IL-4-Induced Priming of Human Intestinal Mast Cells for Enhanced Survival and Th2 Cytokine Generation Is Reversible and Associated with Incr
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免疫学杂志 2005年第11期
Abstract
In synergy with stem cell factor (SCF), IL-4 strongly enhances mast cell proliferation and shifts IgE-dependent cytokine production in mature human mast cells toward an increased release of Th2 cytokines such as IL-3, IL-5, and IL-13 and a decreased IL-6 expression. In this study we analyzed the kinetics and the mechanisms of these IL-4 effects on mast cells purified from intestinal tissue. If the cells were first cultured with IL-4 for 14 days and then without IL-4 for another 14 days, mast cells lost the capacity of producing higher amounts of Th2 cytokines and regained the capacity of producing IL-6. The IL-4-induced up-regulation of mast cell proliferation and FcRI expression was also reversible if IL-4 was withdrawn for 14 days. Interestingly, in contrast to IL-4, proliferation and phenotype of human intestinal mast cells were not affected by IL-13 although both cytokines were capable of inducing STAT6 activation. Instead, IL-4 treatment (but not IL-13 treatment) was associated with an increased activity of ERK1/2 and c-Fos, the downstream target of ERK1/2 and component of the transcription factor AP-1. Consistently, mast cell proliferation and cytokine expression in response to IL-4 was blocked by the MEK inhibitor PD98059. In summary, our data show that the IL-4 effects on human intestinal mast cell functions are reversible and accompanied by an increased activity of ERK1/2 and c-Fos.
Introduction
Mast cells are of particular importance in immediate-type allergic and other inflammatory reactions. They are a potent cellular source of multiple cytokines suggesting an important role in immunoregulation and host defense. Moreover, they release a number of preformed and de novo-synthesized inflammatory mediators such as histamine, leukotrienes, proteases, PGs, and TNF- upon stimulation with IgE-dependent and IgE-independent agonists (1, 2, 3). Cytokines were also identified as regulators of human mast cell function, of which stem cell factor (SCF)3 and IL-4 seem to be the most important ones (4, 5, 6, 7, 8, 9, 10, 11). We reported recently that IL-4, known to regulate B and T lymphocyte differentiation, strongly enhances growth and IgE-dependent mediator release of human intestinal mast cells (5). Moreover we found that IL-4 shifts human intestinal mast cell cytokine production toward a Th2 profile (6, 7). Long-term challenge for 14 days with IL-4 and SCF compared with SCF alone enhanced the expression of IL-3, IL-5, and IL-13, which became detectable even without stimulation by FcRI cross-linking, whereas suppressing the production of IL-6.
The transcription factor STAT6 is well known to be activated following cell stimulation with IL-4 and this activation of STAT6 was found to be critical for the enhanced expression of many IL-4 responsive genes (12). This finding was confirmed in vivo by mice deficient in STAT6 showing that STAT6 plays a central role in exerting IL-4-mediated effects such as the production of Th2 cytokines from T cells (13). STAT6 also acts as an antagonist of NF-B binding and transcriptional activation found to be responsible for the IL-4-dependent suppression of TNF--induced E-selectin gene expression (14). IL-13 shares many biologic characteristics with IL-4. Both cytokines use the IL-4R -chain as a common receptor component and activate STAT6 (15). Therefore, we compared IL-4 and IL-13 with regard to signal transduction and function in human mast cells. Moreover, we studied the reversibility of the IL-4-dependent mast cell priming for a Th2 dominated cytokine profile and enhanced proliferation. We found that IL-4 affects human mast cell functions by a signaling pathway involving rather ERK1/2 and c-Fos than STAT6.
Statistics
Data from multiple experiments were expressed as mean + SD, if not indicated otherwise. Differences between groups were analyzed for statistical significance defined as p < 0.05 (one-tailed paired t test).
