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The Journal of Immunology, 2005, 175: 5430-5438.
Copyright © 2005 by The American Association of Immunologists

IL-4 Regulates MEK Expression Required for Lysophosphatidic Acid-Mediated Chemokine Generation by Human Mast Cells1

Debby A. Lin* and Joshua A. Boyce2,*,{dagger}

* Departments of Medicine and Pediatrics, Harvard Medical School, and Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA 02115; and {dagger} Partners Asthma Center, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-4 and mast cells (MCs) mediate mucosal defense against helminths and are central to allergic inflammation. Lysophosphatidic acid (LPA), an abundant, potent lipid growth factor, stimulates the growth of cultured human MCs (hMCs) in vitro through a pathway involving LPA receptors 1 and 3 (termed the LPA1 and LPA3 receptors, respectively) and peroxisome proliferator-activated receptor-{gamma}. We now report that LPA potently induces the generation of proinflammatory chemokines (MIP-1{beta}, IL-8, and MCP-1) by hMCs by a mechanism that absolutely requires IL-4. The de novo expression of chemokine mRNA and protein generation involves synergistic actions of calcium flux-dependent NFAT transcription factors and ERK. ERK phosphorylation and chemokine production in response to LPA require IL-4-dependent up-regulation of MEK-1 expression by a pathway involving PI3K. Although receptor-selective agonists for both the LPA2 and LPA3 receptors induce calcium fluxes by hMCs, only the LPA2 receptor-selective agonist fatty alcohol phosphate-12 mimics the IL-4-dependent effect of LPA on chemokine generation. The fact that LPA, an endogenous lipid mediator, activates hMCs by an LPA2 receptor-dependent pathway indicates functional distinctions between different LPA receptor family members that are expressed constitutively by cells of a single hemopoietic lineage. Moreover, the regulation of MEK-dependent signaling is a mechanism by which IL-4 could amplify inflammation in mucosal immune responses through receptor systems for endogenous ligands such as LPA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells (MCs)3 occupy perivascular spaces of mucosal and connective tissues, where they initiate responses to allergic, inflammatory, and infectious stimuli (reviewed in Ref.1). The development of MCs in tissues from circulating bone marrow-derived progenitors requires the interaction of stem cell factor (SCF) with the receptor tyrosine kinase, Kit (2). Specific Ags activate MCs by inducing cross-linkage of the high affinity FcR for IgE, Fc{epsilon}RI, an event that initiates anaphylaxis and also substantially contributes to the pathophysiology of allergic rhinitis and asthma in humans. Cross-linkage of Fc{epsilon}RI initiates calcium flux and signaling pathways that lead to the exocytosis of preformed mediators, generation of eicosanoids, and de novo cytokine gene expression (3). The secretion of several of these mediators by human MCs (hMCs) in vitro is amplified by their priming with IL-4 (4, 5, 6), a Th2 cytokine critical to the induction of mucosal immune responses and allergy. MCs also are activated in vitro or in vivo by stimulation with microbially derived products (7), cysteinyl leukotrienes (cys-LTs) (8), and anaphylatoxins (9), each of which acts through mechanistically distinct receptor systems. Such non-Fc{epsilon}RI-dependent mechanisms of MC activation probably underlie certain aspects of mucosal inflammatory responses, innate responses to infection, and nonallergic inflammatory and fibrotic diseases that involve prominent contributions from MCs (10, 11, 12).

