Secretion by human fibroblasts of monocyte chemoattractant protein-1, the product of gene JE.
We recently purified human monocyte chemoattractant protein-1 (MCP-1) from culture fluids of either human glioma cell lines or mitogen-stimulated human peripheral blood mononuclear leukocytes. It has now been shown that MCP-1 is the product of the gene JE, which was first recognized by its expression in fibroblasts stimulated with platelet-derived growth factor (PDGF). We therefore studied secretion of MCP-1 by three human fibroblast cell lines. Monocyte chemotactic activity was found in culture fluids of all three lines after growth to confluence in DMEM-10% FCS, and the amounts secreted per cell were comparable for the three lines. The MRC-5 line was chosen for further study. Monocyte chemotactic activity secretion by confluent MRC-5 cultures continued after a switch to serum-free medium and was not inhibited by anti-PDGF antibody, indicating that secretion may not have been caused by autocrine release of PDGF. When concentrated serum-free MRC-5 culture fluid was injected into an HPLC gel filtration column, only one chemotactic activity peak was observed, which was in the same location as glioma-derived MCP-1. The activity was completely absorbed out by an anti-MCP-1 affinity column, which indicates that all the chemotactic activity in MRC-5 culture fluid was accounted for by MCP-1. PDGF caused a marked increase in chemotactic activity over that found in serum-free culture fluid of MRC-5 or 501T cells. Immunoprecipitation by anti-human MCP-1 showed two bands, corresponding to the two forms of MCP-1 previously described (MCP-1 alpha and beta); and the amounts increased in response to PDGF stimulation. Thus, the reported increase in human fibroblast JE mRNA in response to PDGF-containing serum stimulation is reflected in increased secretion of the MCP-1 gene product.
Yoshimura
T
T
Immunopathology Section, National Cancer Institute, Frederick, MD 21701.
Leonard
E J
EJ
D001681:Biological Assay; D002460:Cell Line; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D009000:Monocytes / Q000502:physiology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
D018932:Chemokine CCL2; D002630:Chemotactic Factors; D006023:Glycoproteins; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D014415:Tunicamycin