The Type 1 Cysteinyl Leukotriene Receptor Triggers Calcium Influx and Chemotaxis in Mouse - and Effector T Cells1
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免疫学杂志 2005年第14期
Abstract
Linker for activation of T cells (LAT) is essential for T cell activation. Mice with mutations of distinct LAT tyrosine residues (LatY136F and Lat3YF) develop lymphoproliferative disorders involving TCR or T cells that trigger symptoms resembling allergic inflammation. We analyzed whether these T cells share a pattern of gene expression that may account for their pathogenic properties. Both LatY136F and Lat3YF T cells expressed high levels of the type 1 cysteinyl leukotriene receptor (CysLT1). Upon binding to the 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD4) cysteinyl leukotriene, CysLT1 induced Ca2+ flux and caused chemotaxis in both LatY136F and Lat3YF T cells. Wild-type in vitro-activated T cells, but not resting T cells, also migrated toward LTD4 however with a lower magnitude than T cells freshly isolated from LatY136F and Lat3YF mice. These results suggest that CysLT1 is likely involved in the recruitment of activated and T cells to inflamed tissues.
Introduction
The signal transduction cassettes operated by and TCR share numerous functional components. Among them, the adaptor molecule linker for activation of T cells (LAT)4 plays a crucial role in that it coordinates the assembly of signaling complexes through multiple tyrosine residues within its intracytoplasmic segment. Most of the signaling activity of LAT appears funneled through the four C-terminal tyrosine residues found at positions 136, 175, 195, and 235 (1). Upon TCR-induced phosphorylation, these tyrosines show some specificity in the cytoplasmic proteins they recruit. For instance, mutation of tyrosine (Y) 136 selectively eliminates binding of phospholipase C (PLC)-1 (2), whereas the simultaneous mutation of Y175, Y195, and Y235 results in loss of binding of the Grb2/Grap adaptor molecules (2, 3).
A knockin mutation, called LatY136F, where tyrosine 136 of LAT was replaced by a phenylalanine, caused a fatal lymphoproliferative disorder involving polyclonal CD4+ T cells that chronically produced type 2 cytokines such as IL-4, IL-5, and IL-13 (4, 5). A compound knockin mutation, called Lat3YF, where tyrosines 175, 195 and 235 were replaced by phenylalanine, resulted in the selective development and expansion of T cells, which spontaneously deployed a Th2-like effector program (6). The LatY136F CD4+ T cells and Lat3YF T cells had both a CD25–CD44highCD62LlowCD69+ phenotype closely resembling that of activated effector and memory T cells. Operationally they behave as effector T cells in that, upon in vitro stimulation with ionomycin, they have immediate effector functions as exemplified by their ability to produce copious amounts of IL-4, IL-5, and IL-13. Thus, a remarkable convergence exists in the functional phenotype induced in the - and T cell lineages by two distinct mutations of the LAT adaptor.
The molecular mechanisms underlying these mutant phenotypes are as yet unclear. Importantly, the pathology developed in these two mouse models encompasses hypergammaglobulinemia E and G1, massive lymphocytic infiltration of the lungs, and tissue eosinophilia and is thus reminiscent of allergic inflammation. Cross-linking of the TCR-CD3 complexes expressed at the surface of CD4+ T cells from LatY136F mice fails to induce detectable PLC-1 activation and Ca2+ mobilization (5). A similar signaling defect also exists in the T cells that expand in Lat3YF mice (6). Considering that the T cells that expand in LatY136F and Lat3Y mice were largely unresponsive to TCR stimulation, we aimed at identifying through transcriptional profiling whether LatY136F T cells and Lat3YF T cells share a pattern of gene expression that may account for their pathogenic properties.
Cysteinyl leukotrienes (CysLTs) are peptide-conjugated lipids that are produced primarily at sites of inflammation by activated eosinophils, basophils, dendritic cells, mast cells, and macrophages. CysLTs have been identified as potent inducers of bronchial smooth muscle constriction (7) but also display a multitude of additional functions (8, 9, 10). For instance, they are involved in the recruitment of myeloid leukocytes to inflamed tissues (10). Two receptors for CysLTs, termed the type 1 and type 2 CysLTRs (CysLT1 and CysLT2; Ref. 11), have been identified. CysLT1 is expressed on bone marrow-derived cells such as alveolar macrophages, eosinophils, and mast cells, and its expression can be up-regulated by Th2-type cytokines (12). Functional CysLT1 expression has not been reported previously in T cells. In this article, we show that both LatY136F T and Lat3YF T cells expressed particularly high levels of CysLT1. Upon binding to its 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD4) ligand, CysLT1 induced Ca2+ flux and consequential chemotaxis in both LatY136F CD4+ T cells and Lat3YF T cells.
