Although stem cell factor (SCF) has been identified as a critical cytokine for the development of human mast cells from their progenitors, the effects of other cytokines on human mast cells are less well understood. We examined the effects of several cytokines on the survival of human mast cells of 100% purity generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of 100 ng/mL recombinant human (rh) SCF and interleukin-6 (IL-6). Mast cells suspended in conventional serum-containing medium died over a period of 2 to 6 days after the withdrawal of SCF and IL-6. The cells became pyknotic and underwent DNA fragmentation characteristic of apoptosis. The addition of SCF, IL-3, IL-4, IL-5, or IL-6 to the cultures in both serum-containing and serum-free medium prolonged their survival in a dose-dependent manner. Some other cytokines, such as IL- 2, IL-9, IL-10, IL-11, tumor necrosis factor-alpha, transforming growth factor-beta 1, and nerve growth factor, had no survival-promoting effect at 100 ng/mL. Preincubation of mast cells with SCF, IL-4, IL-5, or IL-6 for 24 hours during sensitization with IgE enhanced IgE/anti- IgE antibody-induced histamine release from mast cells, whereas IL-3 showed a negligible effect. Polymerase chain reaction amplification of alpha-chains of IL-3 receptor (R), IL-4 R, IL-5 R, and IL-6 R yielded products of the correct size predicted from the sequence of each receptor. The binding assay using 125I-labeled IL-3 indicated that these mast cells bear receptors for IL-3. These findings suggest that IL-3, Il-4, IL-5, and IL-6, which are mainly produced by T-helper 2 lymphocytes, might regulate the functions of human mast cells in vivo via specific receptors in allergic reactions.

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We generated > 10⁷ mast cells by culturing 10⁷ cord blood mononuclear cells for > 10 weeks in the presence of Steel factor, interleukin-6 and prostaglandin E₂. 99% of the cultured cells had tryptase-positive granules, while 18% had chymase-positive granules. Cultured mast cells contained 3.6 μg histamine and 3.5 μg tryptase per 10⁶ cells. Cells sensitized with 1 μg/ml human IgE released 58.5% histamine and 1.55 ng tumor necrosis factor (TNF)-α per 10⁶ cells when challenged with 1 μg/ml antihuman IgE, whereas the control cells spontaneously released 3.7% histamine and 0.18ng TNF-α. Analysis for surface antigens revealed that cultured mast cells expressed the following CD molecules: 9, 13, 14, 29, 33, 38, 43, 44, 45RA, 45RB, 46, 47, 48, 49d, 50, 51, 53, 54, 55, 58, 59, 60, 61 and 117 (c-Kit). Taken together, these cultured cells seem to be functionally mature mast cells.

Summary. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 mg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-b1 (TGF-b1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7-and 2-fold, respectively. The elevated levels of TGF-b1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 mg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-b1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

Human cultured mast cells (HCMCs) grown from cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6) expressed tryptase but no or low chymase in their cytoplasm. The addition of IL-4 to these cells strikingly increased chymase expression. Consequently, the activity of chymase was significantly higher in IL-4–treated mast cells than that in IL-4–nontreated mast cells, whereas the activity of tryptase and histamine content were comparable in both cells. Electron microscopic immunocytochemistry also showed that secretary granules containing chymase increased in IL-4–treated mast cells. Interestingly, the IL-4–induced increase of chymase expression in HCMCs was accompanied by morphological maturation of the cells. Cytoplasmic projections were few in IL-4–nontreated HCMCs, and a small number of secretary granules were observed, most of which were empty or partially filled with discrete scrolls with rough particles showing immaturity. In contrast, IL-4–treated HCMCs had extremely abundant cytoplasmic projections and had many secretary granules filled with electron-dense crystal materials. Taken together, immature HCMCs grown only with SCF and IL-6 expressed tryptase with no or a low amount of chymase, and addition of IL-4 promoted cell maturation together with the expression of both tryptase and a high amount of chymase. Our findings will raise a possibility of a linear pathway of human mast cell development from tryptase single positive mast cells into tryptase and chymase double positive mast cells as the cells mature and will suggest that this maturation process is promoted by IL-4.