Results
The IL-4-dependent priming of human intestinal mast cells concerning survival, FcRI expression, and cytokine expression is reversible
To analyze whether the IL-4-dependent mast cell priming is reversible, mast cells purified from intestinal tissue were cultured for 14 days in the presence or absence of IL-4, harvested, washed, and then cultured for additional 14 days in the presence or absence of IL-4. The experimental design is shown in Fig. 1A. After 14 or 28 days, mast cell recovery was determined. After 14 days, mast cell numbers were two to three times higher if the cells were cultured with IL-4 in addition to SCF compared with cells cultured with SCF alone. After 28 days, we found a pronounced enhancement of mast cell numbers if IL-4 was present the 14 days before measurement (1-2 or 2-2). Vice versa, the mast cell numbers were similar if IL-4 was absent the 14 days before analysis (1, 1-1, or 2-1) (Fig. 1B). Real-time RT-PCR analysis revealed that the enhancement of Th2-type cytokines (IL-3, IL-5, IL-13) and the suppression of IL-6 in response to IL-4 were also reversible (Fig. 1, C–F). If IL-4 was present the 14 days before measurement, the expression of Th2 cytokines was up-regulated whereas IL-6 expression was down-regulated (2, 1-2, or 2-2). If the cells were cultured without IL-4 before cytokine measurement, IL-6, but not Th2 cytokines, was up-regulated (1, 1-1, or 2-1). If the cells were first cultured with IL-4 for 14 days and then without IL-4 for another 14 days, mast cells lost the capacity of producing higher amounts of Th2 cytokines and regained the capacity of producing IL-6 (2-1). FACS analysis revealed that the up-regulation of FcRI was also reversible if IL-4 was withdrawn. FcRI was up-regulated on mast cells if the cells were cultured for 14 days with IL-4 in addition to SCF compared with cells cultured with SCF alone. The up-regulation of the receptor expression was reversible if the cells were cultured for another 14 days without IL-4 but was unchanged if the culture with IL-4 was continued (Fig. 1G). Interestingly, the IL-4-dependent decrease of mast cells containing both tryptase and chymase from 50 to 60% (0 days) to 40–20% (14 days) was discontinued when IL-4 was withdrawn, but the tryptase to chymase mast cell ratio was not reversible within 14 days (Fig. 1H).
Mast cell proliferation and mast cell phenotype is not affected by IL-13
IL-13 is known to share the IL-4R -chain with IL-4 (15). Thus, it should be clarified whether IL-13 is also able to affect mast cell proliferation and mast cell phenotype in a similar manner as IL-4. Mast cells express mRNA for the IL-13R components IL-13R1 and IL-13R2 (Fig. 2A). IL-13R1 initially binds IL-13 with subsequent recruitment of the IL-4R to transduce a signal, whereas the IL-13R2 can bind IL-13 in the absence of IL-4R, but its role in IL-13 signaling is still unclear (16). IL-13 alone had no effect on mast cell survival and all mast cells died within 3–7 days of culture (data not shown). Unlike IL-4, IL-13 did not enhance mast cell proliferation after 14 days of culture in combination with SCF (Fig. 2B). Further, IL-13 (in combination with SCF) did not cause phenotypical changes, which are present after culture with IL-4. Mast cell subtype distribution (Fig. 2C), FcRI expression (Fig. 2D), and cytokine expression (Fig. 2, E–H) were unchanged if mast cells were cultured for 14 days with IL-13 and SCF compared with mast cells cultured with SCF alone. Induction of mRNA expression for IL-3, IL-5, and IL-13 in mast cells cultured with IL-13 in addition to SCF was not found. In contrast, if the cells were cultured in the presence of IL-4 in addition to SCF the expression of mRNA for IL-3, IL-5, and IL-13 was induced.