Materials and Methods
Statistical analysis
All data are presented as mean ± SD. The software GraphPad Prism was used to analyze the results by the unpaired t test, one-tailed p values, and confidence interval 95%.
Results
Activated CD4+ T cells from LatY136F mice highly express the CysLT1R
Using oligonucleotide microarrays focusing on immunologically relevant genes and expressed sequence tags (14), we compared the gene expression profiles of CD4+ T cells sorted from LatY136F mice and from wild-type littermates (Table I). The entire microarray data were deposited in the public Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo/) under accession no. GSE2286. Among the probed genes, the most striking difference was found in the expression of CysLT1, indicating that there was substantially more CysLT1 mRNA in LatY136F CD4+ T cells than in wild-type CD4+ T cells. Real-time PCR analysis confirmed this finding and showed that CysLT1 transcripts were 200-fold up-regulated in purified LatY136F CD4+ T cells as compared with wild-type CD4+ T cells (Fig. 1A). Because two CysLT1 isoforms have been described previously (15), we systematically analyzed their respective expression patterns and found that both were highly expressed in LatY136F CD4+ T cells (Fig. 1B). At the same time, there were no significant differences in the expression level of CysLT2 in LatY136F CD4+ T cells compared with wild-type CD4+ T cells (Fig. 1C).
Functional responses of LatY136F T cells to CysLT1 signaling: T cell chemotaxis through CysLT1
We next aimed at determining the functional consequences of CysLT1 signaling in LatY136F T cells. Although binding of LTD4 to CysLT1 caused Ca2+ flux in LatY136F CD4+ T cells (Fig. 2), it failed to induce IL-4 production (Fig. 3A). In contrast, stimulation with a Ca2+ ionophore such as ionomycin was sufficient to induce IL-4 production (Fig. 3A). However, when compared with the ionomycin-induced Ca2+ signals, the ones induced by LTD4 were of lower magnitude and likely insufficient to turn on the production of IL-4 (compare Fig. 2, A and B).
The mechanisms controlling the recruitment of T cells to inflamed lungs constitute an important focus of research on the pathogenesis of asthma (19). A model of Th2 cell trafficking in asthma has been proposed in which the lipid mediators LTB4 and PGD2 direct the earliest phases of T cell recruitment to the airways, whereas chemokines direct the subsequent phases of T cell recruitment that amplify and maintain airway inflammation (19, 20, 21, 22, 23). Therefore, we determined whether CysLT1 mediated LatY136F CD4+ chemotaxis in response to LTD4. As shown in Fig. 3B, LTD4 constituted a potent chemoattractant for freshly isolated LatY136F CD4+ T cells. To control for the specificity of LTD4 effects on CysLT1, we systematically determined whether the specific CysLT1 antagonist MK571 could inhibit chemotaxis of LatY136F CD4+ T cells toward LTD4. The specificity of the pharmacological antagonist MK571 for CysLT1 has been established in previous studies (24), and MK571 is generally used to discriminate whether CysLT-induced effects are mediated trough CysLT1 or through other related receptors such as CysLT2 (24). Moreover, MK571 does not inhibit chemotactic responses to non-CysLT1 ligands, including CCL21 or CXCL12 (25). As expected, the compound MK571 blocked the migration of freshly isolated LatY136F CD4+ T cells toward LTD4 in a dose-dependent manner, demonstrating thus the specificity of this process (Fig. 3C). In these experiments, T cell migration was measured after 2 h, at which time 25% of the LatY136F CD4+ input T cells had migrated to 10–7 M LTD4.
