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Enhancement of macrophage-mediated tumor cell killing by bacterial outer membrane proteins (porins)
Abstract:
Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes.
Insights
Salmonella typhimurium porins enhance macrophage tumoricidal activity in mice, independent of lipid A responses. This suggests porins may interact directly with macrophage membranes to modulate immune function.
Area of Science:
- Immunology
- Microbial Pathogenesis
- Cell Biology
Background:
- Microbial products like lipopolysaccharide (LPS) modulate macrophage activity.
- Lipid A-associated proteins can enhance macrophage tumoricidal effects.
- Lipid A responsiveness varies in different mouse strains (e.g., C3H/HeN vs. C3H/HeJ).
Purpose of the Study:
- To investigate if Salmonella typhimurium porins influence macrophage activity.
- To compare porin effects on macrophages from lipid A-responsive and -unresponsive mice.
- To determine if porin-mediated effects are LPS-dependent.
Main Methods:
- Extraction of porins from Salmonella typhimurium cell walls using sodium dodecyl sulfate-EDTA.
- Assessment of porin purity (LPS-free) via Limulus amebocyte lysate assay and SDS-PAGE.
- Tumor cell killing assays using macrophages from LPS-unresponsive (C3H/HeJ) and responsive (C3H/HeN) mice under LPS-free conditions.
Main Results:
- Porins (1-10 ng/ml) enhanced tumoricidal activity in macrophages from both C3H/HeN and C3H/HeJ mice.
- Protein-free LPS enhanced activity only in C3H/HeN macrophages.
- Polymyxin B sulfate blocked LPS effects but not porin effects, indicating a non-LPS mechanism for porins.
Conclusions:
- Salmonella typhimurium porins can enhance macrophage tumoricidal activity independently of lipid A signaling.
- Porins may interact directly with macrophage membranes to alter their function.
- This suggests a novel mechanism for microbial modulation of innate immune cells.