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In vitro synthesis of factor B of the alternative pathway of complement activation by mouse peritoneal macrophages
Abstract:
Factor B of the alternative pathway of complement activation was shown to be synthesized and secreted by unstimulated mouse peritoneal macrophages. The activity of B in the culture supernatants from macrophage monolayers was detected by consumption of C3 in reaction mixtures containing supernatant and guinea pig factors C3, D and insoluble C3b. Using a monospecific antiserum, factor B in concentrated culture supernatants was shown by immunodiffusion and immunoelectrophoresis to be identical to factor B in mouse plasma and to form a characteristic complex with cobra venom factor in the presence of D. A steady rate of factor B secretion was observed for 4 days providing the medium was changed every 24 h. Cycloheximide (0.5 mug/ml), an inhibitor of protein synthesis, caused inhibition (90%) of factor B production. Incubation of culture medium containing 14C-labeled amino acids with the macrophage monolayer resulted in incorporation of radioactivity into factor B as detected by autoradiography of precipitation lines formed with anti-B antiserum; This indicated that synthesis of factor B had occurred. In the same culture supernatants the presence of newly synthesized C3 was also demonstrated.
Insights
Unstimulated mouse macrophages synthesize and secrete Factor B, a key component of the complement system. This discovery reveals a novel role for macrophages in innate immunity and complement regulation.
Area of Science:
- Immunology
- Complement System Biology
Background:
- The alternative pathway of complement activation is crucial for innate immunity.
- Factor B is a central protein in this pathway, but its cellular source in unstimulated conditions was unclear.
Purpose of the Study:
- To investigate the synthesis and secretion of Factor B by mouse peritoneal macrophages.
- To characterize the functional activity and identity of macrophage-derived Factor B.
Main Methods:
- Macrophage culture and collection of supernatants.
- Functional assays measuring C3 consumption.
- Immunodiffusion and immunoelectrophoresis using monospecific antiserum.
- Protein synthesis inhibition studies with cycloheximide.
- Radiolabeling experiments to confirm de novo synthesis.
Main Results:
- Unstimulated mouse peritoneal macrophages secrete functional Factor B.
- Macrophage-derived Factor B is biochemically identical to plasma Factor B.
- Factor B secretion is a continuous process over several days.
- Protein synthesis inhibition significantly reduces Factor B production.
- Newly synthesized C3 was also detected in macrophage supernatants.
Conclusions:
- Mouse peritoneal macrophages are a significant source of Factor B.
- This finding highlights macrophages' role in regulating complement activation.
- Macrophages contribute to both the alternative pathway and C3 production.