Basic and clinical immunologyReciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-γ☆
Section snippets
Human mast cell culture
CHMCs were generated as previously reported.11, 12 In brief, heparin-treated umbilical cord blood that was obtained (under an informed consent approved by the hospital’s ethical committee) was diluted and layered over lymphocyte separation medium (Organon Teknika Corp) at room temperature. Cord blood mononuclear cells were purified by density gradient centrifugation methods followed by immunomagnetic positive selection with mAb against CD34 (Militenyi Biotec Inc). The purified CD34+ cells were
Effect of IL-4 or IFN-γ on cytokine production by CHMCs
CHMCs were incubated with IgE in the presence or absence of IL-4 for 2 days as concentration indicated. The sensitized CHMCs were washed and resuspended in mast cell culture medium containing 0 to 10 ng/mL IL-4 and 1 μg/mL CRA-1 for 6 hours. Supernatants were harvested and analyzed for various cytokines by using ELISA. IL-4 did not affect spontaneous MIP-1α production by CHMCs. FcϵRI-mediated MIP-1α production by CHMCs was 0.3 ± 0.1 ng/106 cells in the absence of IL-4 (Fig 1, A ).
DISCUSSION
In this study we showed that IL-4 and IFN-γ have opposite effects on human mast cell cytokine production induced by means of FcϵRI cross-linking. For example, not only does IL-4 upregulate FcϵRI-dependent production of IL-3, IL-5, and IL-13 in CHMCs or human intestinal mast cells,9, 20, 21 but it also enhances production of MIP-1α, IL-8, and GM-CSF (Fig. 1, Fig. 3). This occurs at the level of both mRNA and protein synthesis in addition to enhancement of expression of FcϵRI and FcϵRI-dependent
Acknowledgements
We thank Dr Kiyoshi Kawashima, Dr Shigenobu Shoda, and the staff of the Department of Obstetrics, Gyoda Chuo Hospital, for their continuous support by generously providing the umbilical cord blood. We also thank Dr Akira Akasawa, Dr Toshio Katsunuma, Dr Hidetoshi Kawahara, Dr Ichiro Nomura, and Mr Hisashi Tomita for their technical assistance and advice.
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2005, BloodCitation Excerpt :Although IFN-γ and IL-5 had no effect on human mast cell mediator release, IL-4 potentiated spontaneous and antigen-stimulated β-hexosaminidase release by approximately 40% (Figure 5B). The increase in releasability seen in cultures exposed to IL-4 was accompanied by an increase in FcϵRI expression, consistent with previous reports (data not shown;14). Thus, of the TH1 and TH2 cytokines examined, IL-4 was one cytokine that clearly enhanced mast cell function when added for the first time late in cultures.
Reliability of RT-PCR methods for measuring relative gene expression in mast cells
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Reprint requests: Motohiro Ebisawa, MD, PhD, Department of Allergy, National Children’s Medical Research Center 3-35-31, Taishido, Setagaya-ku, Tokyo, 154-8509, Japan.