Lysophosphatidic acid (LPA), an abundant extracellular metabolite of lysophosphatidyl choline, is generated by platelets, adipocytes, fibroblasts, and certain tumor cells. It is typically present in micromolar concentrations in serum and biological fluids and in higher concentrations at sites of inflammation and tumor growth (13, 14). LPA is a well-recognized growth factor, stimulating the proliferation and facilitating the survival of a broad range of cell types in vitro (14). Many of these effects reflect the actions of LPA at cognate G protein-coupled receptors (GPCRs). Three of these GPCRs (the LPA1, LPA2, and LPA3 receptors) (15, 16, 17) are members of the endothelial differentiation and growth receptor family that also includes GPCRs for sphingosine-1 phosphate, a sphingolipid that is structurally similar to LPA. Another GPCR with little homology to the endothelial differentiation and growth family, formerly designated orphan GPR23/p2y9, was recently shown to bind LPA and is now referred to as the LPA4 receptor (18). LPA is also a transcellular agonist for the nuclear transcription factor peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) (19), and its effects are mediated through PPAR-{gamma} in some cell systems. LPA and the LPA1 receptor are crucial for normal neurodevelopment in mice (20). Recent studies with a rat model of atherosclerosis revealed a role for LPA, acting through PPAR-{gamma}, in the formation of neointimal lesions (21). The physiologic functions of the LPA2, LPA3, and LPA4 receptors are not yet known. Direct intratracheal challenge of naive mice with LPA elicits the recruitment of neutrophils to the lung and the production of a neutrophil-active chemokine, MIP-2 (22), suggesting a potential function for LPA as a sentinel endogenous mediator for the induction of genes that control inflammatory or immune responses. However, although eosinophils (23), lymphocytes (24), and macrophages (25) all express LPA receptors, the target cells and receptors responsible for a putative contribution from LPA to inflammation have yet to be defined.

We recently reported that LPA potently stimulates the proliferation of cord blood-derived hMCs cultured in serum-free medium supplemented with SCF, IL-6, and IL-10 and induces their expression of MC-specific tryptases (26). In experiments with selective antagonists, LPA-mediated proliferation of hMCs was shown to involve Gi proteins, LPA1 and/or LPA3 receptors, and PPAR-{gamma}; these studies suggest a mechanism for inducing MC hyperplasia after platelet activation or vascular leakage. Because MCs initiate inflammatory responses from perivascular spaces, where the local concentrations of LPA are likely to be high, we hypothesized that LPA could also activate hMCs. We now demonstrate that LPA is a potent agonist for the expression and secretion of proinflammatory chemokines by hMCs. This response absolutely depends on priming of the hMCs by IL-4. IL-4 does not alter calcium signaling, the expression of any LPA receptor, or the activity of PI3K in response to LPA. However, IL-4 up-regulates the expression of MAPK kinase-1 (MEK-1), augmenting the ligand-induced phosphorylation of ERK, a requisite event for the expression of chemokines by activated MCs (27). The up-regulation of MEK-1 protein is attenuated by inhibition of PI3K during priming with IL-4. Furthermore, in contrast to its mitogenic effect, LPA mediates chemokine production by hMCs independently of the LPA1 and LPA3 receptors and PPAR-{gamma}, but, rather, through LPA2 receptors. The findings reveal an unanticipated role for IL-4 in the control of MEK/ERK signaling and implicate LPA and its cognate LPA2 receptor in a novel IL-4-dependent pathway for the induction of proinflammatory gene expression by MCs. The apparent convergence of a Th2 cytokine, a resident tissue effector cell, and an endogenous mediator may regulate trafficking of blood-borne leukocytes in circumstances of mucosal inflammation where LPA is likely to be abundant due to vascular leakage. Moreover, the findings indicate that IL-4, while inducing adaptive immune responses through STAT6-dependent transcription, may also amplify the effector phase of inflammation via modulation of MEK-1/ERK signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Agonists and antagonists

LPA species containing one (LPA 18:1) and two (LPA 18:2) double bonds and the LPA3 receptor-selective agonist L-sn-1-O-oleoyl-2-O-methyl-glyceryl-3-phosphothionate (2S-OMPT) (28) were purchased from Echelon. The dual-specific LPA1/LPA3 receptor antagonist VPC 32179 and the selective LPA3 receptor antagonist VPC 32210 were both provided by Dr. K. Lynch (University of Virginia, Charlottesville, VA) (29). The selective agonist of the LPA2 receptor, fatty alcohol phosphate (FAP-12) (30), was purchased from BIOMOL. The PPAR {gamma}-selective agonist (rosiglitazone) was purchased from American Radiolabeled Chemicals, and the PPAR antagonist (GW9662) were purchased from Tocris. Selective inhibitors of MEK (UO126) and PI3K (LY294002) were purchased from Promega. LTD4 was obtained from Cayman.