Discussion
During the last years IL-4 has been recognized as an important regulator of human mast cells derived from intestine, lung, skin, or cord blood (5, 6, 7, 8, 9, 10, 11). In synergy with SCF, IL-4 enhances proliferation, mediator release, and Th2 cytokine generation. In this study we show that this IL-4-induced mast cell priming is reversible if IL-4 is withdrawn. Moreover, our data suggest that IL-4 affects human mast cell functions using a signaling pathway involving ERK1/2 and c-Fos.
So far, Th populations were analyzed regarding reversibility of their cytokine profiles (22). It is well known that cytokines are important in the development of different cytokine producing Th1 and Th2 cells. IL-12 promotes Th1 development with IFN- secretion, whereas IL-4 promotes Th2 development (23, 24). Th1 cells resulting after 1 wk in the presence of IL-12 plus anti-IL-4 Abs could convert to Th2 cells when restimulated with IL-4, and Th2 cells resulting after 1 wk with IL-4 could give rise to Th1 cells upon restimulation with IL-12 plus anti-IL-4. Interestingly, the cytokine profiles of long-term Th1 and Th2 populations arising originally from repeated stimulation in IL-12 or IL-4 for 3 wk were not reversible (22). In contrast, we could show that the IL-4-induced phenotype of human intestinal mast cells including the cytokine expression profile was almost reversible following 2 wk.
The most prominent IL-4-dependent transcription factor is STAT6. STAT6-deficient mice have a phenotype similar to IL-4-deficient mice or mice after treatment with Abs to IL-4 or to IL-4R. Thus, it has been suggested that STAT6 plays an essential role in exerting IL-4-mediated biologic responses such as the production of Th2 cytokines from T cells (13). IL-13, which is known to use the IL-4R -chain as a receptor component, shares many biologic characteristics with IL-4 and is known to activate STAT6 as well (15, 16). Interestingly, we found that IL-13 did not influence the cytokine expression profile, the proliferation rate, the expression of FcRI, and the chymase expression in human intestinal mast cells although we found that STAT6 becomes equally activated following short-term stimulation of human intestinal mast cells with IL-4 or IL-13. Maybe, STAT6 is not crucial for the IL-4-mediated regulation of human mast cells. STAT6-independent Th2 development has been observed in vivo and in vitro (25). The transcription factor GATA-3, found to be induced by IL-4 through STAT6 early in the Th2 development, was also shown to induce Th2 development in STAT6-deficient T cells (26). The described GATA-3 autoactivation causing the Th2 development and commitment was however different from our findings concerning mast cell regulation, independent from IL-4. Moreover, we did not detect differences of GATA-3 expression in human mast cells dependent on IL-4 stimulation (our unpublished observations). Interestingly in regard to our data, in mice STAT6 was found to be essential for IL-4-induced proliferation of T cells but not for IL-4-induced proliferation of bone marrow-derived mast cells (13, 27, 28, 29). The IL-4-induced enhancement of mast cell proliferation and survival occurred in a -chain- and a Jak-3-dependent manner, but was independent of STAT6. Noteworthy, IL-4, but not IL-13, was able to induce proliferation and survival of murine bone marrow-derived mast cells (29).
However, IL-4 causes both the shift of the cytokine pattern toward a Th2 phenotype and the increase of proliferation and mediator release in mature human mast cells (5, 6). ERK1/2 is known to be involved in cell proliferation and cytokine expression (17, 18). We found phosphorylation of ERK1/2 in mast cell extracts even 1 wk after the last feeding provided that IL-4 was present during culture. Wery et al. (30) observed increased tyrosine phosphorylation of ERK1/2, which was accompanied by an enhanced catalytic activity of the enzyme following IL-4 stimulation in human keratinocytic cell lines. They also found that in keratinocytes, but not in T lymphocytes, Shc is phosphorylated, suggesting its possible implication in ERK activation in keratinocytes caused by IL-4. The phosphorylation of the adapter protein Shc may be the link between IL-4R and MAPK.
