IL-4 AND TNF-ALPHA-MEDIATED PROLIFERATION OF THE HUMAN MEGAKARYOCYTICLINE M-O7E IS REGULATED BY INDUCED AUTOCRINE PRODUCTION OF GM-CSF

In this study, the authors examined the effects of recombinant human i nterleukin 4 (rhIL-4) and recombinant human tumour necrosis factor alp ha (rhTNF-alpha) alone or in combination on proliferation of the human cytokine dependent myeloid cell line, M-O7e, While rhIL-4 or rhTNF-al pha alone induced only a weak proliferative response, a synergistic pr oliferative signal was clearly evident on stimulation of cells with a combination of both cytokines. The stimulatory effect of rhTNF-alpha i s mediated predominantly by the 55-kDa TNF receptor because the agonis tic monoclonal antibody htr-9 and the Trp(32) Thr(86) TNF-alpha mutant protein specific for this receptor type produced similar results to r hTNF-alpha. In contrast, the Asn(143) Arg(145) TNF-alpha mutant protei n specific for the 75-kDa TNF receptor produced only minimal prolifera tion of M-O7e cells, Using RT-PCR, we found that rhTNF-alpha rapidly a nd strongly induced granulocyte-macrophage colony-stimulating factor ( GM-CSF) mRNA production, while rhIL-4 was a slow and less efficient in ducer of GM-CSF mRNA, However, there was little evidence of the TNF-al pha/IL-3 combination acting synergistically on GM-CSF mRNA production as the levels of GM-CSF mRNA increased only marginally compared with I L-4 or TNF-alpha alone, Thus, the observed synergistic effect of TNF-a lpha/IL-4 costimulation of M-O7e cells seems to be mediated via induct ion of GM-CSF secretion rather than an enhanced production of GM-CSF m RNA, 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 antibo dy against GM-CSF abrogated the observed synergistic effect of rhIL-4 and rhTNF-alpha treatment, indicating that the rhIL-4/TNF-alpha combin ation acts to significantly increase GM-CSF release which then acts in an autocrine manner to enhance the proliferation of M-O7e cells.