Cell-Associated Human Retinal Pigment Epithelium Interleukin-8 and Monocyte Chemotactic Protein-1: Immunochemical and In-situ Hybridization Analyses

Abstract

Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1β) (0.2–20 ng ml−1), tumor necrosis factor (TNF-α) (0.2–20 ng ml−1) or interferon-gamma (IFN-γ) (10–1000 U ml−1) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr.ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1β or TNF-α, but cell associated IL-8 was detectable only after high dose (20 ng ml−1) IL-1β stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1β>TNF-α>IFN-γ stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (>60%) and monocyte (53–57%) chemotactic activity, respectively.Using in situ hybridization IL-8 mRNA was readily detected within IL-1β>TNF-α stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1β>TNF-α> or IFN-γstimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1β(20 ng ml−1) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-β, TNF-α, or IFN-γ. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.