Effects of mechanical stress on expressions of monocyte chemoattractant protein-1 and interleukin-8 of cultured human retinal pigment epithelial cells

Abstract

To investigate the expressions of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) of cultured human retinal pigment epithelial (hRPE) cells under mechanical stress in vitro, so as to mimic and understand the role of these 2 inflammatory cytokines in the early stage of development of primary retinal detachment. Ferric oxide beads coated with collagen were added to the culture plate containing wall-attaching hRPE cells and then the cells were incubated and washed to remove the unbound beads. A magnet was put to the culture plate to provide vertical magnetic force. Cytochalasin D (CD) was added to the culture fluid to inhibit the phagocytizing, secreting, and moving functions of the hRPE. Before the experiment and 15 min, 0.5 h, 1 h, 4 h, and 8 h the beginning of experiment, the MCP-1 mRNA and IL-8 mRNA in the hRPE cells were measured with reverse transcriptase polymerse chain reaction (RT-PCR), and the MCP-1 and IL-8 protein expression in the supernatants was tested with enzyme-linked immunosorbent assay (ELISA) kits. MCP-1 and IL-8 mRNA were expressed at very low levels in the RPE cells and supernatant not exposed to magnetic force. After exposure to magnetic force, both MCP-1 and IL-8 mRNA demonstrated "double peaks" expression. Their first peak levels appeared within 0.5 h, being 3.30 ng/L +/- 0.12 ng/L and 1.88 ng/L +/- 0.08 ng/L respectively. The first peaks of MCP-1 and IL-8 protein expression were within 1 h, being 552.05 ng/L +/- 7.64 ng/Land 236.67 ng/L +/- 14.30 ng/L respectively, and the second peaks appeared about 4 hours later. After cytochalasin D pretreated, the expression and secretion of both MCP-1 and IL-8 of the hRPE cells were significantly decreased to the levels of 2.36 ng/L +/- 0.27 ng/L and 1.64 ng/L +/- 0.08 ng/L, and 353.80 ng/L +/- 16.68 ng/L and 101.86 ng/L +/- 15.92 ng/L respectively. Mechanical stress induces the RPE cells to express MCP-1 and IL-8, and this effect was inhibited in part by pretreatment of CD, indicating that the cytoskeleton may be involved in the effect and that these two inflammatory cytokines take a part in the early stage of development of primary retinal detachment.