Synthesis of complement components C2 and C4 by human monocyte-derived macrophages during in vitro differentiation in serum-free culture conditions.

In order to analyze the kinetics of complement component synthesis by human monocytes/macrophages, we have developed a system of defined culture conditions in the absence of serum. Moreover, the use of the polymerase chain reaction (PCR) provides a high sensitivity for the detection of mRNAs and the study of the regulation of complement component synthesis by these cells. Human blood monocytes were collected and purified by cytapheresis and elutriation, and then cultured in nonadherent cell culture bags for up to 3 weeks. Cells were grown in Iscove's modified Dulbecco's medium supplemented with alpha-phosphatidylcholine, transferrin, insulin, glutamine and antibiotics. The phenotypic and functional properties of macrophages differentiated under these serum-free culture conditions have been previously analyzed [1]. Secretion of complement component was measured by a hemolytic assay. mRNA was prepared using guanidine isothiocyanate extraction followed by cesium gradient ultra-centrifugation. cDNA was obtained by reverse transcription, then amplified by PCR. Fresh monocytes did not display any secretion of C2 as measured by hemolytic assay. However, C2 secretion was detected on and after the 3rd day of serum-free cultured unstimulated monocytes. The rate of C2 production increased along with macrophage differentiation up to the 3rd week of culture. We were not able to detect any functional C4 secretion. In preliminary studies, C2 and C4 mRNAs were detected in macrophages and in unstimulated fresh monocytes. These preliminary studies show that the serum-free culture conditions we have developed allow very satisfactory survival and differentiation of human monocytes, and provide optimal conditions for the study of their secretory activity.