Functional CysLT1 expression on T cells derived from Lat3YF mice
Considering that Lat3YF T cells displayed a Th2-like phenotype conspicuously similar to that of LatY136F CD4+ T cells (6), we also analyzed their gene expression profile using oligonucleotide microarrays (Table I). The entire microarray data were deposited in the public GEO database (www.ncbi.nlm.nih.gov/geo/), under accession no. GSE2287. Because T cells are the only T cells able to mature in Lat3YF mice, we compared their gene expression profile with that of wild-type T cells purified from TCR enhancer-deficient mice (E–/– mice) (13). These constitute a particularly appropriate control for T cells present in Lat3YF mice because they also matured in an environment deprived of T cells. Among the genes that were differentially expressed when Lat3YF T cells were compared with wild-type T cells, CysLT1 constituted one of the most differentially expressed genes. Real-time PCR analysis confirmed that transcripts of both CysLT1 isoforms were 50-fold more abundant in Lat3YF T cells as compared with wild-type T cells derived from E–/– mice (Fig. 4, A and B), while there were no differences in the expression level of CysLT2 (data not shown). Next, we determined whether CysLT1 also mediated Lat3YF T cell chemotaxis in response to LTD4. LTD4 constituted a potent chemoattractant for freshly isolated Lat3YF T cells but not for freshly isolated wild-type T cells (Fig. 4C and data not shown). To control for the specificity of LTD4 effects on CysLT1, we determined whether the specific CysLT1 antagonist MK571 could inhibit chemotaxis of Lat3YF T cells toward LTD4. Consistent with the results for the LatY136F CD4+ T cells, this compound blocked the migration of the Lat3YF effector T cells toward LTD4, demonstrating the specificity of this process. In these experiments, we measured T cell migration after 2 h, at which time 10% of the Lat3YF input T cells had migrated to 10–7 M LTD4. Therefore, the strong induction of CysLT1 mRNA and functional CysLT1 protein expression in LatY136F CD4+ T cells and Lat3YF T cells enabled both types of cells to migrate along an LTD4 gradient.
Functional CysLT1 expression on wild-type in vitro-activated T cells
To extend our results from the LatY136F and Lat3YF knockin mice to the wild-type situation, we next determined whether TCR-mediated T cell activation induced CysLT1 expression and thereby conferred the ability to respond chemotactically to CysLTs. Wild-type CD4+ T cells were activated in vitro under Th1- or Th2-polarizing conditions. As shown in Fig. 6A, real-time PCR analysis of CysLT1 expression revealed an 10-fold higher expression in both Th1- and Th2-polarized cells as compared with unstimulated CD4+ T cells. The expression of the short isoform of CysLT1 was also induced (Fig. 6B). The reason why ex vivo LatY136F CD4+ T cells expressed levels of CysLT1 20 times higher than those reached by in vitro-activated wild-type CD4+ T cells remains to be determined. However, CysLT1 expression on T cells after stimulation through the TCR and CD28 was sufficient to effect their migration toward LTD4, with a lower intensity than the effector T cells isolated from LatY136F and Lat3YF mice (Fig. 6, C and D). CD4+ and CD8+ T cells showed no significant differences in their chemotactic response to LTD4. Consistent with the results for the T cells of LatY136F and Lat3YF knockin mice, the specific CysLT1 inhibitor MK571 blocked the migration of the activated T cells toward LTD4, demonstrating the specificity of this process. In these experiments, 5% of the input T cells had migrated to 10–7 M LTD4. In contrast, wild-type CD4+ T cells freshly isolated from spleen failed to migrate to LTD4 (Fig. 6C). In conclusion, T cells that have differentiated in vitro to effector phenotypes had increased levels of CysLT1 encoding mRNA as compared with naive T cells, which expressed little or no CysLT1 mRNA. This CysLT1 expression correlated with specific chemotactic responses to the CysLT LTD4.