Culture and priming of hMCs

Human MCs were derived from human umbilical cord blood under serum-free conditions as previously described (26). The mononuclear cells were cultured at a concentration of 106 cells/ml in serum-free AIM V medium (Invitrogen Life Technologies) supplemented with SCF (100 ng/ml; R&D Systems), IL-6 (50 ng/ml; R&D Systems), IL-10 (10 ng/ml; Endogen), and LPA 18:1 (5 µM) at 37°C and 5% CO2. The cells were transferred to fresh medium and cytokines weekly to maintain a concentration of 106 cells/ml. Cells were harvested for experiments at 3–4 wk, when >90% stained metachromatically with toluidine blue dye and exhibited strong cytofluorographic expression of Kit (26). All hMCs were passaged into fresh medium containing the triad of cytokines with LPA (5 µM) 5 days before stimulation, with some cells receiving additional supplementation with IL-4 (1–50 ng/ml; Endogen) to augment Fc{epsilon}RI{alpha} expression (31) and to prime them for cytokine production. For experiments in which involvement of IL-4-induced PI3K activation was tested in priming, LY294002 was added to the cells concomitantly with IL-4, was maintained throughout the priming period, and was washed out before cell activation. In some experiments, hMCs grown for 6–8 wk in standard serum-containing medium using the same cytokine triad (32) were used to examine the effect of IL-4 on MEK expression and ERK phosphorylation. The IL-4 responses of these hMCs and those grown in the absence of serum did not differ.

Calcium flux

Human MCs were washed and loaded with fura 2-AM (Molecular Probes) for 30 min at 37°C. Samples of 106 hMCs were stimulated with LPA 18:1, FAP-12, 2S-OMPT (1–50 µM for each agonist), or LTD4 (0.1 µM). In some experiments cells were preincubated for 12 h with pertussis toxin (PTX; 100 ng/ml) or were treated with VPC 32179 (100 nM) or VPC 32210 (100 nM) for 5 min before stimulation. Changes in the intracellular levels of free Ca2+ were measured using excitation at 340 and 380 nm in a fluorescence spectrophotometer (Hitachi F-4500) after cell stimulation with LPA or its mimics. The relative ratios of fluorescence emitted at 510 nm were recorded and displayed as a reflection of intracellular concentrations of Ca2+.

Activation of hMCs

Human MCs were passively sensitized with human myeloma IgE (2 µg/ml; Chemicon International) overnight. On the day of stimulation, hMCs were washed and distributed in aliquots of 1 x 105 cells/well of 96-well, flat-bottom microtiter assay plates (Costar) at a concentration of 5 x 105 cells/ml in AIM V medium containing SCF (100 ng/ml). IL-4 was not maintained in the culture medium during any of the stimulations. Human MCs were stimulated in triplicate at 37°C with LPA 18:1 or 18:2 (1–50 µM), FAP-12 (1–50 µM), 2S-OMPT (1–50 µM), rosiglitazone (10 µM), {alpha}-IgE (1 µg/ml), or LTD4 (0.1 µM). The dose range of LPA was chosen so as to exceed the concentration of LPA used during the culture derivation of the cells and to overcome potential inhibition of the LPA effects by extracellular lipid phosphate phosphatases reported in some in vitro studies (33). To determine the contributions of various signaling events to the primed chemokine production in response to LPA, the cells were stimulated after preincubation with PTX (100 ng/ml) for 4–12 h; with VPC32179, VPC32210 (100 nM each), or GW9662 (5 µM) for 30 min; or with the selective inhibitor of UO126 (5 µg/ml), the selective calcineurin inhibitor, cyclosporin A (CsA; 1 µg/ml; Bedford Laboratories) for 5 min. In some experiments, LY294002 (10 µM) was added to the cells 30 min before activation to test the role of PI3K in the response to LPA. Cell samples were harvested at 30 min (for the measurement of histamine release and eicosanoid production) and 6 h (for measurement of cytokine generation). Histamine content was measured by a commercial ELISA (Cayman) in both supernatants and pellets and was expressed as the percent release, as detailed previously (5). Supernatants from hMCs stimulated for 30 min were assayed for PGD2 (Cayman) and cys-LTs (Amersham Biosciences) using ELISAs. The concentrations of MIP-1{beta}, MCP-1, TNF-{alpha}, IL-8, IL-13 (all from Endogen), and IL-5 (eBioscience) were measured with ELISAs from the indicated vendors.

