Human macrophage colony-stimulating factor induces macrophage colonies after L-phenylalanine methylester treatment of human marrow

Abstract

Macrophage-colony stimulating factor (M-CSF) has well-known effects on murine bone marrow, but its colony stimulating activity for human bone marrow is controversial. After treatment of human bone marrow with L- phenylalanine methylester (PME), macrophage-colonies (CFU-M) were induced by M-CSF in a dose-dependent fashion. The optimal concentration of recombinant human-macrophage colony stimulating factor (rhM-CSF) was 1,000 U/mL. Purified human urine M-CSF had colony stimulating activity similar to rhM-CSF. Further studies were performed to determine the factors responsible for the enhanced CFU-M formation from PME treated marrow. Compared with nylon wool and carbonyl iron monocyte depletion methods, PME eliminated significantly more monocytes and myeloid cells. This observation suggested that these cells may release hematopoietic inhibitory factors for CFU-M. Low concentrations (1%) but not normal (10%) concentrations of blood monocytes were inhibitory (mean inhibition, 48%) to CFU-M. High concentrations of monocytes (50%) augmented CFU-M colonies. HL-60 conditioned media was used to simulate secretory products of early myeloid cells. HL-60 conditioned media (1%) inhibited CFU-M formation but not granulocyte macrophage or granulocyte colonies. We conclude that M-CSF has colony stimulating activity for human marrow that can be recognized after removal of inhibitory cells by PME treatment.

Effect of L‐Phenylalanine methyl ester on the colony formation of hematopoietic progenitor cells from human bone marrow

“…Differential counts showed that monocytes, as well as the majority of cells in the neutrophilic maturation series were lysed, further supporting the hypothesis of the removal of inhibitory cells. Our results with BM differential counts were similar to those published by Rosenfeld et al [7]. Depletion of cytotoxic cells from BM by amino acid methyl esters has been explored as a possible method of eliminating cells that cause GvHD [8-111.…”

“…Our earlier report demonstrated that depletion of monocytes by PME was an effective and labor-saving step in the enrichment of hematopoietic cells from adult PB or cord blood [5, 6]. The growth of macrophage colony-forming units (CFU-m) from low density ( S 1.077 g/ml) human bone marrow (BM) cells was found to increase with increasing concentrations of macrophage colonystimulating factor (M-CSF) after the cells were incubated with PME [7]. Leu-Leu-OMe had been successfully used to prevent graft-versus-host disease (GvHD) in animal BM transplantation models f8-111.…”

Transforming growth factor‐β1 augments macrophage‐colony stimulating factor activity on human marrow

“…Bone marrow cells were further treated with PME as described previously [9]. Briefly, cells were resuspended in RPMI at a final concentration of 1 x lo7 cells/ml.…”

“…The assay used in this laboratory is capable of assessing CFU-M from all normal donors. This assay utilizes marrow treated with phenylalanine methyl ester (PME) and cultured in agar at 7.5% C02/21% O2 [9]. PME eliminates monocytes, myeloid cells and fibroblasts from human marrow [9].…”

“…This assay utilizes marrow treated with phenylalanine methyl ester (PME) and cultured in agar at 7.5% C02/21% O2 [9]. PME eliminates monocytes, myeloid cells and fibroblasts from human marrow [9]. An assay described by Broxmeyer for CFU-M utilizes density gradient separated marrow that is cultured in agarose at 5 % These reliable assays for the CSA of M-CSF can be used to investigate the regulation of human monocytopoiesis in vitro.…”