Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-γ

doi:10.1067/mai.2000.107043

Journal of Allergy and Clinical Immunology

Volume 106, Issue 1, Part 1, July 2000, Pages 141-149

https://doi.org/10.1067/mai.2000.107043 Get rights and content

Abstract

Background: T_H1 and T_H2 cytokines are thought to regulate allergic inflammation. Objective: Two key regulatory cytokines, IL-4 and IFN-γ, were examined for their effects on cytokine production by cultured human mast cells (CHMCs). Methods: CHMCs were obtained by culturing cord blood–derived CD34⁺ cells in the presence of stem cell factor and IL-6 for 14 to 16 weeks. CHMCs were passively sensitized with human myeloma IgE and supplemented with or without IL-4 or IFN-γ. After the sensitization, CHMCs were stimulated with anti-FcϵRIα mAb. Concentrations of secreted cytokines were measured by using ELISA, and cytokine messenger RNA was analyzed by using quantitative competitive RT-PCR. Results: IL-4 profoundly enhanced FcϵRI-mediated production of macrophage inflammatory protein (MIP) 1α, IL-8, and GM-CSF. For example, the enhancement by IL-4 (10 ng/mL) of the production of MIP-1α, IL-8, and GM-CSF was 25-, 7-, and 90-fold, respectively, after 6 hours. IL-4 also enhanced levels of FcϵRI-induced cytokine messenger RNA but to a lesser degree. In contrast, IFN-γ inhibited FcϵRI-induced production of MIP-1α, IL-8, and GM-CSF. For example, the inhibition by IFN-γ (10 ng/mL) of FcϵRI-mediated production of MIP-1α, IL-8, and GM-CSF was 80%, 75%, and 95%, respectively. IFN-γ also suppressed FcϵRI-induced messenger RNA expression of these cytokines. Neither IL-4 nor IFN-γ affected the kinetics of cytokine production by CHMCs. Conclusion: These data suggest that IL-4 and IFN-γ may influence allergic reactions by modulating human mast cell cytokine production. (J Allergy Clin Immunol 2000;106:141-9.)

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 ).

