title: Production of monocyte chemoattractant protein-1 by bovine glomerular endothelial cells.
abstract: 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, 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-1 beta (IL-1 beta) 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-1 beta and tumor necrosis factor-alpha (TNF-alpha) 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-1 beta and TNF-alpha 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-alpha. 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-alpha. 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.
authors.affiliation: Department of Pediatrics, Hirosaki University School of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kakizaki
authors.forename: S
authors.initials: S
authors.lastname: Waga
authors.forename: K
authors.initials: K
authors.lastname: Sugimoto
authors.forename: H
authors.initials: H
authors.lastname: Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Nukii
authors.forename: M
authors.initials: M
authors.lastname: Takeya
authors.forename: T
authors.initials: T
authors.lastname: Yoshimura
authors.forename: M
authors.initials: M
authors.lastname: Yokoyama
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis* / Q000235:genetics; D016207:Cytokines / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism / Q000473:pathology; D005921:Glomerulonephritis / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D007678:Kidney Glomerulus / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D018932:Chemokine CCL2; D016207:Cytokines; D012333:RNA, Messenger