In contrast to the findings observed in keratinocytes we did not detect activation of ERK1/2 in response to short-term stimulation with IL-4. Noteworthy in this respect, Levings et al. (31) reported that the inability of IL-4 to activate Ras/Raf/ERK pathway and subsequently to promote cell cycle progression is complemented by expression of activated N-Ras or conditionally active mutants of Raf1. Moreover, they found that IL-4 and Raf1 not only synergize to promote proliferation, but also to increase JNK activity. Maybe, SCF is responsible for the activation of Ras/Raf in human mast cells that synergize with IL-4 to enhance proliferation and cytokine expression. In accordance with the activation of ERK1/2 we found increased mRNA expression for c-Fos, the downstream target of ERK1/2 and component of the transcription factor AP-1, in response to culture with IL-4. To examine a causal link of IL-4 effects and ERK activation we treated mast cells with the ERK kinase inhibitor PD98059 (21). We found that mast cell proliferation as well as cytokine expression in response to IL-4 was inhibited by PD98059. These findings further suggest a role of ERK1/2 and c-Fos in IL-4-dependent effects although we cannot exclude an inhibitory effect of PD98059 on other kinases and thus a lack of specificity.
Noteworthy, the increased activity of ERK1/2 and c-Fos corresponds not only with the induced Th2 cytokine expression in response to IL-4 but also with the cytokine expression in response to FcRI cross-linking suggesting a general role of the ERK1/2 branch of the MAPK pathway for cytokine expression in human mast cells (32).
In conclusion, our data show that IL-4-induced priming of human intestinal mast cells for enhanced survival and Th2 cytokine generation is reversible and that ERK1/2 and c-Fos might be involved in these IL-4-dependent effects. These observations further emphasize that mast cell functions are subject to a considerable diversity and plasticity depending on the cytokine milieu.
Acknowledgments
We thank Antje Radke, Nicole Steegmann, Birgit Hegemann, and Gisela Weier for excellent technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Deutsche Forschungsgemeinschaft SFB621-C8 and LO581/3-1.
2 Address correspondence and reprint requests to Dr. Axel Lorentz, Institute of Nutritional Medicine, University of Hohenheim, Fruwirthstrasse 12, D-70599 Stuttgart, Germany. E-mail address: lorentz.axel{at}mh-hannover.de
3 Abbreviation used in this paper: SCF, stem cell factor.
Received for publication July 16, 2004. Accepted for publication March 21, 2005.
References
Metcalfe, D. D., D. Baram, Y. A. Mekori. 1997. Mast cells. Physiol. Rev. 77: 1033-1079.
Maurer, M., T. Theoharides, R. D. Granstein, S. C. Bischoff, J. Bienenstock, B. Henz, P. Kovanen, A. M. Piliponsky, N. Kambe, H. Vliagoftis, et al 2003. What is the physiological function of mast cells?. Exp. Dermatol. 12: 886-910.
Wedemeyer, J., M. Tsai, S. J. Galli. 2000. Roles of mast cells and basophils in innate and acquired immunity. Curr. Opin. Immunol. 12: 624-631.
Bischoff, S. C., S. Schwengberg, R. Raab, M. P. Manns. 1997. Functional properties of human intestinal mast cells cultured in a new culture system: enhancement of IgE receptor-dependent mediator release and response to stem cell factor. J. Immunol. 159: 5560-5567.[
Bischoff, S. C., G. Sellge, A. Lorentz, W. Sebald, R. Raab, M. P. Manns. 1999. IL-4 enhances proliferation and mediator release in mature human mast cells. Proc. Natl. Acad. Sci. USA 96: 8080-8085.
Lorentz, A., S. Schwengberg, G. Sellge, M. P. Manns, C. S. Bischoff. 2000. Human intestinal mast cells are capable of producing different cytokine profiles: role of IgE-receptor cross-linking and IL-4. J. Immunol. 164: 43-48.