Real-time quantitative PCR

The expressions of MIP-1{beta}, MEK-1, and MEK-2 mRNA were determined using real-time PCR performed on ABI PRISM 7700 Sequence Detection System (Applied Biosystems) (34). Samples of 5–10 x 106 primed or unprimed, sensitized hMCs were stimulated with anti-IgE (1 µg/ml), LPA (50 µM), or medium alone for 2 h at 37°C in the presence of SCF at a constant concentration of 100 ng/ml. RNA was prepared using an RNeasy Mini Kit (Qiagen) and was treated with RNase-free DNase (Qiagen) according to the manufacturer’s protocols. RT was performed on samples of 2 µg of DNase-treated RNA using TaqMan RT reagents (Applied Biosystems). Primers and FAM-labeled probes for the amplification of human {beta}2-microglobulin and the corresponding VIC dye were purchased from Applied Biosystems. Primers for MIP-1{beta} (forward, 5' TGTTCCTGAGCC ACCCTACTG 3'; reverse, 5'-TGTTTCCTTGGGCATGAAGAG-3'), MEK-1 (forward 5'-ACCAGCCCAGCACACCAA-3'; reverse, 5'-GGGACTCGCTCTTTGTTGCTT-3'), and MEK-2 (forward, 5' TGCTCACAAACCACACCTTCA-3'; reverse, 5'-ACACAACCAGCCGGCAAA-3') were purchased from Geneprobe. FAM-labeled probes for MIP-1{beta} (5'-CTGGCCCTCCGCACATCCAATGCTGG-3'), MEK-1 (5'-CAAACACTTAGACGCCAGCAGCATGG-3'), and MEK-2 (5'-CCACTTCTTCCACCTCGGACCGCT-3') were purchased from Applied Biosystems.

Flow cytometry

For detection of Kit and LPA receptors, 3-wk-old hMCs were fixed in paraformaldehyde and permeabilized with saponin as detailed previously (35). Samples of 0.2 x 106 cells were incubated with rabbit anti-human polyclonal Abs directed against the LPA1, LPA2, and LPA3 receptors (all from Orbigen), the LPA4 receptor (Lifespan Biosciences), or a mouse anti-human IgG1 against Kit (BioSource International). Nonspecific rabbit IgG and mouse IgG1 (BioSource International) were used as respective negative controls. For cytofluorographic assessment of MAPK phosphorylation, hMCs were washed and incubated in AIM V medium alone for 1 h. Human MCs were then stimulated with medium, LPA (50 µM), FAP-12 (50 µM), 2S-OMPT (50 µM), or LTD4 (0.1 µM) for 5 min at 37°C. The cells were fixed in paraformaldehyde and permeabilized by methanol. Samples of 0.5 x 106 cells were stained with rabbit anti-human polyclonal Abs specific for phospho-p44/42 MAPK, total p44/42 MAPK (both from Cell Signaling Technology), or rabbit IgG control (Cayman). For all flow cytometry experiments, replicate cell samples were stained with a rabbit IgG (Cayman) or mouse IgG1 (BioSource International) as negative controls. The cells were then stained with FITC-conjugated donkey anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories) and analyzed by FACS (BD Biosciences) using overlaid histograms.

SDS-PAGE immunoblotting

Phosphorylated and total ERK, p38, JNK, and Akt were assessed by immunoblotting. SDS-PAGE immunoblots were probed with Abs specific for ERK, p38, and JNK MAPKs and their respective phosphorylated isoforms as previously described (8). All MAPK Abs were purchased from Cell Signaling Technologies. Abs against phospho- and total Akt were purchased from BD Pharmingen.

