Constitutive and lps-induced expression of mcp-1 and il-8 by human uveal melanocytes in vitro and relevant signal pathways

Abstract

PURPOSE. Melanocytes are one of the major cellular components in the uvea. Interleukin-8/ CXCL8 and monocyte chemoattractant protein-1 (MCP-1/CCL2) are the two most important proinflammatory chemokines. We studied the constitutive and lipopolysaccharide (LPS)-induced expression of IL-8 and MCP-1 in cultured human uveal melanocytes (UM) and explored the relevant signal pathways. METHODS. Conditioned media and cells were collected from UM cultured in medium with and without stimulation of LPS. Interleukin-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Nuclear factor (NF)-jB in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases1/2 (ERK1/2), and c-Jun N-terminal kinase1/2 (JNK1/2) in cells cultured with and without LPS were measured by ELISA kits. Inhibitors of p38 (SB203580), ERK1/2 (UO1026), JNK1/2 (SP600125), and NF-jB (BAY11-7082) were added to the cultures to evaluate their effects. RESULTS. Low levels of IL-8 and MCP-1 proteins were detected in the conditioned media in UM cultured without serum. Lipopolysaccharide (0.01-1 lg/mL) increased IL-8 and MCP-1 mRNAs and proteins levels in a dose-and time-dependent manner, accompanied by a significant increase of phosphorylated JNK1/2 in cell lysates and NF-jB in nuclear extracts. Nuclear factor-jB and JNK1/2 inhibitors significantly blocked LPS-induced expression of IL-8 and MCP-1. CONCLUSIONS. This is the first report on the expression and secretion of chemokines by UM. The data suggest that UM may play a role in the pathogenesis of ocular inflammatory diseases.