Bischoff, S. C., G. Sellge, M. P. Manns, A. Lorentz. 2001. Interleukin-4 induces a switch of human intestinal mast cells from proinflammatory cells to Th2-type cells. Int. Arch. Allergy Immunol. 124: 151-154.(Axel Lorentz2,*,, Mikosch)
In synergy with stem cell factor (SCF), IL-4 strongly enhances mast cell proliferation and shifts IgE-dependent cytokine production in mature human mast cells toward an increased release of Th2 cytokines such as IL-3, IL-5, and IL-13 and a decreased IL-6 expression. In this study we analyzed the kinetics and the mechanisms of these IL-4 effects on mast cells purified from intestinal tissue. If the cells were first cultured with IL-4 for 14 days and then without IL-4 for another 14 days, mast cells lost the capacity of producing higher amounts of Th2 cytokines and regained the capacity of producing IL-6. The IL-4-induced up-regulation of mast cell proliferation and FcRI expression was also reversible if IL-4 was withdrawn for 14 days. Interestingly, in contrast to IL-4, proliferation and phenotype of human intestinal mast cells were not affected by IL-13 although both cytokines were capable of inducing STAT6 activation. Instead, IL-4 treatment (but not IL-13 treatment) was associated with an increased activity of ERK1/2 and c-Fos, the downstream target of ERK1/2 and component of the transcription factor AP-1. Consistently, mast cell proliferation and cytokine expression in response to IL-4 was blocked by the MEK inhibitor PD98059. In summary, our data show that the IL-4 effects on human intestinal mast cell functions are reversible and accompanied by an increased activity of ERK1/2 and c-Fos.
Introduction
Mast cells are of particular importance in immediate-type allergic and other inflammatory reactions. They are a potent cellular source of multiple cytokines suggesting an important role in immunoregulation and host defense. Moreover, they release a number of preformed and de novo-synthesized inflammatory mediators such as histamine, leukotrienes, proteases, PGs, and TNF- upon stimulation with IgE-dependent and IgE-independent agonists (1, 2, 3). Cytokines were also identified as regulators of human mast cell function, of which stem cell factor (SCF)3 and IL-4 seem to be the most important ones (4, 5, 6, 7, 8, 9, 10, 11). We reported recently that IL-4, known to regulate B and T lymphocyte differentiation, strongly enhances growth and IgE-dependent mediator release of human intestinal mast cells (5). Moreover we found that IL-4 shifts human intestinal mast cell cytokine production toward a Th2 profile (6, 7). Long-term challenge for 14 days with IL-4 and SCF compared with SCF alone enhanced the expression of IL-3, IL-5, and IL-13, which became detectable even without stimulation by FcRI cross-linking, whereas suppressing the production of IL-6.
The transcription factor STAT6 is well known to be activated following cell stimulation with IL-4 and this activation of STAT6 was found to be critical for the enhanced expression of many IL-4 responsive genes (12). This finding was confirmed in vivo by mice deficient in STAT6 showing that STAT6 plays a central role in exerting IL-4-mediated effects such as the production of Th2 cytokines from T cells (13). STAT6 also acts as an antagonist of NF-B binding and transcriptional activation found to be responsible for the IL-4-dependent suppression of TNF--induced E-selectin gene expression (14). IL-13 shares many biologic characteristics with IL-4. Both cytokines use the IL-4R -chain as a common receptor component and activate STAT6 (15). Therefore, we compared IL-4 and IL-13 with regard to signal transduction and function in human mast cells. Moreover, we studied the reversibility of the IL-4-dependent mast cell priming for a Th2 dominated cytokine profile and enhanced proliferation. We found that IL-4 affects human mast cell functions by a signaling pathway involving rather ERK1/2 and c-Fos than STAT6.
Statistics
Data from multiple experiments were expressed as mean + SD, if not indicated otherwise. Differences between groups were analyzed for statistical significance defined as p < 0.05 (one-tailed paired t test).