Statistics

Data are expressed as the mean ± SEM except where indicated otherwise. Significance was determined using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of LPA on cytokine generation, histamine release, and eicosanoid generation by hMCs

To determine whether LPA could activate hMCs for secretion of their characteristic product profile, hMCs were stimulated with LPA at various concentrations. Because priming by IL-4 up-regulates Fc{epsilon}RI{alpha} expression by cord blood-derived hMCs (31) and amplifies their generation of cytokines in response to diverse activating stimuli (4, 8, 36), both unprimed and IL-4-primed cells were studied. Unprimed hMCs stimulated for 6 h with LPA (50 µM) did not secrete MIP-1{beta}, IL-8, or MCP-1 (n = 4). Unprimed cells stimulated with anti-IgE secreted small amounts of each product. In contrast, hMCs primed with IL-4 and stimulated with LPA secreted robust amounts of MIP-1{beta}, nearly equivalent to the amounts secreted by replicate cells stimulated with anti-IgE (n = 5; Fig. 1A). The secretion of MIP-1{beta} was associated with an IL-4-dependent induction of the corresponding mRNA transcript 2 h after stimulation with LPA (n = 3; Fig. 1B). Primed hMCs also produced IL-8 and MCP-1 at levels comparable to those generated in response to Fc{epsilon}RI cross-linkage (n = 3 and 4, respectively; Fig. 1C); again, this response was completely dependent on IL-4 priming (not shown). Priming with IL-4 significantly increased the quantities of TNF-{alpha} generated by LPA-stimulated hMCs (14 ± 5 vs 2.1 ± 1 pg/106 hMCs; p = 0.03; n = 4, not shown). IL-4 also enhanced the secretion of cytokines and the expression of MIP-1{beta} mRNA by hMCs stimulated by Fc{epsilon}RI cross-linkage, although this enhancement was relative rather than absolute (Fig. 1, A and B). At concentrations of 5–50 µM, LPA 18:1 and LPA 18:2 were virtually equipotent agonists for MIP-1{beta} generation by IL-4-primed hMCs (n = 3; data not shown). Priming of hMCs for either LPA- or anti-IgE-mediated chemokine production required a minimum of 3 days of culture in the presence of IL-4 (10 ng/ml) and required a minimum concentration of 5 ng/ml IL-4 (n = 3; as shown for MIP-1{beta} in individual experiments; Fig. 1, D and E). In contrast to IL-4, neither IL-5 (10 ng/ml) nor IL-13 (50 ng/ml) primed hMCs for LPA-mediated production of any cytokine or chemokine tested (not shown). As anticipated on the basis of our previous studies (8), LTD4 induced secretion of the same profile of chemokines as did LPA, again dependent on IL-4 priming (not shown). Human MCs stimulated with LPA for 30 min did not undergo exocytosis of histamine, and did not produce either cys-LTs or PGD2 regardless of priming (n = 3; not shown).



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  		FIGURE 1. Chemokine generation by IL-4-primed and unprimed hMCs stimulated with LPA. A, Human MCs developed under serum-free conditions in the presence of SCF, IL-6, and IL-10 with LPA were maintained for an additional 5 days of culture with or without IL-4 as detailed in Materials and Methods, passively sensitized, and stimulated with anti-IgE or LPA (50 µM). MIP-1{beta} concentrations were measured in supernatants collected after 6 h of stimulation. Data depicted are the mean ± SEM (n = 5). The p values indicate a significant effect of IL-4. B, Real-time PCR showing relative levels of MIP-1{beta} transcript expression by IL-4-primed and unprimed hMCs stimulated with LPA (50 µM) or anti-IgE. Data are the mean ± SEM (n = 3). C, Secretion of IL-8 and MCP-1 by hMCs primed with IL-4 and stimulated with LPA (50 µM) or anti-IgE for 6 h. Data are the mean ± SEM (n = 3 for IL-8; n = 4 for MCP-1). The p values indicate significance compared with medium alone. D and E, Dose-response (D) and time course (E) of the effect of priming hMCs with IL-4 on the generation of MIP-1{beta} in response to stimulation LPA (50 µM) or anti-IgE (1 µg/ml). The time-course experiments used IL-4 at a dose of 10 ng/ml. Results depicted are the mean ± SD of triplicate samples from a single experiment representative of three performed.

