Determination of Subpopulations of Leukocytes Involved in the Synthesis of α- 1 -Antitrypsin in Vitro 1

Abstract

Summary

Cell-free culture medium from stimulated and unstimulated 6-day cultures of mononuclear leukocytes (MNL), nonadherent MNL (lymphocytes), and adherent MNL (monocytes) was evaluated for evidence of in vitro synthesis of protein and of α₁-antitrypsin (A₁AT). Phytohemagglutinin (PHA, 2.0 μg/ml) promoted significant increases in ³H-labeled protein content in both MNL and lymphocyte cultures, whereas endoxotin (25.0 μg/ml) but not PHA caused significant increases in the amount of protein synthesized by monocytes. A₁AT was found to be made by MNL and monocytes in culture but was not newly synthesized by lymphocytes. Evidence for the presence of de novo synthesized A₁AT was obtained by Sephadex G-200 chromatography, immuno-precipitation with antibody specific for A₁AT, and SDS-polyacrylamide gel electrophoresis. Evidence was also found for the release of small amounts of A₁AT from the surface of lymphocytes after 6 days in culture, presumably complexed to labeled protease; however, this accounted for less than 10% of the A₁AT made by endotoxin-stimulated monocytes.

We thank Charles L. Smith for editorial assistance, Dr. H. Hugh Fudenberg for continuing support, and Dr. P. Arnaud for performing SDS-acrylamide gel electrophoresis.