Production of MCP-1 and RANTES in Cultured Human Peritoneal Mesothelial Cells Stimulated with IL-1beta

Song, In Suk; Yang, Won Seok; Lee, Sang Koo; Yoon, Kyun Il; Park, Jung Sik; Kim, Soon Bae

Abstract

Human peritoneal mesothelial cells may have a great potential to secrete chemokines, growth factors, adhesion molecules, and various cytokines stimulated with proinflammatory cytokines during peritoneal infection. In the course of peritonitis, rapid neutrophil cell influx and subsequent monocytic cell influx can be observed. It has been demonstrated that human peritoneal mesothelial cells secrete a C-X-C chemokine, IL-8, which contributes to the recruitment of neutrophil influx during peritoneal infection. However, the production and role of C-C chemokines have not been fully defined in human peritoneal mesothelial cells. This study was performed to evaluate the production of MCP-1 and RANTES and their influence on the chemotaxis of monocytes when human peritoneal mesothelial cells were stimulated with IL-1beta. Mesothelial cells obtained by enzymatic digestion of pieces of human omentum and stimulated with a various doses and times of IL-1beta. The expression of MCP-1 and RANTES mRNA was measured by Northern blot assay and the expression of their proteins was analyzed by ELISA. To evaluate their function, monocytes chemotaxis assay was performed using a 48-well chemotactic chamber. Cultured human peritoneal mesothelial cells appeared to be polygonal at confluence using phase contrast microscope. Indirect immunofluorescent staining demonstrated that the mesothelial cells reacted positively with anti-cytokeratin antibody and anti-vimentin antibody. The expression of MCP-1 and RANTES mRNA increased in response to IL-1beta in time and dose dependent manner. The protein levels of MCP-1 and RANTES with stimulation of 1.0ng/mL of IL-1beta for 24 hours were higher than those without(30.0+/-2.22 vs 3.55+/-0.74ng/105cells and 1.53+/-0.41 vs 0.11+/-0.02ng/105cells respectively, p<0.05, n=6). Chemotaxis assay showed that the supernatants from human peritoneal mesothelial cells with stimulation of IL-1beta for 24 hours had significantly higher chemotaxis of monocytes than those without(71+/-3.4% vs 50+/-2.9%, p<0.05, n=6). Coincubation of supernatants with stimulation and antibodies to MCP-1 or RANTES(20 micro L/mL, 10 micro L/mL, respectively) resulted in a significant inhibition of chemotaxis of monocytes by 33% and 12%(47+/-3.1% and 62+/-3.0% respectively, p<0.05, n=6). Human peritoneal mesothelial cells are capable of the expression of MCP-1 and RANTES mRNA and the production of their proteins in response to IL-1beta. Functionally, mesothelial cells derived Mand RANTES may contribute to the recruitment of monocytes and amplify the inflammatory process. Thus, human peritoneal mesothelial cells play an important role during peritoneal infection.