title: IL-4 and TNF-alpha-mediated proliferation of the human megakaryocytic line M-O7E is regulated by induced autocrine production of GM-CSF.
abstract: In this study, the authors examined the effects of recombinant human interleukin 4 (rhIL-4) and recombinant human tumour necrosis factor alpha (rhTNF-alpha) alone or in combination on proliferation of the human cytokine dependent myeloid cell line, M-O7e. While rhIL-4 or rhTNF-alpha alone induced only a weak proliferative response, a synergistic proliferative signal was clearly evident on stimulation of cells with a combination of both cytokines. The stimulatory effect of rhTNF-alpha is mediated predominantly by the 55-kDa TNF receptor because the agonistic monoclonal antibody htr-9 and the Trp32 Thr86 TNF-alpha mutant protein specific for this receptor type produced similar results to rhTNF-alpha. In contrast, the Asn143 Arg145 TNF-alpha mutant protein specific for the 75-kDa TNF receptor produced only minimal proliferation of M-O7e cells. Using RT-PCR, we found that rhTNF-alpha rapidly and strongly induced granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA production, while rhIL-4 was a slow and less efficient inducer of GM-CSF mRNA. However, there was little evidence of the TNF-alpha/IL-4 combination acting synergistically on GM-CSF mRNA production as the levels of GM-CSF mRNA increased only marginally compared with IL-4 or TNF-alpha alone. Thus, the observed synergistic effect of TNF-alpha/IL-4 costimulation of M-O7e cells seems to be mediated via induction of GM-CSF secretion rather than an enhanced production of GM-CSF mRNA. Higher levels of GM-CSF were detectable in supernatants of cells treated with both rhIL-4 and rhTNF-alpha than in cells stimulated with either cytokine alone. Furthermore, addition of a neutralising antibody against GM-CSF abrogated the observed synergistic effect of rhIL-4 and rhTNF-alpha treatment, indicating that the rhIL-4/TNF-alpha combination acts to significantly increase GM-CSF release which then acts in an autocrine manner to enhance the proliferation of M-O7e cells.
authors.affiliation: Division of Immunobiology, National Institute for Biological Standards and Control, Herts, UK.
authors.forename: M
authors.initials: M
authors.lastname: Wadhwa
authors.forename: P
authors.initials: P
authors.lastname: Dilger
authors.forename: A
authors.initials: A
authors.lastname: Meager
authors.forename: B
authors.initials: B
authors.lastname: Walker
authors.forename: R
authors.initials: R
authors.lastname: Gaines-Das
authors.forename: R
authors.initials: R
authors.lastname: Thorpe
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D003470:Culture Media; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology*; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor / Q000276:immunology; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D003470:Culture Media; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor