Quantitative determination of complement components produced by purified hepatocytes

Abstract

In this report we describe, on a quantitative basis, the secretion of complement components by hepatocytes. Primary cultures were established after isolation of the cells from guinea-pig liver and the synthesis of C3, C5, C4 and C2 was measured. The cells were isolated by collagenase perfusion of the liver followed by differential centrifugation. The contamination of the hepatocyte suspension with non-parenchymal cells was less than 1%. At 24 h after plating the cells the kinetics of complement production were measured. C3 and C5 content in the culture medium harvested at different time intervals was determined by a sensitive ELISA. Secretion of C2 and C4 was measured haemolytically using C2 or C4 deficient guinea-pig serum. Under the conditions used hepatocytes secreted C3 at a rate of about 100 ng/10(6) cells/h with a plateau of secretion after 24 h of culture corresponding to about 350,000 molecules/cell/h. C5 secretion was detectable after 3-6 h of culture. The C5 secretion rate was about 15 ng/10(6) cells/24 h. The functional activity of C4 and C2 in the supernatants amounted to about 80 SFU/cell/h if the culture medium was changed every 3 h but dropped significantly if the medium was changed every 12 h. The decrease of the haemolytic activity became stronger if the medium was changed every 24 h. Cycloheximide reversibly inhibited the complement production. Our results show that guinea-pig hepatocytes synthesize considerably more C3 and C5 compared to peritoneal macrophages supporting the hypothesis that hepatocytes provide the major source of plasma complement.