 
Signaling requirements for LPA-dependent chemokine production by hMCs

Selective inhibitors of calcineurin/NFAT (CsA), MEK (UO126), and PI3K (LY294002) were used at their maximally effective concentrations (1 µg/ml, 5 µg/ml, and 10 µM, respectively) to determine the signaling pathways responsible for LPA-dependent chemokine generation by the primed hMCs. After a 5-day period of priming by IL-4, pretreatment of hMCs with either CsA or UO126 for 30 min before stimulation blocked MIP-1{beta} production in response to LPA by 95 ± 4 and 85 ± 1%, respectively, and also blocked Fc{epsilon}RI-mediated MIP-1{beta} production to similar extents (mean ± SEM; n = 3; Fig. 2A). When added to the cells 30 min before activation, the PI3K inhibitor LY294002 decreased LPA-mediated MIP-1{beta} generation by 43 ± 15% and blocked 62 ± 11% of the MIP-1{beta} produced in response to anti-IgE (n = 3; Fig. 2A). The same effects were observed for MCP-1 production (not shown).



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  		FIGURE 2. Signaling pathways used by IL-4-primed hMCs for chemokine production. A, Effects of CsA (1 µg/ml), UO126 (5 µg/ml), and LY294002 (10 µM) on LPA- and anti-IgE-mediated production of MIP-1{beta} by hMCs. Inhibitors were added to the cells after IL-4 was washed out and 30 min before the addition of activating agonists. Data are expressed as the percent inhibition and are the mean ± SEM for three experiments. B, Effects of LPA on the accumulation of intracellular calcium in hMCs. Three-week-old hMCs were maintained for 5 additional days with or without IL-4, loaded with fura 2 dye, and stimulated with the indicated concentrations of LPA. Data are from a single experiment representative of three performed. C, SDS-PAGE analysis of phosphorylated Akt and total Akt in lysates of unprimed or primed hMCs stimulated with LPA (50 µM) for 5 min. Results are from one of three experiments with similar results. D, Flow cytometric analysis was performed on LPA (50 µM)- or LTD4 (100 nM)-stimulated, permeabilized hMCs using an Ab specific for phosphorylated ERK (dark tracings) or an isotype-matched control (light tracings). MFI, log mean fluorescence intensity. E, SDS-PAGE immunoblotting was performed on cell lysates obtained after 5 min of cell stimulation with the indicated agonists, using Abs specific for total and phosphorylated (phospho) ERK and p38, respectively. Data depicted for each assay are from single experiments representative of three performed.

 
Because LPA-mediated chemokine generation required IL-4 priming and involved calcium-dependent transcription, MEK/ERK signaling, and PI3K activation, we determined whether priming the hMCs with IL-4 altered any of these signals in response to LPA. Primed and unprimed 3-wk-old hMCs were loaded with fura 2 dye and stimulated with various concentrations of LPA. LPA induced a sustained rise in cytosolic free calcium in a dose-dependent manner (Fig. 2B). Priming with IL-4 did not change the magnitude of the LPA-induced calcium signal and did not alter the effective concentration of LPA necessary to elicit calcium flux. SDS-PAGE immunoblotting revealed that primed and unprimed hMCs showed the same degree of phosphorylated Akt, a signature of PI3K activation, at 5 min after stimulation with LPA (Fig. 2C). Stimulation of unprimed hMCs with LPA for 5 min failed to elicit phosphorylation of ERK MAPK, as measured by either FACS analysis of permeabilized hMCs (Fig. 2D) or SDS-PAGE immunoblotting (Fig. 2E). In contrast, LPA strongly induced phosphorylation of ERK in IL-4-primed hMCs (Fig. 2, D and E). This phosphorylation was evident at the earliest time point examined after stimulation with LPA (2 min) and persisted above the baseline for as long as 15 min (n = 3; as shown for one experiment; Fig. 2D). As determined by SDS-PAGE immunoblotting, IL-4 priming also increased the baseline levels of phosphorylated ERK as well as LTD4-mediated ERK phosphorylation, although IL-4 was not required for ERK phosphorylation in response to LTD4 (n = 3; as shown for one experiment; Fig. 2E). Although p38 MAPK was constitutively phosphorylated in these experiments, LPA did induce some incremental phosphorylation; however, p38 phosphorylation (in contrast to that of ERK) in response to LPA did not depend on IL-4 priming (n = 3; as shown for one experiment; Fig. 2E). Phosphorylation of JNK was not detected under any of the conditions tested (n = 2; not shown).

