Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells

Abstract

We have previously shown the development in vitro of tryptase+ human mast cells from fetal liver cells cocultured with murine 3T3 fibroblasts. In this study, recombinant human stem cell factor (rhuSCF), the ligand for the c-kit proto-oncogene product called Kit, stimulated the growth and differentiation primarily of mast cells from dispersed fetal liver cells, whereas recombinant human interleukin-3 (rhuIL-3) stimulated the differentiation of basophils along with other cell types. Cultures of fetal liver cells were initiated and maintained in the presence of rhuSCF or rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified as mast cells primarily on the basis of tryptase expression, and as MCT or MCTC by immunohistochemistry using monoclonal antibodies against tryptase and chymase, whereas basophils were metachromatic, polymorphonuclear, and lacked these proteases. Levels of tryptase and histamine were measured by radioimmunoassay, tryptase and chymase activities by peptide hydrolysis, and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8. The predominant presence of mast cells occurred only in the cultures supplemented with rhuSCF. The percentage and total number of mast cells increased over time with increasing concentrations of rhuSCF and reached a plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first appeared by day 7; by day 42, 106% of the starting number of cells were present and 85% of these were tryptase+, 31% being weakly chymase+. These mast cells appeared immature by ultrastructural criteria; most cells were mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was demonstrated by incorporation of bromodeoxyuridine. Calculated levels of histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar to those determined previously in coculture experiments with murine 3T3 fibroblasts. Chymase activity was undetectable in most cell extracts. On day 0, 4% to 20% of fetal liver cells expressed cell surface Kit. In the presence of rhuSCF, the percentages and total numbers of Kit+ cells and the apparent concentration of Kit per cell increased along with the number of tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase- cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage of these toluidine blue+ cells then declined to about 6% by days 21 and 35, while the total number of positive cells declined over 10-fold. Kit+ cells in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day 35.(ABSTRACT TRUNCATED AT 400 WORDS)

IL‐6 attenuates apoptosis, while neither IL‐6 nor IL‐10 affect the numbers or protease phenotype of fetal liver‐derived human mast cells

“…5 and Table 1. In all culture conditions, tryptase-positive cells increased in parallel to those stained metachromatically with toluidine blue (data not shown) as reported previously [12,15,27]. After 1 week of culture, no chymase-positive cells were detected with the mAb against chymase and less than 5% of the cells were labelled with the rabbit pAb against chymase, regardless of the added cytokine mixture, while about half of the cells already were tryptase-positive and metachromatic.…”

“…Stem cell factor (SCF), which is constitutively expressed by fibroblasts and other cell types, is the ligand for Kit [5±8], and the only growth factor that by itself supports the growth and differentiation in vitro of human mast cells from haematopoietic precursors in bone marrow and peripheral blood placed into suspension cultures [9], cord blood [10,11] and fetal liver [12]. The MC T phenotype appears to predominate when protease expression is examined in such in vitro-derived mast cells by immunocytochemistry.…”

Systemic mastocytosis associated with acute myeloid leukaemia: report of two cases and detection of the c‐kit mutation Asp‐816 to Val

“…However, c-kit is also involved in the regulation of growth of normal and neoplastic MC. Thus, SCF, the ligand of c-kit, induces differentiation of human MC from their progenitor cells (Irani et al, 1992;Valent et al, 1992;Kirshenbaum et al, 1992;Mitsui et al, 1993). Moreover, functional defects in the SCF or c-kit gene in mice are associated with an 'MCdeficient' phenotype (Kitamura et al, 1989).…”

Mast cell cultures: bench to bedside

“…The studies on stem cell factor in the murine system opened the way to human primary mast cell cultures. In fact, in recent years SCF was shown to be a major growth and differentiation factor for human mast cell progenitors in cord blood mononuclear cells [17], bone marrow [18,19] and fetal liver [20]. However, in order to fully obtain differentiated human mast cells, co-culture with murine skin fibroblasts [21] or additional growth factors (IL-6, prostaglandin E 2 ) [22] may be needed.…”

“…IL-3 does not bind to human lung mast cells [23], although it may support SCF-induced human mast cell differentiation [18,20], acting probably on SCF-responsive progenitors [24]. Recently it has been reported that IL-3 prolongs the survival of cultured human mast cells that appear to bear receptors for this cytokine [25].…”