Discussion
In this study, we took advantage of two knockin models with mutations affecting tyrosine residues of the LAT adaptor where almost all peripheral T cells (4) or T cells (6) adopt a Th2 effector phenotype. We showed that CysLT1 transcripts were highly expressed on these and mouse effector T cells. Moreover, binding of CysLT1 to its physiological ligand LTD4 resulted in Ca2+ mobilization and in T cell chemotaxis. CysLT1 was also expressed on in vitro-activated effector T cells from wild-type mice, suggesting a role for CysLT1 in normal T cell physiology. To our knowledge, this constitutes the first report that CysLT1 is expressed functionally on mouse-activated T cells. In this context, a recent study using knockin mice expressing a GFP-Foxp3 fusion protein established that the transcription factor Foxp3 constitutes a genuine marker for T regulatory cells (27). In that process, they also identified a population of CD4+CD25+Foxp3– T cells that represents in vivo-activated/effector T cells. Because the analyzed mice were not submitted to intended immunization, these CD4+CD25+Foxp3– T cells were likely the result of responses to environmental microbes. Importantly, transcriptional profiling experiments showed that CysLT1 expression is up-regulated in the activated/effector CD4+CD25+Foxp3– T cells as compared with the naive CD4+CD25–Foxp3– T cells and to the CD4+CD25+Foxp3+ T regulatory cells. The presence of CysLT1 on wild-type T cells activated in vivo is further supported by a recent transcriptional profiling study of human T cells where CysLT1 was listed, without additional comment, in a compilation of genes that were up-regulated on various human effector T cell subsets (28). Importantly, our results suggest that targeting the CysLT1-operated signaling pathway through selective antagonists, which are effective drugs in the treatment of human asthma, not only affect myeloid leukocytes but may also have a profound effect on adaptive immune cells. Our finding included CD4+ and CD8+, Th1, Th2, and unpolarized effector T cells and comprised both and T cells, implying a general mechanism that leads to the expression of CysLT1 on effector T cells. Several reports that have examined the regulation of CysLT1 expression in mouse and human suggested that cytokines, including IL-4, IL-5, IL-13, and TGF-, have an inducing role on CysLT1 expression on myeloid leukocytes and bronchial smooth muscle cells (29). Considering that expression of IL-4, IL-5, and IL-13 is increased dramatically in LatY136F and Lat3YF T cells, it is likely that such cytokines also contribute to the induction of high expression levels of CysLT1 observed in these T cells.
What is the functional relevance of CysLT1 expression on effector T cells? CysLT1 can initiate signaling through the Gq subunit, thereby activating PLC- isoforms and inducing an increase in inositol phosphate and diacylglycerol (16, 17). These secondary messengers may synergize with those triggered by TCR engagement and thus lower the threshold for Ag-specific T cell activation at sites of inflammation. Despite the Ca2+ flux and chemotaxis that followed LTD4 binding to the CysLT1R expressed on LatY136F CD4+ T cells, no IL-4 production was triggered. Furthermore, when the TCR present on the surface of CD4+ T cells from LatY136F was cross-linked in the presence of LTD4, no IL-4 production ensued. This contrasts with findings in human mast cells, where LTD4 treatment induces de novo expression of proinflammatory cytokines (30), and in transfected Jurkat T cells, where the G protein-coupled muscarinic subtype 1 receptor is capable of inducing IL-2 production (31). Thus, CysLT1 expressed on T cells is likely important for the migration of effector T cells to sites of CysLT production, i.e., sites of inflammation. However, CysLT1 signals alone are not sufficient to induce a "surrogate signal 1," resulting in cytokine production.
At this point, it is difficult to decide whether CysLT1 expression is the chicken or the egg in the development of the allergic inflammation that occurs in LatY136F and Lat3YF mice. Mice lacking CysLT1 have been derived (32). However, considering the many cell types expressing CysLT1, it would be difficult to define individual roles for each in the pathogenesis of allergic inflammation using these knockouts. In contrast, use of the Cre-loxP site-specific recombinase system should permit T cell-specific ablation of CysLT1 and answer how CysLTs contribute to control T cell trafficking to inflamed organs.
Acknowledgments
We thank M. Kursar, H.-W. Mittrücker, C. A. Stewart, A. Magnan, E. Mamessier, Pierre Grenot, and Anne Gillet for discussion and Pierre Ferrier for donating E-deficient mice.
Disclosures
The authors have no financial conflict of interest.
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 has been supported by institutional grants from Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique and a specific grant from the European Community (MUGEN Network of Excellence). I.P. was supported by a fellowship from Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche and by an EIF Marie Curie fellowship from the European Community.
2 Current address: Department of Biochemistry, Molecular Biology B and Immunology, University of Murcia, Murcia, Spain.
3 Address correspondence and reprint requests to Dr. Bernard Malissen, Centre d’Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille, Cedex 9, France. E-mail address: bernardm@ciml.univ-mrs.fr
4 Abbreviations used in this paper: LAT, linker for activation of T cells; PLC, phospholipase C; CysLT, cysteinyl leukotriene; LTD4, 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid; PG IP, prostaglandin receptor IP1; CTP, cytidine 5'-triphosphate; GEO, Gene Expression Omnibus.
Received for publication December 10, 2004. Accepted for publication April 29, 2005.
References
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Lin, J., A. Weiss. 2001. Identification of the minimal tyrosine residues required for linker for activation of T cell function. J. Biol. Chem. 276: 29588-29595.
