Characterization and partial purification of a high molecular weight tumoricidal activity secreted by murine bone marrow macrophages

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Abstract
Cultured murlne bone marrow-derived macrophage (BMMΦ) can be Induced to secrete tumoricidal activity in vitro when activated with recomblnant IFN-γ and bacterial lipopolysaccharide (LPS). We have analyzed this activity for tumor specificity, relationship to tumor necrosis factor-α (TNF-α), 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 BMMΦ treated with a combination of IFN-γ 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-α (r-muTNF-α), but did not significantly affect non-tumorigenic cells of either species. It was inactivated by 56°C, trypsin, and neuraminldase treatment, but could not be inhibited by neutralizing antibodies against r-muTNF-α or serine protease inhibitors. Tumoricidal activity was purified -10-fold by gel filtration and eluted as a major peak with a Mr of 170 kDa, containing a single predominant protein band of -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 BMMΦ by a combination of IFN-γ 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 BMMΦ culture supernatants, whereas no such correlation was found with respect to the amount of secreted TNF-α or reactive nitrogen intermediates. These data demonstrate that activated murine BMMΦ secrete a tumoricidal activity, which is not related to TNF-α, 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.

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