Complement production by mononuclear phagocytes has been identified as a maker of cellular maturation. In humans, production of hemolytically active C2 and factor B is characteristic of the tissue from which the cells are isolated. These differences in complement production may be accounted for by changes in synthetic rates, in secretion rates, or in extracellular catabolism. The availability of techniques for the establishment and maintenance of human monocyte and macrophage monolayers, the availability of methods for functional and immunochemical measurement of picogram amounts of these proteins, and the availability of a cDNA probe for human factor B and the expected availability of a similar probe for C2 will permit examination of these differences at several specific biosynthetic steps. First, synthesis and secretion rates of C2 and factor B will be estimated by pulse-chase experimentation and compared to C3 and total protein synthesis and secretion. If differences in synthesis rates in culture are found, mRNA from freshly isolated monocytes, freshly isolated macrophages, and monocytes maintained for two weeks in culture will be quantitatied by Northern blot and dot hybridization and functionally by cell-free translation in an endogenous system. If differences in secretion are detected, glycosylation and other possible regulatory steps in post-translational processing will be examined. Extracelluar catabolism will also be investigated using peptide mapping, recovery of hemolytically active, purified complement proteins, mixing experiments with conditions media, recovery of internally (35S methionine) and 125I labelled complement proteins. Using this approach, the specific steps in complement biosynthesis which account for maturational differences in complement secretions will be determined.
