Basic and Clinical Immunology
Role of activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells in IgE receptor-mediated cytokine expression in mature human mast cells,☆☆

https://doi.org/10.1067/mai.2003.1342Get rights and content

Abstract

Background: On activation by cross-linking the high-affinity IgE receptor (FcϵRI), expression of TNF-α, IL-3, IL-5, and IL-13 is induced in human intestinal mast cells. Objective: We sought to examine, for the first time, FcϵRI signaling in mature human mast cells. Methods: Mast cells were isolated from intestinal tissue and cultured in the presence of stem cell factor. The cells were treated with specific inhibitors before stimulation by means of FcϵRI cross-linking. Cytokine mRNA expression was analyzed by means of RT-PCR, and activation of signaling molecules was determined by means of immunocytochemistry, RT-PCR, Western blotting, and protein kinase C (PKC) assay. Results: We found that nuclear factor-κB (NF-κB), as well as c-Fos and c-Jun, the components of activator protein 1 (AP-1), are activated after FcϵRI cross-linking in human intestinal mast cells. Treatment of the cells with specific inhibitors revealed an involvement of NF-κB and nuclear factor of activated T cells, as well as the necessity of extracellular signal-regulated kinase 1/2 (ERK-1/2), PKC, and AP-1 for the induced cytokine gene expression. Consistently, we found that activation of c-Fos corresponds with the induced cytokine gene expression and that ERK-1/2, an activator of c-Fos, was activated in response to FcϵRI cross-linking. Conclusion: Our data on human mast cells show that the activity of ERK-1/2, PKC, and subsequent activation of AP-1 are necessary for the FcϵRI-mediated cytokine expression. Nuclear factor of activated T cells and NF-κB seem to be necessary for the induction of TNF-α, IL-3, and IL-13 but are less important for the transcription of IL-5. (J Allergy Clin Immunol 2003;111:1062-8.)

Section snippets

Isolation and culture of human intestinal mast cells

Human intestinal mast cells were isolated from surgical tissue specimens (macroscopically normal border sections free of tumor cells, as determined by means of histologic examination of the tissue) derived from patients who underwent bowel resection because of cancer. Permission to conduct the study was obtained from the local ethical committee of the Medical School of Hannover. The methods of mechanical and enzymatic tissue dispersion yielding single-cell preparations containing 4% ± 2% (mean

NF-κB, c-Fos, and c-Jun are activated after FcϵRI cross-linking in human intestinal mast cells

FcϵRI-mediated cytokine production in mast cells is regulated at the transcriptional level.4, 6, 19 The transcription factors NF-κB and AP-1 are known to regulate the expression of multiple inflammatory genes.20 Mast cells were treated with 100 ng/mL mAb 29C6 and centrifugated on slides to access activation of NF-κB in response to FcϵRI cross-linking. Immunostaining of the cells revealed a translocation of NF-κB into the nucleus in response to FcϵRI stimulation (Fig 1, A ).

. Activation of NF-κB,

DISCUSSION

Mast cells play a central role in inflammatory and particularly in allergic reactions. Recently, we reported that FcϵRI cross-linking induces the expression of multiple cytokines in mature human mast cells isolated from intestinal mucosa.6 Several transcription factors are known to be involved in allergic inflammation, including NF-κB, AP-1, and NF-AT.20 In this study we show that NF-κB and AP-1 become activated in response to FcϵRI cross-linking in human intestinal mast cells and that the

Acknowledgements

We thank Antje Radke, Nicole Steegmann, Birgit Hegemann, and Gisela Weier for excellent technical assistance.

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    Supported by the Deutsche Forschungsgemeinschaft (SFB621-A8).

    ☆☆

    Reprint requests: Axel Lorentz, PhD, Department of Gastroenterology, Hepatology and Endocrinology, Medical School of Hannover, D-30623 Hannover, Germany.

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