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C. Blondin, P. Hamard, F. Brignole, C. Baudouin; Human Trabecular Meshwork Cells Produce the Pro-inflammatory Chemokines Interleukin-8 (IL-8) and Monocyte Chemoattractant Protein-1 (MCP-1) in vitro . Invest. Ophthalmol. Vis. Sci. 2003;44(13):679.
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Purpose: To investigate the production of chemokines by human trabecular meshwork (HTM) cells. Methods: Normal (HTM5) or glaucomatous (HTM3) trabecular cell lines were treated either with 10 ng/ml human recombinant TNFa for 1, 6, 12, 24, 48 and 72 hr, or with increasing concentrations of TNFa (0.1 to 100 ng/ml) for 48 hr. At each time point, culture supernatants were harvested and assayed for IL-8 and MCP-1 proteins by ELISA. Adherent cells were trypsinized, stained for intracellular IL-8 and MCP-1 with fluorescent antibodies and analyzed by flow cytometry. In addition, chemokine production was assessed after stimulation by various neuropeptides or pro-inflammatory stimuli for 48 hr. Results: No IL-8 was produced by unstimulated HTM5 and HTM3 cells. Addition of 10 ng/ml TNFa resulted in secretion of IL-8 by both cell types in a time-dependent and dose-dependent manner (from 630 ± 135 pg/ml at 6 hr to 4462 ± 909 pg/ml at 72 hr and to 6695 ± 783 pg/ml in response to 100 ng/ml TNFa for HTM5 cells). This increased production of extracellular IL-8 was concomitant with a decreased intracellular IL-8 expression. Similarly, HTM5 and HTM3 cells responded to TNFa by secreting MCP-1 in a time- and dose-dependent fashion. However, these cells constitutively produced MCP-1 and secreted 10- to 12-times more MCP-1 than IL-8 in response to TNFa. Production of IL-8 and MCP-1 by HTM3 cells was significantly (p<0.001) higher than that of HTM5 cells. Moreover, both cell types produced chemokines in response to phorbol myristate acetate (PMA) and to a mixture of PMA and ionomycine. None of the other stimuli tested (lipopolysaccharide, calcium ionophore A23187, IL-2, IL-4, GM-CSF, IFNg, VIP, CGRP and substance P) induced chemokine production. Conclusions:: This study provides direct evidence for the production of IL-8 and MCP-1 by human trabecular cells. Strikingly, glaucomatous HTM cells produced significantly more chemokines than non-glaucomatous cells. These data may point to a biological function of chemokines in the pathogenesis of chronic open angle glaucoma.
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