Involvement of PI3K and MEK in priming by IL-4

To identify potential candidate mechanisms to explain the absolute requirement for IL-4 priming for LPA-mediated phosphorylation of ERK in hMCs, we screened a profile of mRNA transcripts expressed by hMCs that were induced or up-regulated in response to treatment with IL-4 (37). Among >100 genes with expression levels that were up-regulated by IL-4 priming, MEK-1 was the only MAPK family member (2.5- and 3.7-fold increases in MEK-1 transcript levels in two separate experiments with cells from different donors; not shown). Cell priming with IL-4 did not alter levels of transcripts encoding PI3K and NF-AT family members in either experiment. Real-time PCR performed in two consecutive experiments confirmed that the hMCs primed with IL-4 for 5 days increased their expression of steady-state levels of mRNA encoding MEK-1 by ~2-fold, whereas the low level of MEK-2 transcript was unchanged (not shown). IL-4 priming for 3 days strikingly enhanced MEK-1 protein expression (n = 4; as shown for one experiment; Fig. 3A). The MEK-2 protein was not detected in these experiments. Phosphorylated MEK-1 increased in parallel with total MEK-1. The time and dose requirements for the up-regulation of MEK-1 by IL-4 paralleled the requirements for functional priming (Fig. 1, D and E), being maximal at 3 days (n = 3; as shown for one experiment; Fig. 3C) and at a concentration range that reached a plateau at 5 ng/ml (Fig. 3A). IL-4 transiently induced the phosphorylation of Akt 5 min after exposure (Fig. 3B). When added concomitantly with IL-4 and maintained throughout the priming period, the PI3K inhibitor LY294002 attenuated the IL-4-dependent up-regulation of MEK-1 (n = 3; as shown for one experiment; Fig. 3C) and also interfered with priming for LPA-induced MIP-1{beta} production (51 ± 21% inhibition; n = 3; Fig. 3D). In contrast, inhibition of PI3K during IL-4 priming had no effect on Fc{epsilon}RI-induced MIP-1{beta} generation (Fig. 3D), although it did interfere with this response when added just before activation (Fig. 2A).



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  		FIGURE 3. Involvement of MEK-1 and PI3K in the priming effect of IL-4 for LPA-induced chemokine generation by hMCs. A, Dose-dependent effects of IL-4 on total and phosphorylated MEK-1 protein. SDS-PAGE analysis was performed on lysates of 3-wk-old hMCs treated with the indicated concentrations of IL-4 for 3 days (the time of maximal induction). The same blot was stripped and reprobed with an Ab against {beta}-actin as a control for loading. B, Phosphorylation of the PI3K substrate Akt in response to IL-4 (10 ng/ml). C, Time course for IL-4-dependent induction of MEK-1 expression and effect of LY294002 (10 µM) on this induction. Results in A–C are from single experiments representative of three performed for each assay. D, Effect of LY294002 on priming by IL-4 for LPA- and Fc{epsilon}RI-mediated MIP-1{beta} generation. The inhibitor was added to the cells at the same time as IL-4 and was washed out before cell activation with the indicated agonists 3 days later. Results are expressed as the percent inhibition compared with the untreated controls and are the mean ± SEM from three independent experiments. The p value indicates a significant difference in effect on LPA- vs anti-IgE-mediated responses.

 
Determination of LPA receptor subtypes responsible for activation

To determine the profile of LPA receptor proteins expressed on the surface of hMCs, cells were harvested for FACS analysis after 3–4 wk of culture in serum-free medium in the presence of SCF, IL-6, and IL-10 supplemented with 5 µM LPA 18:1. These cells were strongly positive for Kit and also expressed LPA1, LPA2, and LPA3 receptor proteins (n = 3; as shown for one experiment; Fig. 4A). The LPA4 receptor protein was not detected. Priming the cells with IL-4 did not change the profile or the level of LPA receptor expression, but did decrease the cytofluorographic levels of surface Kit expression (n = 3; not shown). SDS-PAGE immunoblotting revealed constitutive expression of PPAR-{gamma}, which was not changed by priming the cells with IL-4 for 5 days (not shown).