Results
The IL-4-dependent priming of human intestinal mast cells concerning survival, FcRI expression, and cytokine expression is reversible
To analyze whether the IL-4-dependent mast cell priming is reversible, mast cells purified from intestinal tissue were cultured for 14 days in the presence or absence of IL-4, harvested, washed, and then cultured for additional 14 days in the presence or absence of IL-4. The experimental design is shown in Fig. 1A. After 14 or 28 days, mast cell recovery was determined. After 14 days, mast cell numbers were two to three times higher if the cells were cultured with IL-4 in addition to SCF compared with cells cultured with SCF alone. After 28 days, we found a pronounced enhancement of mast cell numbers if IL-4 was present the 14 days before measurement (1-2 or 2-2). Vice versa, the mast cell numbers were similar if IL-4 was absent the 14 days before analysis (1, 1-1, or 2-1) (Fig. 1B). Real-time RT-PCR analysis revealed that the enhancement of Th2-type cytokines (IL-3, IL-5, IL-13) and the suppression of IL-6 in response to IL-4 were also reversible (Fig. 1, C–F). If IL-4 was present the 14 days before measurement, the expression of Th2 cytokines was up-regulated whereas IL-6 expression was down-regulated (2, 1-2, or 2-2). If the cells were cultured without IL-4 before cytokine measurement, IL-6, but not Th2 cytokines, was up-regulated (1, 1-1, or 2-1). If the cells were first cultured with IL-4 for 14 days and then without IL-4 for another 14 days, mast cells lost the capacity of producing higher amounts of Th2 cytokines and regained the capacity of producing IL-6 (2-1). FACS analysis revealed that the up-regulation of FcRI was also reversible if IL-4 was withdrawn. FcRI was up-regulated on mast cells if the cells were cultured for 14 days with IL-4 in addition to SCF compared with cells cultured with SCF alone. The up-regulation of the receptor expression was reversible if the cells were cultured for another 14 days without IL-4 but was unchanged if the culture with IL-4 was continued (Fig. 1G). Interestingly, the IL-4-dependent decrease of mast cells containing both tryptase and chymase from 50 to 60% (0 days) to 40–20% (14 days) was discontinued when IL-4 was withdrawn, but the tryptase to chymase mast cell ratio was not reversible within 14 days (Fig. 1H).
Mast cell proliferation and mast cell phenotype is not affected by IL-13
IL-13 is known to share the IL-4R -chain with IL-4 (15). Thus, it should be clarified whether IL-13 is also able to affect mast cell proliferation and mast cell phenotype in a similar manner as IL-4. Mast cells express mRNA for the IL-13R components IL-13R1 and IL-13R2 (Fig. 2A). IL-13R1 initially binds IL-13 with subsequent recruitment of the IL-4R to transduce a signal, whereas the IL-13R2 can bind IL-13 in the absence of IL-4R, but its role in IL-13 signaling is still unclear (16). IL-13 alone had no effect on mast cell survival and all mast cells died within 3–7 days of culture (data not shown). Unlike IL-4, IL-13 did not enhance mast cell proliferation after 14 days of culture in combination with SCF (Fig. 2B). Further, IL-13 (in combination with SCF) did not cause phenotypical changes, which are present after culture with IL-4. Mast cell subtype distribution (Fig. 2C), FcRI expression (Fig. 2D), and cytokine expression (Fig. 2, E–H) were unchanged if mast cells were cultured for 14 days with IL-13 and SCF compared with mast cells cultured with SCF alone. Induction of mRNA expression for IL-3, IL-5, and IL-13 in mast cells cultured with IL-13 in addition to SCF was not found. In contrast, if the cells were cultured in the presence of IL-4 in addition to SCF the expression of mRNA for IL-3, IL-5, and IL-13 was induced.
Discussion
During the last years IL-4 has been recognized as an important regulator of human mast cells derived from intestine, lung, skin, or cord blood (5, 6, 7, 8, 9, 10, 11). In synergy with SCF, IL-4 enhances proliferation, mediator release, and Th2 cytokine generation. In this study we show that this IL-4-induced mast cell priming is reversible if IL-4 is withdrawn. Moreover, our data suggest that IL-4 affects human mast cell functions using a signaling pathway involving ERK1/2 and c-Fos.
