28 mAb produced the greatest increases in IL-8 and MCP-1 mRNA. IFN-gamma induced dose-dependent increases in HRPE MCP-1, but not IL-8, IFN-gamma potentiated IL-1 beta and TNF-alpha-induced MCP-1 production, but showed little modulation of IL-1 beta and TNF-alpha-induced IL-8 production. IL-2 did not induce HRPE IL-8 or MCP-1, nor did it modulate the effects of the other cytokines. Northern blot analysis confirmed the ELISA results. CONCLUSIONS: T-lymphocyte secretions induce HRPE IL-8 and MCP-1 gene expression and secretion. TNF and IFN-gamma appear to be necessary components of T-lymphocyte CM for the induction of HRPE IL-8 and MCP-1. IFN-gamma alone induces HRPE MCP-1, albeit to a lesser extent than would IL-1 beta or TNF-alpha, and potentiates IL-1 beta- and TNF-alpha-induced HRPE MCP-1. IL-2 does not appear to modulate cytokine-induced HRPE IL-8 or MCP-1.
PURPOSE: The purpose of the study was to examine the effect of T-lymphocyte products on human retinal pigment epithelial (HRPE) cell interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and gene expression. METHODS: HRPE cells were stimulated for 2, 4, 8, or 24 hours with 20% conditioned media (CM) from T-lymphocytes stimulated with CD3 or CD28 monoclonal antibodies (mAbs) or phorbol myristic acid. In some experiments, CM from CD3 mAb-stimulated T-lymphocytes was preincubated with neutralizing anti-(alpha)-tumor necrosis factor (TNF), alpha-interferon-gamma (IFN-gamma), or alpha-interleukin-1 (IL-1) mAb (control) to determine the contributions of each of these cytokines to HRPE chemokine induction by stimulated T-lymphocyte CM. HRPE cells were stimulated for 8 and 24 hours with IL-1 beta (0.2 to 20.0 ng/ml) (positive control), TNF-alpha (0.2 to 20.0 ng/ml) (positive control), IFN-gamma (1 to 1000 U/ml), IFN-gamma + IL-1 beta, IFN-gamma + TNF-alpha. Interleukin-2 (IL-2; 100 ng/ml) alone or in combination with IL-1 beta, TNF-alpha, or IFN-gamma also was tested. Enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses were performed to determine secreted IL-8 and MCP-1 and their steady state mRNA expression, respectively. RESULTS: ELISA showed significant increases in HRPE IL-8 and MCP-1 secretion by CM from T-lymphocytes stimulated with CD3 or CD3 + CD28 mAb. Smaller, but significant, increases in IL-8 and MCP-1 resulted from CM phorbol myristic acid-stimulated T-lymphocytes. CM preincubated with neutralizing alpha-TNF or alpha-IFN-gamma mAb induced significantly less HRPE IL-8 and MCP-1, whereas preincubation of CM with neutralizing alpha-IL-1 mAb failed to inhibit CM-induced IL-8 or MCP-1. Northern blot analysis showed increased HRPE IL-8 and MCP-1 mRNA expression within 2 hours of stimulation and was maintained up to 24 hours. CM from T-lymphocytes stimulated with CD3 mAb or CD3 + CD28 mAb produced the greatest increases in IL-8 and MCP-1 mRNA. IFN-gamma induced dose-dependent increases in HRPE MCP-1, but not IL-8, IFN-gamma potentiated IL-1 beta and TNF-alpha-induced MCP-1 production, but showed little modulation of IL-1 beta and TNF-alpha-induced IL-8 production. IL-2 did not induce HRPE IL-8 or MCP-1, nor did it modulate the effects of the other cytokines. Northern blot analysis confirmed the ELISA results. CONCLUSIONS: T-lymphocyte secretions induce HRPE IL-8 and MCP-1 gene expression and secretion. TNF and IFN-gamma appear to be necessary components of T-lymphocyte CM for the induction of HRPE IL-8 and MCP-1. IFN-gamma alone induces HRPE MCP-1, albeit to a lesser extent than would IL-1 beta or TNF-alpha, and potentiates IL-1 beta- and TNF-alpha-induced HRPE MCP-1. IL-2 does not appear to modulate cytokine-induced HRPE IL-8 or MCP-1.