. Effect of

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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IFNγ is strongly related to mast cell-associated diseases. There are many reports that IFNγ inhibits mast cell degranulation. However, inflammatory cytokine production in mast cells stimulated with IFNγ has not yet been clearly investigated. Therefore, we aimed to investigate the signaling pathways of cytokine production in mast cells stimulated with IFNγ. Human mast cell line (HMC)-1 or mouse bone marrow-derived mast cells (BMMCs) were stimulated with IFNγ (100 units) for time periods indicated. Expressions of proteins and mRNAs of cytokines were determined by ELISA and RT-PCR, respectively, activities of MAP kinases, PKC, JAK1/2, and STAT1 on tyrosine 701 and serine 727 by immunoblotting, the DNA-binding activity of the transcription factors by electrophoretic mobility shift assay. IFNγ-stimulated mast cells showed increase in expressions of proteins and mRNAs of inflammatory cytokines, phosphorylations of MAP kinases, PKCα and βI, JAK1/2, and STAT1 on tyrosine 701 and serine 727. JAK inhibitor or PKC inhibitors inhibited the phosphorylations of p38 kinase, STAT1 on serine 727, and activities of NF-κB and AP-1 compared to IFNγ stimulation alone. These data suggest that IFNγ-stimulated mast cells induce productions of inflammatory cytokines through PKC/p38/NF-κB and AP-1 pathways, not through classical JAK/STAT1 pathway, in both mast cells.
Progress in allergy signal research on mast cells: Systemic approach to mast cell biology in allergic diseases
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At the end of the last century, microarray technology that examines the total genes and transcripts present in a cell became available as a laboratory tool. Mast cells are known to play a pivotal role in initiating allergic inflammation by releasing various mediators and cytokines. According to the recent microarray-based studies, mast cells have been found to be much more versatile functional molecules than we ever thought. Also, genes that are exclusively expressed in mast cells have been identified in comparison with other cell types. In this article, the outcome of microarray-based analyses on the role of mast cells in allergic inflammation will be reviewed by focusing on the mast cell–specific genes as drug targets.
Identification of a lysophosphatidylserine receptor on mast cells
2006, Biochemical and Biophysical Research Communications
Citation Excerpt :
After 4 weeks of culture, >98% of the cells were mast cells as demonstrated by staining with Alcian blue and safranin. Cultured human mast cells (CHMCs) were generated as previously described [22–24]. Briefly, mononuclear cells were separated from umbilical cord blood samples derived from three healthy mothers using density gradient centrifugation followed by immunomagnetic positive selection with mAb against CD34 (MACS II; Miltenyi Biotech, Bergisch Gladbach, Germany).
Lysophosphatidyl-l-serine (lysoPS) is thought to be an immunological regulator because it dramatically augments the degranulation of rat peritoneal mast cells (RPMCs). This stimulatory effect may be mediated by a lysoPS receptor, but its molecule has not been identified yet. During a ligand fishing study for the orphan G-protein-coupled receptor 34 (GPR34), we found that lysoPS caused a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in human GPR34-expressing Chinese hamster ovary (CHO/hGPR34) cells. The CHO/hGPR34 cells were unresponsive to other structurally related phospholipids examined. Quantitative real-time-PCR demonstrated that mRNAs of GPR34 are particularly abundant in mast cells. The effective lysoPS concentration for RPMC degranulation was similar to that required for GPR34 activation, and the structural requirement of lysoPS for RPMC degranulation was in good agreement with that observed in CHO/hGPR34 cells. These results suggest that GPR34 is the functional mast cell lysoPS receptor.
Production of matrix metalloproteinases in human cultured mast cells: Involvement of protein kinase C-mitogen activated protein kinase kinase-extracellular signal-regulated kinase pathway
2006, Allergology International
Matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components. To confirm the role of mast cells as a source of MMPs, we investigated the production of MMP and its pathway in human cultured mast cells (HCMC). We also investigated the production of tissue inhibitors of metalloproteinase (TIMPs).
HCMC was stimulated with phorbor 12-miristate 13-acetate (PMA) and/or calcium ionophore A23187 (A23187), and the resulting MMP production was evaluated by gelatin zymography and western blotting. Expression of MMP and TIMP mRNA was also examined. Granulocyte macrophage-colony stimulating factor (GM-CSF) was measured by ELISA and activation of extracellular signal-regulated kinase (ERK) was evaluated by western blotting.
We detected the de novo synthesis of MMP-9 in HCMC after stimulation with PMA and found that the synthesis was mediated through protein kinase C–mitogen activated protein kinase kinase (MEK)–ERK pathway. The MMP-9 production induced by PMA was suppressed by simultaneous treatment with A23187, whereas GM-CSF production was potentiated. We also detected the expression of mRNA for membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 after stimulation with PMA. Glucocorticoids and flavonoids inhibited MMP-9 production, and TIMPs and MMP inhibitors inhibited the gelatinolytic activity of mast cell-derived MMP-9. Furthermore, phenylmethylsulfonyl fluoride, a protease inhibitor, inhibited the conversion from proMMP-9 to active MMP-9.
These results suggest that the human mast cell is a leading member of MMP production, and the production, activation and activity are controllable by pharmacological agents.
High-resolution tracking of cell division demonstrates differential effects of T<inf>H</inf>1 and T<inf>H</inf>2 cytokines on SCF-dependent human mast cell production in vitro: Correlation with apoptosis and Kit expression
2005, Blood
Citation 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.
T-helper 1 (T_H1) (interferon-γ [IFN-γ]) and T_H2 (interleukin-4 [IL-4] and IL-5) cytokines have been variably reported to alter human mast cell numbers in complex culture systems. The effects of these cytokines on the kinetics of cell division and cell death are unknown, and their effect on mast cell behavior is relevant to anticipate the consequences of in vivo strategies that alter cytokine levels. To determine the effect of these cytokines on stem cell factor (SCF)–dependent human mast cell production, we used highresolution tracking of cell division and correlated the results with cell apoptosis, expression of Kit, and mast cell degranulation. When IFN-γ, IL-5, or IL-4 was administered over 8 weeks, we found each cytokine decreased the mast number through a different mechanism. IFN-γ inhibited early progenitor cell division, IL-4 down-regulated early Kit expression, and IL-5 blocked later cell division. Further, IL-4 and IFN-γ had the greatest suppressive effect on degranulation and FcϵRI expression. When these cytokines were administered to mature mast cells, IFN-γ and IL-5 had no effect on degranulation and cell division, but IL-4 induced division and potentiated FcϵRI-mediated degranulation. Thus, exposure of human mast cells to IL-4, IL-5, and IFN-γ during growth and differentiation generally down-regulated mast cell number and function, whereas IL-4 increased mature mast cell division and degranulation.
Reliability of RT-PCR methods for measuring relative gene expression in mast cells
2004, Veterinary Immunology and Immunopathology
Three methods to quantify gene transcript levels in mast cells, real-time RT-PCR, competitive RT-PCR and conventional RT-PCR analyses, were compared. Linear regression analysis on five gene transcripts revealed that the mRNA levels measured by real-time RT-PCR analysis were minimally correlated with those by conventional RT-PCR analysis. In addition, differences in the mRNA level between samples measured by conventional RT-PCR analysis were smaller than those by real-time RT-PCR analysis, suggesting that conventional RT-PCR analysis is less sensitive at measuring mRNA levels. Results from competitive RT-PCR analysis correlated closely with those from real-time RT-PCR analysis. When the differences in mRNA level between samples are relatively smaller, however, the correlation tended to be weaker. Real-time RT-PCR analysis has higher reliability, but is expensive. In contrast, competitive RT-PCR analysis is inexpensive, but is weaker at detecting smaller differences in gene transcript level between samples. Therefore, the most appropriate analytical method to measure mRNA levels should be chosen, depending on the experimental conditions.

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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.

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Basic and clinical immunologyReciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-γ☆

Abstract

Section snippets

Human mast cell culture

Effect of IL-4 or IFN-γ on cytokine production by CHMCs

DISCUSSION

Acknowledgements

J Allergy Clin Immunol

New concepts about the mast cell

N Engl J Med

Human mast cells produce IL-8

J Immunol

Interleukin-13 expression in the nasal mucosa of perennial allergic rhinitis

Am J Respir Crit Care Med

Presence of tumour necrosis factor or a related factor in human basophil/mast cells

Immunology

Interleukin 4 is localized to and released by human mast cells

J Exp Med

Immunolocalization of cytokines in the nasal mucosa of normal and perennial rhinitic subjects. The mast cell as a source of IL-4, IL-5, and IL-6 in human allergic mucosal inflammation

J Immunol

Interleukin-4, -5, and -6 and tumor necrosis factor-α in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines

Am J Respir Cell Mol Biol

Heterogeneity of human mast cells based on cytokine content

J Immunol

IL-4 enhances proliferation and mediator release in mature human mast cells

Proc Natl Acad Sci USA

Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the FcϵRI, CD40L, IL-4, and IL-13, and can induce IgE synthesis in B cells

J Clin Invest

Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells

J Immunol

Activation human mast cells release factors supporting eosinophil survival in vitro

Int Arch Allergy Immunol

Basic and clinical immunology
Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-γ☆