So far, Th populations were analyzed regarding reversibility of their cytokine profiles (22). It is well known that cytokines are important in the development of different cytokine producing Th1 and Th2 cells. IL-12 promotes Th1 development with IFN- secretion, whereas IL-4 promotes Th2 development (23, 24). Th1 cells resulting after 1 wk in the presence of IL-12 plus anti-IL-4 Abs could convert to Th2 cells when restimulated with IL-4, and Th2 cells resulting after 1 wk with IL-4 could give rise to Th1 cells upon restimulation with IL-12 plus anti-IL-4. Interestingly, the cytokine profiles of long-term Th1 and Th2 populations arising originally from repeated stimulation in IL-12 or IL-4 for 3 wk were not reversible (22). In contrast, we could show that the IL-4-induced phenotype of human intestinal mast cells including the cytokine expression profile was almost reversible following 2 wk.
The most prominent IL-4-dependent transcription factor is STAT6. STAT6-deficient mice have a phenotype similar to IL-4-deficient mice or mice after treatment with Abs to IL-4 or to IL-4R. Thus, it has been suggested that STAT6 plays an essential role in exerting IL-4-mediated biologic responses such as the production of Th2 cytokines from T cells (13). IL-13, which is known to use the IL-4R -chain as a receptor component, shares many biologic characteristics with IL-4 and is known to activate STAT6 as well (15, 16). Interestingly, we found that IL-13 did not influence the cytokine expression profile, the proliferation rate, the expression of FcRI, and the chymase expression in human intestinal mast cells although we found that STAT6 becomes equally activated following short-term stimulation of human intestinal mast cells with IL-4 or IL-13. Maybe, STAT6 is not crucial for the IL-4-mediated regulation of human mast cells. STAT6-independent Th2 development has been observed in vivo and in vitro (25). The transcription factor GATA-3, found to be induced by IL-4 through STAT6 early in the Th2 development, was also shown to induce Th2 development in STAT6-deficient T cells (26). The described GATA-3 autoactivation causing the Th2 development and commitment was however different from our findings concerning mast cell regulation, independent from IL-4. Moreover, we did not detect differences of GATA-3 expression in human mast cells dependent on IL-4 stimulation (our unpublished observations). Interestingly in regard to our data, in mice STAT6 was found to be essential for IL-4-induced proliferation of T cells but not for IL-4-induced proliferation of bone marrow-derived mast cells (13, 27, 28, 29). The IL-4-induced enhancement of mast cell proliferation and survival occurred in a -chain- and a Jak-3-dependent manner, but was independent of STAT6. Noteworthy, IL-4, but not IL-13, was able to induce proliferation and survival of murine bone marrow-derived mast cells (29).
However, IL-4 causes both the shift of the cytokine pattern toward a Th2 phenotype and the increase of proliferation and mediator release in mature human mast cells (5, 6). ERK1/2 is known to be involved in cell proliferation and cytokine expression (17, 18). We found phosphorylation of ERK1/2 in mast cell extracts even 1 wk after the last feeding provided that IL-4 was present during culture. Wery et al. (30) observed increased tyrosine phosphorylation of ERK1/2, which was accompanied by an enhanced catalytic activity of the enzyme following IL-4 stimulation in human keratinocytic cell lines. They also found that in keratinocytes, but not in T lymphocytes, Shc is phosphorylated, suggesting its possible implication in ERK activation in keratinocytes caused by IL-4. The phosphorylation of the adapter protein Shc may be the link between IL-4R and MAPK.