Zhang, W., R. P. Trible, M. Zhu, S. K. Liu, C. J. McGlade, L. E. Samelson. 2000. Association of Grb2, Gads, and phospholipase C-1 with phosphorylated LAT tyrosine residues: effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling. J. Biol. Chem. 275: 23355-23361.
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Sommers, C. L., C. S. Park, J. Lee, C. Feng, C. L. Fuller, A. Grinberg, J. A. Hildebrand, E. Lacana, R. K. Menon, E. W. Shores, L. E. Samelson, P. E. Love. 2002. A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 296: 2040-2043.(Immo Prinz*, Claude Grego)
Linker for activation of T cells (LAT) is essential for T cell activation. Mice with mutations of distinct LAT tyrosine residues (LatY136F and Lat3YF) develop lymphoproliferative disorders involving TCR or T cells that trigger symptoms resembling allergic inflammation. We analyzed whether these T cells share a pattern of gene expression that may account for their pathogenic properties. Both LatY136F and Lat3YF T cells expressed high levels of the type 1 cysteinyl leukotriene receptor (CysLT1). Upon binding to the 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD4) cysteinyl leukotriene, CysLT1 induced Ca2+ flux and caused chemotaxis in both LatY136F and Lat3YF T cells. Wild-type in vitro-activated T cells, but not resting T cells, also migrated toward LTD4 however with a lower magnitude than T cells freshly isolated from LatY136F and Lat3YF mice. These results suggest that CysLT1 is likely involved in the recruitment of activated and T cells to inflamed tissues.
Introduction
The signal transduction cassettes operated by and TCR share numerous functional components. Among them, the adaptor molecule linker for activation of T cells (LAT)4 plays a crucial role in that it coordinates the assembly of signaling complexes through multiple tyrosine residues within its intracytoplasmic segment. Most of the signaling activity of LAT appears funneled through the four C-terminal tyrosine residues found at positions 136, 175, 195, and 235 (1). Upon TCR-induced phosphorylation, these tyrosines show some specificity in the cytoplasmic proteins they recruit. For instance, mutation of tyrosine (Y) 136 selectively eliminates binding of phospholipase C (PLC)-1 (2), whereas the simultaneous mutation of Y175, Y195, and Y235 results in loss of binding of the Grb2/Grap adaptor molecules (2, 3).
A knockin mutation, called LatY136F, where tyrosine 136 of LAT was replaced by a phenylalanine, caused a fatal lymphoproliferative disorder involving polyclonal CD4+ T cells that chronically produced type 2 cytokines such as IL-4, IL-5, and IL-13 (4, 5). A compound knockin mutation, called Lat3YF, where tyrosines 175, 195 and 235 were replaced by phenylalanine, resulted in the selective development and expansion of T cells, which spontaneously deployed a Th2-like effector program (6). The LatY136F CD4+ T cells and Lat3YF T cells had both a CD25–CD44highCD62LlowCD69+ phenotype closely resembling that of activated effector and memory T cells. Operationally they behave as effector T cells in that, upon in vitro stimulation with ionomycin, they have immediate effector functions as exemplified by their ability to produce copious amounts of IL-4, IL-5, and IL-13. Thus, a remarkable convergence exists in the functional phenotype induced in the - and T cell lineages by two distinct mutations of the LAT adaptor.
The molecular mechanisms underlying these mutant phenotypes are as yet unclear. Importantly, the pathology developed in these two mouse models encompasses hypergammaglobulinemia E and G1, massive lymphocytic infiltration of the lungs, and tissue eosinophilia and is thus reminiscent of allergic inflammation. Cross-linking of the TCR-CD3 complexes expressed at the surface of CD4+ T cells from LatY136F mice fails to induce detectable PLC-1 activation and Ca2+ mobilization (5). A similar signaling defect also exists in the T cells that expand in Lat3YF mice (6). Considering that the T cells that expand in LatY136F and Lat3Y mice were largely unresponsive to TCR stimulation, we aimed at identifying through transcriptional profiling whether LatY136F T cells and Lat3YF T cells share a pattern of gene expression that may account for their pathogenic properties.
Cysteinyl leukotrienes (CysLTs) are peptide-conjugated lipids that are produced primarily at sites of inflammation by activated eosinophils, basophils, dendritic cells, mast cells, and macrophages. CysLTs have been identified as potent inducers of bronchial smooth muscle constriction (7) but also display a multitude of additional functions (8, 9, 10). For instance, they are involved in the recruitment of myeloid leukocytes to inflamed tissues (10). Two receptors for CysLTs, termed the type 1 and type 2 CysLTRs (CysLT1 and CysLT2; Ref. 11), have been identified. CysLT1 is expressed on bone marrow-derived cells such as alveolar macrophages, eosinophils, and mast cells, and its expression can be up-regulated by Th2-type cytokines (12). Functional CysLT1 expression has not been reported previously in T cells. In this article, we show that both LatY136F T and Lat3YF T cells expressed particularly high levels of CysLT1. Upon binding to its 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD4) ligand, CysLT1 induced Ca2+ flux and consequential chemotaxis in both LatY136F CD4+ T cells and Lat3YF T cells.
Materials and Methods
Statistical analysis
All data are presented as mean ± SD. The software GraphPad Prism was used to analyze the results by the unpaired t test, one-tailed p values, and confidence interval 95%.
Results
Activated CD4+ T cells from LatY136F mice highly express the CysLT1R
Using oligonucleotide microarrays focusing on immunologically relevant genes and expressed sequence tags (14), we compared the gene expression profiles of CD4+ T cells sorted from LatY136F mice and from wild-type littermates (Table I). The entire microarray data were deposited in the public Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo/) under accession no. GSE2286. Among the probed genes, the most striking difference was found in the expression of CysLT1, indicating that there was substantially more CysLT1 mRNA in LatY136F CD4+ T cells than in wild-type CD4+ T cells. Real-time PCR analysis confirmed this finding and showed that CysLT1 transcripts were 200-fold up-regulated in purified LatY136F CD4+ T cells as compared with wild-type CD4+ T cells (Fig. 1A). Because two CysLT1 isoforms have been described previously (15), we systematically analyzed their respective expression patterns and found that both were highly expressed in LatY136F CD4+ T cells (Fig. 1B). At the same time, there were no significant differences in the expression level of CysLT2 in LatY136F CD4+ T cells compared with wild-type CD4+ T cells (Fig. 1C).
Functional responses of LatY136F T cells to CysLT1 signaling: T cell chemotaxis through CysLT1
We next aimed at determining the functional consequences of CysLT1 signaling in LatY136F T cells. Although binding of LTD4 to CysLT1 caused Ca2+ flux in LatY136F CD4+ T cells (Fig. 2), it failed to induce IL-4 production (Fig. 3A). In contrast, stimulation with a Ca2+ ionophore such as ionomycin was sufficient to induce IL-4 production (Fig. 3A). However, when compared with the ionomycin-induced Ca2+ signals, the ones induced by LTD4 were of lower magnitude and likely insufficient to turn on the production of IL-4 (compare Fig. 2, A and B).
The mechanisms controlling the recruitment of T cells to inflamed lungs constitute an important focus of research on the pathogenesis of asthma (19). A model of Th2 cell trafficking in asthma has been proposed in which the lipid mediators LTB4 and PGD2 direct the earliest phases of T cell recruitment to the airways, whereas chemokines direct the subsequent phases of T cell recruitment that amplify and maintain airway inflammation (19, 20, 21, 22, 23). Therefore, we determined whether CysLT1 mediated LatY136F CD4+ chemotaxis in response to LTD4. As shown in Fig. 3B, LTD4 constituted a potent chemoattractant for freshly isolated LatY136F CD4+ T cells. To control for the specificity of LTD4 effects on CysLT1, we systematically determined whether the specific CysLT1 antagonist MK571 could inhibit chemotaxis of LatY136F CD4+ T cells toward LTD4. The specificity of the pharmacological antagonist MK571 for CysLT1 has been established in previous studies (24), and MK571 is generally used to discriminate whether CysLT-induced effects are mediated trough CysLT1 or through other related receptors such as CysLT2 (24). Moreover, MK571 does not inhibit chemotactic responses to non-CysLT1 ligands, including CCL21 or CXCL12 (25). As expected, the compound MK571 blocked the migration of freshly isolated LatY136F CD4+ T cells toward LTD4 in a dose-dependent manner, demonstrating thus the specificity of this process (Fig. 3C). In these experiments, T cell migration was measured after 2 h, at which time 25% of the LatY136F CD4+ input T cells had migrated to 10–7 M LTD4.
Functional CysLT1 expression on T cells derived from Lat3YF mice
Considering that Lat3YF T cells displayed a Th2-like phenotype conspicuously similar to that of LatY136F CD4+ T cells (6), we also analyzed their gene expression profile using oligonucleotide microarrays (Table I). The entire microarray data were deposited in the public GEO database (www.ncbi.nlm.nih.gov/geo/), under accession no. GSE2287. Because T cells are the only T cells able to mature in Lat3YF mice, we compared their gene expression profile with that of wild-type T cells purified from TCR enhancer-deficient mice (E–/– mice) (13). These constitute a particularly appropriate control for T cells present in Lat3YF mice because they also matured in an environment deprived of T cells. Among the genes that were differentially expressed when Lat3YF T cells were compared with wild-type T cells, CysLT1 constituted one of the most differentially expressed genes. Real-time PCR analysis confirmed that transcripts of both CysLT1 isoforms were 50-fold more abundant in Lat3YF T cells as compared with wild-type T cells derived from E–/– mice (Fig. 4, A and B), while there were no differences in the expression level of CysLT2 (data not shown). Next, we determined whether CysLT1 also mediated Lat3YF T cell chemotaxis in response to LTD4. LTD4 constituted a potent chemoattractant for freshly isolated Lat3YF T cells but not for freshly isolated wild-type T cells (Fig. 4C and data not shown). To control for the specificity of LTD4 effects on CysLT1, we determined whether the specific CysLT1 antagonist MK571 could inhibit chemotaxis of Lat3YF T cells toward LTD4. Consistent with the results for the LatY136F CD4+ T cells, this compound blocked the migration of the Lat3YF effector T cells toward LTD4, demonstrating the specificity of this process. In these experiments, we measured T cell migration after 2 h, at which time 10% of the Lat3YF input T cells had migrated to 10–7 M LTD4. Therefore, the strong induction of CysLT1 mRNA and functional CysLT1 protein expression in LatY136F CD4+ T cells and Lat3YF T cells enabled both types of cells to migrate along an LTD4 gradient.
Functional CysLT1 expression on wild-type in vitro-activated T cells
To extend our results from the LatY136F and Lat3YF knockin mice to the wild-type situation, we next determined whether TCR-mediated T cell activation induced CysLT1 expression and thereby conferred the ability to respond chemotactically to CysLTs. Wild-type CD4+ T cells were activated in vitro under Th1- or Th2-polarizing conditions. As shown in Fig. 6A, real-time PCR analysis of CysLT1 expression revealed an 10-fold higher expression in both Th1- and Th2-polarized cells as compared with unstimulated CD4+ T cells. The expression of the short isoform of CysLT1 was also induced (Fig. 6B). The reason why ex vivo LatY136F CD4+ T cells expressed levels of CysLT1 20 times higher than those reached by in vitro-activated wild-type CD4+ T cells remains to be determined. However, CysLT1 expression on T cells after stimulation through the TCR and CD28 was sufficient to effect their migration toward LTD4, with a lower intensity than the effector T cells isolated from LatY136F and Lat3YF mice (Fig. 6, C and D). CD4+ and CD8+ T cells showed no significant differences in their chemotactic response to LTD4. Consistent with the results for the T cells of LatY136F and Lat3YF knockin mice, the specific CysLT1 inhibitor MK571 blocked the migration of the activated T cells toward LTD4, demonstrating the specificity of this process. In these experiments, 5% of the input T cells had migrated to 10–7 M LTD4. In contrast, wild-type CD4+ T cells freshly isolated from spleen failed to migrate to LTD4 (Fig. 6C). In conclusion, T cells that have differentiated in vitro to effector phenotypes had increased levels of CysLT1 encoding mRNA as compared with naive T cells, which expressed little or no CysLT1 mRNA. This CysLT1 expression correlated with specific chemotactic responses to the CysLT LTD4.
Discussion
In this study, we took advantage of two knockin models with mutations affecting tyrosine residues of the LAT adaptor where almost all peripheral T cells (4) or T cells (6) adopt a Th2 effector phenotype. We showed that CysLT1 transcripts were highly expressed on these and mouse effector T cells. Moreover, binding of CysLT1 to its physiological ligand LTD4 resulted in Ca2+ mobilization and in T cell chemotaxis. CysLT1 was also expressed on in vitro-activated effector T cells from wild-type mice, suggesting a role for CysLT1 in normal T cell physiology. To our knowledge, this constitutes the first report that CysLT1 is expressed functionally on mouse-activated T cells. In this context, a recent study using knockin mice expressing a GFP-Foxp3 fusion protein established that the transcription factor Foxp3 constitutes a genuine marker for T regulatory cells (27). In that process, they also identified a population of CD4+CD25+Foxp3– T cells that represents in vivo-activated/effector T cells. Because the analyzed mice were not submitted to intended immunization, these CD4+CD25+Foxp3– T cells were likely the result of responses to environmental microbes. Importantly, transcriptional profiling experiments showed that CysLT1 expression is up-regulated in the activated/effector CD4+CD25+Foxp3– T cells as compared with the naive CD4+CD25–Foxp3– T cells and to the CD4+CD25+Foxp3+ T regulatory cells. The presence of CysLT1 on wild-type T cells activated in vivo is further supported by a recent transcriptional profiling study of human T cells where CysLT1 was listed, without additional comment, in a compilation of genes that were up-regulated on various human effector T cell subsets (28). Importantly, our results suggest that targeting the CysLT1-operated signaling pathway through selective antagonists, which are effective drugs in the treatment of human asthma, not only affect myeloid leukocytes but may also have a profound effect on adaptive immune cells. Our finding included CD4+ and CD8+, Th1, Th2, and unpolarized effector T cells and comprised both and T cells, implying a general mechanism that leads to the expression of CysLT1 on effector T cells. Several reports that have examined the regulation of CysLT1 expression in mouse and human suggested that cytokines, including IL-4, IL-5, IL-13, and TGF-, have an inducing role on CysLT1 expression on myeloid leukocytes and bronchial smooth muscle cells (29). Considering that expression of IL-4, IL-5, and IL-13 is increased dramatically in LatY136F and Lat3YF T cells, it is likely that such cytokines also contribute to the induction of high expression levels of CysLT1 observed in these T cells.
What is the functional relevance of CysLT1 expression on effector T cells? CysLT1 can initiate signaling through the Gq subunit, thereby activating PLC- isoforms and inducing an increase in inositol phosphate and diacylglycerol (16, 17). These secondary messengers may synergize with those triggered by TCR engagement and thus lower the threshold for Ag-specific T cell activation at sites of inflammation. Despite the Ca2+ flux and chemotaxis that followed LTD4 binding to the CysLT1R expressed on LatY136F CD4+ T cells, no IL-4 production was triggered. Furthermore, when the TCR present on the surface of CD4+ T cells from LatY136F was cross-linked in the presence of LTD4, no IL-4 production ensued. This contrasts with findings in human mast cells, where LTD4 treatment induces de novo expression of proinflammatory cytokines (30), and in transfected Jurkat T cells, where the G protein-coupled muscarinic subtype 1 receptor is capable of inducing IL-2 production (31). Thus, CysLT1 expressed on T cells is likely important for the migration of effector T cells to sites of CysLT production, i.e., sites of inflammation. However, CysLT1 signals alone are not sufficient to induce a "surrogate signal 1," resulting in cytokine production.
At this point, it is difficult to decide whether CysLT1 expression is the chicken or the egg in the development of the allergic inflammation that occurs in LatY136F and Lat3YF mice. Mice lacking CysLT1 have been derived (32). However, considering the many cell types expressing CysLT1, it would be difficult to define individual roles for each in the pathogenesis of allergic inflammation using these knockouts. In contrast, use of the Cre-loxP site-specific recombinase system should permit T cell-specific ablation of CysLT1 and answer how CysLTs contribute to control T cell trafficking to inflamed organs.
Acknowledgments
We thank M. Kursar, H.-W. Mittrücker, C. A. Stewart, A. Magnan, E. Mamessier, Pierre Grenot, and Anne Gillet for discussion and Pierre Ferrier for donating E-deficient mice.
Disclosures
The authors have no financial conflict of interest.
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 has been supported by institutional grants from Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique and a specific grant from the European Community (MUGEN Network of Excellence). I.P. was supported by a fellowship from Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche and by an EIF Marie Curie fellowship from the European Community.
2 Current address: Department of Biochemistry, Molecular Biology B and Immunology, University of Murcia, Murcia, Spain.
3 Address correspondence and reprint requests to Dr. Bernard Malissen, Centre d’Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille, Cedex 9, France. E-mail address: bernardm@ciml.univ-mrs.fr
4 Abbreviations used in this paper: LAT, linker for activation of T cells; PLC, phospholipase C; CysLT, cysteinyl leukotriene; LTD4, 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid; PG IP, prostaglandin receptor IP1; CTP, cytidine 5'-triphosphate; GEO, Gene Expression Omnibus.
Received for publication December 10, 2004. Accepted for publication April 29, 2005.
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