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Characterization and partial purification of a high molecular weight tumoricidal activity secreted by murine bone
G Schwamberger1, I Flesch, E Ferber
1Max-Planck-Institut für Immunbiologie, Freiburg, Germany.
Abstract:
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.
Insights
Activated macrophages secrete a novel tumoricidal factor, gp170, a glycoprotein distinct from TNF-alpha, serine proteases, or reactive nitrogen intermediates. This finding offers new insights into macrophage-mediated anti-tumor immunity.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Research
Background:
- Murine bone marrow-derived macrophages (BMM phi) can be activated to exhibit tumoricidal activity.
- This activity is typically induced by combinations of recombinant interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS).
Purpose of the Study:
- To characterize the tumoricidal activity secreted by activated BMM phi.
- To determine the relationship of this activity to known cytotoxic factors like tumor necrosis factor-alpha (TNF-alpha), serine proteases, and reactive nitrogen intermediates.
- To identify and partially purify the molecule responsible for the observed tumoricidal effect.
Main Methods:
- Cultured BMM phi were activated with IFN-gamma and LPS.
- Tumoricidal activity was assessed against various tumor and non-tumorigenic cell lines.
- Purification of the active component was performed using high-pressure liquid chromatography (HPLC) and gel filtration.
- Protein characterization involved SDS-PAGE and analysis of subunit composition.
Main Results:
- Activated BMM phi secreted a potent tumoricidal activity selective for tumor cells of murine and human origin.
- This activity was heat-labile and sensitive to trypsin and neuraminidase, but not inhibited by anti-TNF-alpha antibodies or serine protease inhibitors.
- The tumoricidal activity was purified and found to be associated with a 170 kDa glycoprotein (gp170), composed of 110 kDa and 58 kDa subunits.
- gp170 expression strongly correlated with tumoricidal activity, independent of TNF-alpha, serine proteases, or reactive nitrogen intermediates.
Conclusions:
- Activated murine macrophages secrete a novel tumoricidal factor, gp170.
- This glycoprotein is distinct from previously identified cytotoxic mediators like TNF-alpha, serine proteases, and reactive nitrogen intermediates.
- gp170 represents a significant finding in understanding macrophage-mediated anti-tumor mechanisms.