In contrast to the findings observed in keratinocytes we did not detect activation of ERK1/2 in response to short-term stimulation with IL-4. Noteworthy in this respect, Levings et al. (31) reported that the inability of IL-4 to activate Ras/Raf/ERK pathway and subsequently to promote cell cycle progression is complemented by expression of activated N-Ras or conditionally active mutants of Raf1. Moreover, they found that IL-4 and Raf1 not only synergize to promote proliferation, but also to increase JNK activity. Maybe, SCF is responsible for the activation of Ras/Raf in human mast cells that synergize with IL-4 to enhance proliferation and cytokine expression. In accordance with the activation of ERK1/2 we found increased mRNA expression for c-Fos, the downstream target of ERK1/2 and component of the transcription factor AP-1, in response to culture with IL-4. To examine a causal link of IL-4 effects and ERK activation we treated mast cells with the ERK kinase inhibitor PD98059 (21). We found that mast cell proliferation as well as cytokine expression in response to IL-4 was inhibited by PD98059. These findings further suggest a role of ERK1/2 and c-Fos in IL-4-dependent effects although we cannot exclude an inhibitory effect of PD98059 on other kinases and thus a lack of specificity.
Noteworthy, the increased activity of ERK1/2 and c-Fos corresponds not only with the induced Th2 cytokine expression in response to IL-4 but also with the cytokine expression in response to FcRI cross-linking suggesting a general role of the ERK1/2 branch of the MAPK pathway for cytokine expression in human mast cells (32).
In conclusion, our data show that IL-4-induced priming of human intestinal mast cells for enhanced survival and Th2 cytokine generation is reversible and that ERK1/2 and c-Fos might be involved in these IL-4-dependent effects. These observations further emphasize that mast cell functions are subject to a considerable diversity and plasticity depending on the cytokine milieu.
Acknowledgments
We thank Antje Radke, Nicole Steegmann, Birgit Hegemann, and Gisela Weier for excellent technical assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Deutsche Forschungsgemeinschaft SFB621-C8 and LO581/3-1.
2 Address correspondence and reprint requests to Dr. Axel Lorentz, Institute of Nutritional Medicine, University of Hohenheim, Fruwirthstrasse 12, D-70599 Stuttgart, Germany. E-mail address: lorentz.axel{at}mh-hannover.de
3 Abbreviation used in this paper: SCF, stem cell factor.
Received for publication July 16, 2004. Accepted for publication March 21, 2005.
References
Metcalfe, D. D., D. Baram, Y. A. Mekori. 1997. Mast cells. Physiol. Rev. 77: 1033-1079.
Maurer, M., T. Theoharides, R. D. Granstein, S. C. Bischoff, J. Bienenstock, B. Henz, P. Kovanen, A. M. Piliponsky, N. Kambe, H. Vliagoftis, et al 2003. What is the physiological function of mast cells?. Exp. Dermatol. 12: 886-910.
Wedemeyer, J., M. Tsai, S. J. Galli. 2000. Roles of mast cells and basophils in innate and acquired immunity. Curr. Opin. Immunol. 12: 624-631.
Bischoff, S. C., S. Schwengberg, R. Raab, M. P. Manns. 1997. Functional properties of human intestinal mast cells cultured in a new culture system: enhancement of IgE receptor-dependent mediator release and response to stem cell factor. J. Immunol. 159: 5560-5567.[
Bischoff, S. C., G. Sellge, A. Lorentz, W. Sebald, R. Raab, M. P. Manns. 1999. IL-4 enhances proliferation and mediator release in mature human mast cells. Proc. Natl. Acad. Sci. USA 96: 8080-8085.
Lorentz, A., S. Schwengberg, G. Sellge, M. P. Manns, C. S. Bischoff. 2000. Human intestinal mast cells are capable of producing different cytokine profiles: role of IgE-receptor cross-linking and IL-4. J. Immunol. 164: 43-48.
Bischoff, S. C., G. Sellge, M. P. Manns, A. Lorentz. 2001. Interleukin-4 induces a switch of human intestinal mast cells from proinflammatory cells to Th2-type cells. Int. Arch. Allergy Immunol. 124: 151-154.(Axel Lorentz2,*,, Mikosch)