Production of monocyte chemoattractant protein-1 by bovine glomerular endothelial cells

To explore the role of glomerular endothelial cells (GEN) in the pathogenesis of glomerulonephritis, the in vitro production of monocyte chemoattractant protein-1 (MCP-1) by bovine GEN was determined by chemotaxis assay, Northern and Western blot analysis, and immunocytochemistry. Monocyte chemotactic activity of GEN-conditioned media was detectable by a chemotaxis assay using human peripheral blood monocytes. Exposure to human recombinant interleukin-1β (IL-1β) and phorbol myristate acetate (PMA) significantly increased the chemotactic activity of GEN-conditioned media. A checkerboard analysis showed that the response of monocytes to GEN-conditioned media was truly chemotactic. Immunoadsorption with a monoclonal antibody to human MCP-1 reduced the chemotactic activity of GEN-conditioned media by 85%. Northern blot analysis revealed that MCP-1 mRNA was constitutively expressed by GEN and that IL-1β and tumor necrosis factor-α (TNF-α) increased MCP-1 mRNA levels in a dose- and time-dependent manner. Furthermore, PMA induced an increase in MCP-1 mRNA levels, whereas dibutyryl cyclic AMP and forskolin had minimal effects. Inhibition study using protein kinase inhibitors revealed that MCP-1 mRNA expression induced by IL-1β and TNF-α was suppressed by the tyrosine kinase inhibitor genistein, not by the protein kinase C inhibitors staurosporine or H-7, or the protein kinase A inhibitor H-89, suggesting an important role of tyrosine kinase in the cytokine-induced MCP-1 gene expression. Dexamethasone had a small inhibitory effect on constitutive MCP-1 mRNA expression, but no effect on the induction by TNF-α. By immunoperoxidase staining and Western blot analysis using an anti-MCP-1 monoclonal antibody, MCP-1 protein was detected in untreated GEN and increased by exposure to TNF-α. These results demonstrate the cytokine-induced production of MCP-1 by GEN at gene and protein levels as well as bioactivity, and suggest that GEN may participate in the development of glomerulonephritis through the production of MCP-1.