Tumor necrosis factor and c-fos expression in human peripheral-blood monocytes: expression is dependent on stage of
J A Turpin1, M Blick, J P Hester
1Department of Clinical Immunology and Biological Therapy, University of Texas M.D. Anderson Cancer Center, Houston.
Abstract:
The differentiation of monocytes and macrophages both in vitro and in vivo can be characterized by the modulation of specific functional and molecular phenotypes. We have determined in human peripheral-blood monocytes (HPBM) the role of in vitro differentiation on the expression of nonspecific tumoricidal activity, induction of soluble tumor necrosis factor (TNF) activity and TNF-specific mRNA transcription. HPBM were activatable by bacterial lipopolysaccharide (LPS; 1 microgram/ml) for nonspecific cytotoxicity to A375M (human malignant melanoma cell line) only during the first 24 h of in vitro differentiation. Activated supernatants of HPBM were found to be partially neutralizable (75 +/- 7%) by rabbit polyclonal anti-TNF antibody and, in freshly isolated HPBM, the release of soluble TNF activity determined by the L929 assay was found to occur only after activation with LPS. Maximal TNF release occurred at 8 h of LPS stimulation, and required both protein and RNA synthesis as evidenced by the ability of both actinomycin D and cycloheximide to inhibit its release. Neither control untreated HPBM nor recombinant interferon-gamma (rIFN-gamma; 1 U/ml)-treated HPBM alone released soluble TNF activity. Further in vitro culture determined that HPBM were activatable for TNF release out to 72 h of culture after which HPBM became resistant to LPS-mediated TNF release. The expression of TNF and c-fos mRNA was also determined during in vitro differentiation. Both TNF and c-fos mRNA were expressed in freshly isolated HPBM, and returned to baseline by 24 h of in vitro culture. Treatment of HPBM with LPS induced TNF transcription as late as 5 days of in vitro culture with maximal induction occurring during the first 48 h. rIFN-gamma significantly induced TNF transcription at 24 h in the absence of soluble TNF activity, but did not increase transcription at later times. The expression of nonspecific cytolytic activity, the release of soluble bioactive TNF and the induction of TNF and c-fos mRNA are regulated in HPBM by differentiation-determined processes.
