Biosynthesis of the subcomponents Clq, Clr and Cls of the first component of complement (Cl) by guinea pig hepatocyte primary cultures

Abstract Thus far, the synthesis of Clq by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (10 6 cells/ml) were cultured under serumfree conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4–8) antibody directed against a nonpolymorphic la determinant and preabsorbed rabbit serum. The hemolytic activity of Cl and its subcomponents Clq and Clr/Cls was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to 3 × 10 3 effective Clq molecules/cell/24 h and 34 ± 10 3 effective Clr/Cls molecules/cell/24 h. The synthesis of Clq and Clr/Cls could be reversibly inhibited by cycloheximide (50 μg/ml). Furthermore, to demonstrate de novo synthesis of the Clq subcomponent, endogeneous labeling with 3 H‐proline (or 14 C‐proline) was performed. The immunoprecipitated Clq from cellular lysates and culture medium was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography. Compared to biosynthetically labeled guinea pig Clq from peritoneal macrophages, three corresponding bands (30, 28 and 24 kDa, respectively) were detectable in the fluorograph. The data show that guinea pig hepatocytes are able to synthesize Cl subcomponents, whereby the synthesis of Clq and Clr/Cls occurs independently.