Synthesis of the second component of complement by long-term primary cultures of human monocytes.

Abstract

A method has been developed for preparation of confluent monolayers of human monocytes from small volumes of blood and for maintenance of these pure monocyte cultures for up to 16 wk in vitro. These cells phagocytosed 5.7 mum diameter latex beads, rosetted with erythrocytes coated with IgG or with C3, killed Listeria monocytogenes, and synthesized both lysozyme and the second component of complement. Lysozyme was secreted at a rate of approximately 50,000 mol/min per cell for at least 12 wk in cultures. The maximal rate of C2 synthesis and secretion was considerably less; i.e., approximately 30 mol/min per cell between the 2nd and 12th wk in culture. Monocytes produced little C2 during the first 6 days in culture after which a marked increase in the rate of C2 production was noted. This increase was coincident with morphologic evidence of monocyte maturation.

Biosynthesis of the third component of complement (C3) in vitro by monocytes from both normal and homozygous C3-deficient humans.

“…Long-term primary cultures of human monocytes were prepared using a previously described method (7). Preliminary studies indicated that hemolytically active partially purified C3 (Cordis Laboratories, Miami, Fla.) was stable for at least 48 h in Medium 199 (Microbiological Associates, Bethesda Md.)…”

“…Parallel gels with standard molecular weight markers were run and stained with Coomassie Blue according to reference (20 Fluorimetric assay of DNA. The number of monocytes per cover slip was determined using a previously described fluorimetric assay for DNA (7).…”

“…Although it seemed that the liver, and specifically the hepatocvte, was the major site of synthesis of C3, the demonstration that human rheumatoid, but not normal, synovitim produced functionally active C3 in vitro (6) In view of these considerations and the inaccessibility of liver for routine longitudinal study of human C3 synthesis in genetic and acquired abnormalities of C3 production, blood monocytes were examined for their capacity to synthesize C3 in vitro. A recently developed method (7) for the preparation and maintenance of human monocytes for up to 6 mo in culture was utilized for these studies. The cells can be obtained from small volumes of blood, synthesize lysozyme and the second component of complement (C2), phagocytose latex beads, rosette with IgG-or C3-coated erythrocytes, and kill Listeria monocytogenes.…”

Effects of gold sodium thiomalate on functional correlates of human monocyte maturation

“…Human monocytes do not spontaneously synthesize or secrete C2 (2). This capacity is a consequence of maturation or differentiation of monocytes into macrophage-like cells (3).…”

“…Peroxidase activity is lost as a result of maturation of monocytes into macrophages (7). Spontaneous C2 synthesis occurs after 48-72 hours of incubation and reaches a maximum level, provided these cells are not further stimulated with lymphokine, after 122-144 hours of culture (2,8). Monocytes acquire the ability to lyse chicken erythrocytes after 4-5 days of incubation, and this activity reaches a peak at day 7 (9).…”