Characterization and partial purification of a high molecular weight tumoricidal activity secreted by murine bone marrow macrophages.
Cultured murine bone marrow-derived macrophage (BMM phi) can be induced to secrete tumoricidal activity in vitro when activated with recombinant IFN-gamma and bacterial lipopolysaccharide (LPS). We have analyzed this activity for tumor specificity, relationship to tumor necrosis factor-alpha (TNF-alpha), serine proteases, and reactive nitrogen intermediates, and partially purified this activity by high pressure liquid chromatography. Cytolytic activity was recovered in conditioned culture supernatants of serum-free cultivated BMM phi treated with a combination of IFN-gamma and LPS but was not inducible by either stimulant alone. It selectively affected tumor cells of murine as well as human origin irrespective of sensitivity towards recombinant murine TNF-alpha (r-muTNF-alpha), but did not significantly affect non-tumorigenic cells of either species. It was inactivated by 56 degrees C, trypsin, and neuraminidase treatment, but could not be inhibited by neutralizing antibodies against r-muTNF-alpha or serine protease inhibitors. Tumoricidal activity was purified approximately 10-fold by gel filtration and eluted as a major peak with a Mr of 170 kDa, containing a single predominant protein band of approximately 170 kDa on SDS-PAGE analysis, which is shown to be a disulfide linked glycoprotein heterodimer of 110 and 58 kDa subunits (gp170). Expression of this glycoprotein was strongly dependent on activation of BMM phi by a combination of IFN-gamma and LPS but was only marginally induced by either stimulant alone. Furthermore, the level of gp170 expression was quantitatively correlated with the tumoricidal activity of BMM phi culture supernatants, whereas no such correlation was found with respect to the amount of secreted TNF-alpha or reactive nitrogen intermediates. These data demonstrate that activated murine BMM phi secrete a tumoricidal activity, which is not related to TNF-alpha, serine proteases, or reactive nitrogen intermediates, but is closely associated with a 170 kDa glycoprotein composed of two subunits with Mr's of 110 and 58 kDa.
Schwamberger
G
G
Max-Planck-Institut für Immunbiologie, Freiburg, Germany.
Flesch
I
I
Ferber
E
E
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000276:immunology
D006023:Glycoproteins; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma