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The Type 1 Cysteinyl Leukotriene Receptor Triggers Calcium Influx and Chemotaxis in Mouse - and Effector T Cells¹

Immo Prinz^*, Claude Gregoire^*, Hans Mollenkopf, Enrique Aguado^2,*, Ying Wang^*, Marie Malissen^*, Stefan H.E. Kaufmann and Bernard Malissen^3,*

^* 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, Marseille, France; and Microarray Core Facility, and Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany

	Abstract

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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

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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^–CD44^highCD62L^lowCD69⁺ 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

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Mice

LatY136F (4), Lat3YF (6), and E^–/– (13) mice were maintained in the specific pathogen-free animal facility of Centre d’Immunologie de Marseille-Luminy. All experiments were performed in accordance with protocols approved by French law and European directives.

Real-time quantitative PCR gene expression analysis

CD4⁺ T cells were positively sorted from LatY136F or from wild-type spleen using MACS (Miltenyi Biotec) and anti-CD4 beads. T cells were sorted from the spleen of Lat3YF and of E^–/– mice using MACS and anti-CD5 beads (6). In vitro-differentiated Th1 and Th2 cells were generated by culturing naive wild-type CD4⁺ T cells for 4 days in complete RPMI 1640 medium in the presence of anti-CD3 mAb (1 µg/ml; 2C11) plus anti-CD28 mAb (1 µg/ml; H37.51) with the addition of IL-12 (10 ng/ml) and of anti-IL-4 mAb (5 µg/ml) for Th1-polarizing conditions, and of IL-4 (20 ng/ml) plus anti-IFN- mAb (10 µg/ml) for Th2-polarizing conditions. RNA was prepared using the TRIzol method (Invitrogen Life Technologies) followed by DNase digestion. cDNA was prepared using the Superscript II system (Invitrogen Life Technologies) according to the manufacturer’s instructions. Quantitative real-time PCRs were performed in duplicate using the Abi Prism 5700 Sequence Detection System (Applied Biosystems) and the QuantiTect SYBR Green PCR Master Mix (Qiagen). The same cDNA served as template for the genes of interest and, as an internal control, for -actin. A nontemplate control was also routinely performed in duplicate for each primer pair. Amplification curves were analyzed using software provided with the Abi Prism 5700 Sequence Detection System. A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified, and PCR products were additionally tested on an agarose gel. The relative expression levels of the genes of interest were calculated as fold difference = 2^(–Ct), where Ct is threshold cycle normalized to -actin internal control. The primers used were as follows: -actin forward, 5'-TGGAATCCTGTGGCATCCATGAAAC-3', and -actin reverse, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'; CysLT₁ long isoform forward, 5'-TTAAATTCACCATCTTCCTGCTTTGG-3', CysLT₁ short isoform forward, 5'-GGAGACTAGCAGGTTGGAATTTCTGA-3', and CysLT₁ long + short isoforms reverse, 5'-GAGATGTCGTCAGATTTTCAGTTCCA-3'; CysLT₂ forward, 5'-TGCTTTTGGAAGAGAGAAGAGTCCA-3', and CysLT₂ reverse, 5'-AATGATACACATCCTTCCCCAGGA-3'; prostagladin receptor IP1 (PG IP) forward, 5'-GGATGAAGTTTACCACCTGATTCTGC-3', and PG IP reverse, 5'-AGCCTTTCGGAAAAGGATGAAGAC-3'.

Microarray gene expression analysis

Microarray experiments were based on two-color ratio hybridizations and a Fluorescent Linear Amplification kit (Agilent) for RNA labeling. In brief, cDNA was reverse transcribed from 4 µg of total RNA with an oligo(dT)-T7 promoter primer and Moloney murine leukemia virus-reverse transcriptase. Second-strand synthesis was conducted with random hexamers. Fluorescent antisense cRNA was synthesized with T7 polymerase, using either cyanine 3-cytidine 5'-triphosphate (CTP) or cyanine 5-CTP. The fluorescent-labeled antisense cRNA was precipitated overnight with LiCl, washed with ethanol, and resuspended in water. The purified products were quantified by absorbance at A_{552 nm} for cyanine 3-CTP and A_{650 nm} for cyanine 5-CTP, and labeling efficiency was verified with a Nanodrop photometer (Kisker). Before hybridization, 1.25 µg of each labeled cRNA product were fragmented and mixed with control targets and hybridization buffer according to the Supplier’s protocol (Agilent). Hybridizations were done overnight for 17 h at 60°C. The slides were washed according to the manufacturer’s manual, and scanning of microarrays was performed with 5-µm resolution using a DNA microarray laser scanner (Agilent). To compensate for dye specific effects and to ensure statistically relevant data, a color swap dye reversal was performed. Features were extracted with an image analysis tool version A 6.1.1 (Agilent) using default settings. Data analysis was conducted on the Rosetta Inpharmatics Platform Resolver Built 4.0. Expression patterns were identified by stringent data analysis using anticorrelation of the dye reversal ratio profiles and a 2-fold expression cutoff. By combining the first and the second criteria of analysis, we filtered out data points with a low p value (p < 0.01), making the analysis robust and reproducible. By using this strategy, data selection was independent of error models implemented in the Rosetta Resolver system.

Intracellular Ca²⁺ mobilization

Lymph node single-cell suspensions were loaded for 60 min at 37°C with 5 µM Indo-1 (Molecular Probes), resuspended at 2 x 10⁷ cells/ml, and stained with PE-labeled anti-CD4 mAb (clone L3T4 RM4-5; BD Pharmingen). The baseline 500- to 400-nm fluorescence ratio was established using a LSR cytometer (BD Biosciences). Effector T cells consistently showed elevated baseline Ca²⁺ levels compared with freshly isolated wild-type T cells. The addition of anti-CD3 mAb (10 µg/ml final) plus streptavidin (100 ng/ml final) was used to initiate TCR ligation. The CysLT₁ agonist LTD₄ (Cayman Chemical) was added at final concentrations ranging from 10^–6 to 10^–11 M. Subsequent Ca²⁺ fluxes were recorded in gated CD4⁺ T cells. Where indicated, samples were preincubated for 1 min with the CysLT₁ antagonist MK571 (Cayman Chemical) at a final concentration of 10^–5 M. To exclude artifacts, controls were performed with the addition of 10-fold higher concentrations of the organic solvents (ethanol and DMSO) used to vehicle LTD₄ and MK571. Ionomycin was used as a positive control at 1 µg/ml final concentration.

ELISA

Single-cell suspensions from spleens were cultured for 48 h in 96-well plates (2 x 10⁵ spleen cells/well) in complete RPMI 1640 medium. The cells were stimulated with either 5 µg/ml anti-CD3 (clone 145 2C11) plus 2 µg/ml anti-CD28 (clone H37.51), with LTD₄ (10^–6 to 10^–7 M) or with 50 µM ionomycin ± 10 ng/ml PMA. After 2 days, supernatants were collected and analyzed for IL-4 using an OptEIA mouse ELISA kit (BD Biosciences), according to the manufacturer’s instructions. Plates were developed by the addition of 100 µl of 0.3 mg/ml ABTS substrate (Sigma-Aldrich) dissolved in 0.05 M acidic citrate buffer adjusted to pH 4 and containing 0.03% H₂O₂.

Chemotaxis assay

Migration assays were performed in 24-well Transwell plates (Corning Costar) with 5-µm pore polycarbonate filters. Spleen cells stimulated for 4 days with anti-CD3 mAb (1 µg/ml; 2C11), anti-CD28 mAb (1 µg/ml; H37.51), and IL-2 or freshly prepared single-cell suspensions from LatY136F, Lat3YF, or wild-type lymph nodes were resuspended at 2 x 10⁶ cells/ml in RPMI 1640 medium prewarmed at 37°C and containing 0.5% BSA. Cell suspensions (100 µl) were placed in the upper chamber and 600 µl of medium with or without LTD₄ (10^–7 M) in the lower chamber. For specific inhibition, the cells were preincubated for 3 min with dilutions of MK571 ranging from 10^–5 to 10^–8 M, and the same concentration of MK571 was added to the corresponding lower chambers. In all experiments, all media contained equal concentrations of 0.1% ethanol and 0.1% DMSO to exclude artifacts resulting from the organic solvent of LTD₄ and MK571, respectively. After incubation for 2 h at 37°C, the upper chamber was removed, and the cells in the lower chamber resuspended and transferred to tubes. After centrifugation, cells were collected and stained with anti-CD4 and anti-CD8 and analyzed by flow cytometry. The migration index was calculated as the fold difference in the numbers of specified T cells that migrated toward LTD₄ or medium alone.

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

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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).

View this table: [in this window] [in a new window]   Table I. RNA sequences that are highly differentially expressed in LATY136F CD4+ compared with wild-type CD4+ T cells (fold change A) and in LAT3YF compared with TcrE+/– (fold change B)

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Real-time PCR analysis reveals high expression of CysLT1 but not of CysLT2 in LatY136F CD4+ T cells. Bar diagrams show the relative expression of full-length CysLT1 (long isoform) (A), of the short isoform of CysLT1 (B), and of CysLT2 (C) in ex vivo-purified CD4+ T cells derived from LatY136F mice compared with ex vivo-purified wild-type CD4+ T cells. Values are given as fold difference of expression (mean + SD) normalized to -actin. Results are representative of at least three independent experiments.

We next assessed whether CysLT₁ mRNA gave rise to functional proteins via monitoring the intracellular Ca²⁺ mobilization through CysLT₁. The mutation of LAT tyrosine 136 in LatY136F mice inhibits PLC-1 binding and activation (4, 5), which is illustrated in Fig. 2A where cross-linking of the TCR present on the surface of CD4⁺ T cells isolated from LatY136F mice failed to stimulate Ca²⁺ influx. In contrast, CysLT₁ can initiate signaling through the G_q subunit and thereby mediates PLC-1 activation and calcium mobilization (16, 17). Therefore, we determined whether LatY136F CD4⁺ T cells were capable of Ca²⁺ mobilization in response to LTD₄, the physiological ligand of CysLT₁. Consistent with the pattern of CysLT₁ mRNA expression (Fig. 1), LTD₄ induced a robust Ca²⁺ signal in CD4⁺ T cells derived from LatY136F mice but not in resting, wild-type CD4⁺ T cells (Fig. 2B). The Ca²⁺ mobilization observed in LatY136F CD4⁺ T cells was dose dependent and was inhibited by MK571 (Fig. 2C), a specific pharmacological inhibitor of CysLT₁ (18). In conclusion, the effector CD4⁺ T cells found in LatY136F mice that expressed high levels of CysLT₁ mobilized intracellular Ca²⁺ in response to the CysLT₁ ligand LTD₄.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. CD4+ T cells from LatY136F mice are unresponsive to TCR cross-linking but respond to LTD4 stimulation. A, Intracellular Ca2+ mobilization in CD4+-gated lymphocytes from LatY136F mice (solid line) or wild-type littermate controls (dotted line) after TCR cross-linking mice was assessed by FACS analysis. Arrows indicate the time points at which anti-CD3 mAb, streptavidin, or ionomycin were added. B, Kinetics of intracellular Ca2+ mobilization on CD4+-gated lymphocytes isolated from wild-type (upper panel) or LatY136F (lower panel) in response to addition of LTD4 at a final concentration of 10–6 M. C, Ca2+ responses in LatY136F CD4+ T cells are dose dependent and are blocked by MK571, a specific pharmacological inhibitor for CysLT1. Left panel, Intracellular Ca2+ mobilization of CD4+-gated LatY136F lymphocytes in response to LTD4 at final concentrations ranging from 10–6 to 10 –1 M was assessed by FACS. Right panel, Response to LTD4 at final concentrations ranging from 10–6 to 10–8 M following preincubation of the cells with 10–5 M of the CysLT1-specific inhibitor MK571. Representative results of two independent experiments are shown.

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).

View larger version (20K): [in this window] [in a new window]   FIGURE 3. LTD4 stimulation of LatY136F lymphocytes induces chemotaxis of CysLT1-expressing effector T cells but is not sufficient to induce IL-4 production. A, IL-4 production of spleen cell suspensions derived from LatY136F mice () or wild-type littermate controls (). At 48 h after stimulation with the indicated reagent, IL-4 concentrations were determined by ELISA and plotted on a logarithmic scale. *, IL-4 levels below detection limit. Error bars represent SD calculated from triplicates. Representative results of at least three independent experiments are shown. B, Single-cell suspensions from lymph nodes of LatY136F mice were added to the upper wells of a migration chamber with or without LTD4 (10–7 M) in the lower wells. C, All the lower wells contained 10–7 M of LTD4, and the specified concentrations of the CysLT1-specific inhibitor MK571 were added to both the upper and lower wells. B and C, After 2 h, the numbers of CD4+ T cells that reached the lower wells were measured by flow cytometry. A migration index was calculated by dividing the number of cells that migrated in the LTD4-containing wells by the number of cells that migrated to medium alone. Data are given as mean of quadruplicates ± SD. One of three representative experiments is shown.

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.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. High levels of CysLT1 expression allow chemotactic responses of Lat3YF T cells to LTD4. Bar diagrams show the relative expression of full length CysLT1 (long isoform) (A) and the short isoform of CysLT1 (B) in ex vivo-purified T cells derived from Lat3YF mice compared with ex vivo-purified wild-type T cells derived from E–[supi]/– mice. Values are given as fold difference of expression (mean + SD) normalized to -actin. Results are representative of at least three independent experiments. C, Single-cell suspensions from lymph nodes of Lat3YF mice were added to the upper wells of a migration chamber with or without LTD4 (10–7 M) in the lower wells. In the specified experiments, MK571 was added to both the upper and lower wells. After 2 h, the number of cells that reached the lower wells was measured by flow cytometry. A migration index was calculated by dividing the number of cells that migrated in the LTD4 containing wells by the number of cells that migrated to medium alone. Data are given as mean of quadruplicates ± SD. One of three representative experiments is shown.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Real-time PCR analysis of PG IP receptor expression in LatY136F and Lat3YF mice. A, Bar diagrams show the relative expression of the PG IP receptor in ex vivo-purified CD4+ T cells derived from LatY136F compared with ex vivo-purified CD4+ T cells from wild-type littermates. B, Bar diagrams show the relative expression of the PG IP receptor in ex vivo-purified T cells from Lat3YF mice compared with ex vivo-purified wild-type T cells from E–/– mice. Values are given as fold difference of expression (mean + SD) and normalized to -actin. They are representative of three independent experiments.

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.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Up-regulation of CysLT1 expression allows chemotactic responses of activated wild-type effector T cells to LTD4. Bar diagrams show the relative expression of full-length CysLT1 (long isoform) (A) and the short isoform of CysLT1 (B) in wild-type CD4+ T cells that were activated in vitro using Th1- or Th2-polarizing conditions compared with purified, resting wild-type CD4+ T cells. Values are given as fold difference of expression (mean + SD) normalized to -actin. Results are representative of at least three independent experiments. C, Wild-type spleen single-cell suspensions or in vitro-stimulated spleen cells (D–F) were added to the upper wells of a migration chamber with or without LTD4 (10–7 M) in the lower wells. In the specified experiments, the CysLT1-specific inhibitor MK571 was added to both the upper and lower wells. After 2 h, the numbers of combined CD4+ and CD8+ T cells (C and D), of CD4+ T cells (E), or of CD8+ T cells (F) that reached the lower wells were measured by flow cytometry. A migration index was calculated by dividing the number of cells that migrated in the LTD4 containing wells by the number of cells that migrated to medium alone. Data are given as mean of quadruplicates ± SD. One representative of three experiments is shown.

	Discussion

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What is the functional relevance of CysLT₁ expression on effector T cells? CysLT₁ can initiate signaling through the G_q 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

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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{at}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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