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.
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.
Since radioiodination of human granulocyte colony-stimulating factor (G-CSF) is difficult, we synthesized a mutein of human G-CSF that retains full biological activity and receptor-binding capacity for at least 2 weeks after radioiodination. Receptors for human G-CSF were characterized in the plasma membrane fraction from the human term placenta (human placental membranes) and trophoblastic cells by using the III-labeled mutein of human G-CSF (KW-2228). The specific binding of III-labeled KW-2228 to placental membranes was pH-dependent, with maximal specific binding at pH 7.8; it increased linearly with protein to 3.7 mg of protein per ml and was both time-and temperature-dependent, with maximal binding at 4°C after a 24-hr incubation. When we examined the ability of hematopoietic growth factors to inhibit 2-5I-labeled KW-2228 binding, we found that KW-2228 and intact human G-CSF inhibited '25I-labeled KW-2228 binding, whereas erythropoietin or granulocyte-macrophage colonystimulating factor did not. Scatchard analysis revealed a single receptor type with a Bn,,., of 210 fmol/mg of protein and a Kd of 480 pM. The human G-CSF receptors on human placental membranes were shown to consist of two molecular species of 150 kDa and 120 kDa that could be specifically cross-linked to
SUMMARYWe examined the effects of interferon-g (IFN-g) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6). When mast cells were suspended in serum-free medium without any cytokine after the withdrawal of SCF and IL-6, they died over a period of 5 days because of apoptosis. IFN-g in the cultures suppressed apoptosis and prolonged their survival in a dose-dependent manner. This survival-promoting effect of IFN-g was blocked by neutralizing antibodies to IFN-g or to IFN-g receptor (IFN-gR). When mast cells were incubated with IFN-g in serum-free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti-IgE antibody-induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the ␣-chain of IFN-gR (IFN-gR␣) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti-IFN-gR monoclonal antibodies (mAbs) indicated that these mast cells bear IFN-gR on their surface. These findings suggested that IFN-g activates human mast cells via specific receptors in certain aspects of inflammatory reactions.
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