RELEASE OF GRANULOCYTE-SPECIFIC COLONY-STIMULATING ACTIVITY BY HUMAN-BONE MARROW EXPOSED TO PHORBOL ESTERS

Abstract

Granulocyte-macrophage colony growth depends on the presence of colony-stimulating activity (CSA). Phorbol esters induce concentration-dependent colony formation in the absence of exogenous CSA. Whether phorbol esters mimicked the action of CSA by directly stimulating colony growth, or whether phorbol esters acted indirectly by inducing marrow cells to release CSA was questioned. First, after incubating human B cells with phorbol 12,13-dibutyrate (PDP) for 3 days, PDB was separated from the protein peak of the conditioned medium by Sephadex G-10 gel filtration and tested this peak for the presence of CSA. When diluted 1:10 in the agar colony assay, this material induced 133 .+-. 15 colonies/105 B cells. Second, to determine whether B cells required the continued presence of PDB in order to release CSA, PDB was removed from B cells by washing, and these cells were reincubated in fresh medium in the absence of PDB. CSA was found in the medium of these cultures; its release was maximal after preincubation of B cells with 5 .times. 10-8 M PDB for 3 days, followed by incubation for 3 days in the absence of PDB. This CSA stimulated granulopoiesis out of proportion to monocytopoiesis, with 85% .+-. 17% of the colonies being granulocytic (as indicated by histochemical staining for chloroacetate esterase), and 12% .+-. 3% being monocytic (as indicated by nonspecific esterase). Inhibitors of monocyte colony formation, including PGE1 [prostaglandin E1] were not present in the medium that contained this CSA. Normal human B cells exposed to PDB release CSA and that this CSA selectively stimulates granulopoiesis in vitro.