Both human and mouse c-kit ligand induced differentiation of human mast cells in a long-term culture ofthe mononuclear cells of umbilical cord blood. Growth factor activity for human mast cells present in conditioned medium of BALB/3T3 fibroblasts was due to mouse c-kit ligand. Recombinant c-kit ligand induced differentiation and proliferation of mast cell progenitors in early stages of culture. However, apparent selective growth of mast cells by c-kit ligand in cord blood cell cultures is mainly due to the effect of the cytokine to selectively maintain survival of immature mast cells. Electron microscopic analysis indicated that human mast cells developed by c-kit ligand were similar to human mast cells in the lung and gut mucosa, while those developed in coculture of cord blood cells with Swiss albino/3T3 fibroblasts were similar to skin mast cells. This conclusion was supported by the fact that the majority of mast cells developed by c-kit ligand contained only tryptase in their granules, whereas those developed in the cocultures contained both tryptase and chymase. It was also found that mast cells developed by c-kit ligand were immature even after culture for 14 weeks. Nevertheless, these cells express FceRI, and could be sensitized with human IgE for anti-IgE-induced release of histamine, prostaglandin D2, and leukotriene C4.Several murine cytokines, such as interleukin (IL)-3, IL4, IL-9, and IL-10 promote differentiation and proliferation of mouse mast cells (1-4). In contrast, reproducible growth of human mature mast cells had not been achieved until 1989, when we succeeded in developing morphologically and functionally mature human mast cells in a long-term coculture of mononuclear cells of cord blood with Swiss albino/3T3 fibroblasts (5). Subsequent studies revealed that the culture supernatant of 3T3 fibroblasts contained growth factors that promote differentiation of human mast cells (6). Human mast cell growth-promoting activity could be enriched by fractionation of the culture supernatant with ammonium sulfate precipitation and by ion-exchange chromatography. The molecular size of the factor was estimated by gel filtration to be between 70 and 100 kDa (7).While our studies were in progress, a cytokine, c-kit ligand, was characterized and molecular cloning of the cytokine has been accomplished by several groups of investigators (8)(9)(10)(11)(12). The present experiments show that both human and murine c-kit ligand induce differentiation of cord blood cells to human mast cells in culture and provide evidence that human mast cell growth-promoting activity in culture supernatants of 3T3 fibroblasts is associated with murine c-kit ligand.
MATERIALS AND METHODSCell Cutures and Microscopic Examination. Mononuclear cells were obtained from heparinized umbilical cord blood (5) and suspended in RPMI 1640 medium (GIBCO) supplemented with 10%o fetal bovine serum (FBS; GIBCO), 50 ,M 2-mercaptoethanol, 2 mM L-glutamine, 100 units of penicillin per ml, 50 ,ug of streptomycin per ml, and 25 ,ug of gentami...
Prostaglandin (PG) E 2 induces dendritic cell maturation in cooperation with proinflammatory cytokines [such as tumor necrosis factor (TNF)-␣ and interleukin (IL)-1]. To clarify the involvement of E-prostanoid (EP) receptors in the effect of prostaglandin E 2 on human monocyte-derived dendritic cell (MoDC) maturation, we examined the effect of four types of EP receptor-selective agonists on MoDC maturation. PGE 2 as well as 11,15-O-dimethyl prostaglandin (E 2 ONO-AE1-259-01) (EP2 receptor agonist) and ONO-AE1-329 (EP4 receptor agonist) concentration dependently enhanced the expression of CD80, CD86, CD83, and HLA-DR on MoDCs during maturation, especially in the presence of TNF-␣, whereas 17S-2,5-ethano-6-oxo-17,20-dimethyl prostaglandin E 1 (EP1 receptor agonist) and 16S-9-deoxy-9-chloro-15-deoxy-16-hyfroxy-17,17-trimethylene-19,20-didehydro prostaglandin F 2 (EP3 receptor agonist) showed no effect. The maximal effect of ONO-AE1-259-01 was higher than that of ONO-AE1-329; however, the stimulation with ONO-AE1-259-01 was less effective than that with PGE 2 . Simultaneous stimulation with both EP receptor agonists produced additive effects and 11-deoxy-PGE 1 (EP2/EP4 receptor mixed agonist) mimicked the effects of PGE 2 . Dibutyryl cAMP mimicked the effects of PGE 2 , indicating the mediation of PGE 2 action by cAMP. Matured MoDCs induced by PGE 2 or EP2 and/or EP4 receptor agonists showed a decrease in lipopolysaccharide (LPS)-stimulated IL-12p70, IL-6, and IL-10 production. The coculture of naive T cells with matured MoDCs induced under different conditions showed that EP2/EP4-stimulated MoDCs preferentially induced alloresponsive helper T (Th)2 cells. Together, it was concluded that the cooperative stimulation of EP2 and EP4 receptor subtypes by PGE 2 promoted MoDC maturation and inhibited LPS-induced cytokine production in MoDCs. The matured MoDCs under such conditions preferably induced Th2 polarization, indicating the importance of EP2 and EP4 receptors in the determination of Th1/Th2 development of naive T cells.
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