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  		FIGURE 4. LPA receptor expression by hMCs and determination of function. A, Histograms displaying signals elicited by staining with polyclonal Abs against the LPA1, LPA2, LPA3, and LPA4 receptors (bold tracings) and the corresponding negative control IgG (light tracings). IL-4 priming did not alter the profile or intensity of staining (n = 2; not shown). MFI, log mean fluorescence intensity. B, Calcium fluxes in fura 2-loaded hMCs stimulated with LPA (50 µM), the LPA2 receptor-selective agonist FAP-12 (50 µM), and the LPA3 receptor-selective agonist 2S-OMPT (20 µM). The indicated replicate cell samples were treated overnight with PTX or for 5 min with the LPA3 receptor-selective antagonist VPC 32210 or the dual LPA1/LPA3 receptor-selective antagonist VPC 32179 (100 nM for each) before application of the indicated agonists. C, Cross-desensitization between LPA and FAP-12 or 2S-OMPT. Each agonist was applied at 50 µM, except for 2S-OMPT (20 µM). Results depicted in A–C are from individual experiments representative of three for each procedure.

 
To determine the LPA receptor subtypes responsible for LPA-mediated calcium flux, fura 2-loaded hMCs were stimulated with LPA and its receptor-selective agonists, and changes in intracellular free calcium were assessed. Both the LPA2 receptor-selective agonist FAP-12 (30) and the LPA3 receptor-selective agonist 2S-OMPT (28) stimulated calcium flux in the same concentration range as did LPA (as shown for 50 and 20 µM concentrations, respectively; Fig. 4B). LPA (50 µM) desensitized hMCs to subsequent calcium fluxes in response to FAP-12 (50 µM) or 2S-OMPT (20 µM), whereas each receptor-selective agonist only partly desensitized hMCs to LPA (Fig. 4C). At higher concentrations (50 µM), the 2S-OMPT-mediated calcium flux was not completely inhibited by previous administration of LPA at an equal concentration (n = 2; data not shown). LPA-mediated calcium flux was partly attenuated by pretreatment with PTX (Fig. 4B) and was also reduced by pretreatment with either the LPA3-selective antagonist VPC 32210 or the LPA1/LPA3 receptor-selective antagonist VPC 32179 (n = 2; ~50% inhibition for each inhibitor at 100 nM; as shown for one experiment; Fig. 4B). Both compounds also slightly attenuated calcium fluxes in response to FAP-12 and largely blocked the response to 2S-OMPT (n = 2; as shown for one experiment; Fig. 4B). 2S-OMPT-mediated calcium flux was substantially attenuated by pretreatment with PTX, whereas FAP-12-mediated calcium flux was not (Fig. 4B). Both FAP-12 and 2S-OMPT induced ERK phosphorylation in IL-4-primed hMCs, but not in their unprimed replicates (n = 3; data not shown) and were equipotent in this effect.

To determine the relative contributions of the LPA receptors to chemokine generation, replicate samples of IL-4-primed hMCs were stimulated for 6 h with various concentrations of LPA, 2S-OMPT, FAP-12, or the selective PPAR-{gamma} agonist rosiglitazone in the presence or the absence of PTX, VPC 32210, VPC 32179, or the selective PPAR-{gamma} antagonist GW9662. Of the receptor-selective agonists, only FAP-12 induced concentration-dependent production of MIP-1{beta} and MCP-1, essentially mirroring the effect of LPA (n = 3; as shown for one experiment; Fig. 5A). In contrast, neither 2S-OMPT (at doses up to 50 µM; Fig. 5A) nor rosiglitazone (not shown) induced significant chemokine production. Neither VPC 32210 nor VPC 32179 (n = 3; as shown for one experiment; Fig. 5B) nor GW9662 (not shown) altered LPA-mediated MIP-1{beta} or MCP-1 generation. The production of these chemokines in response to either LPA or FAP-12 was similarly sensitive to partial inhibition by pretreatment of the cells with PTX (n = 3; as shown for one representative experiment; Fig. 5C). PTX completely abrogated MIP-1{beta} and MCP-1 generation by the cells in response to stimulation with LTD4 (n = 3; as shown for one experiment; Fig. 5C).



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  		FIGURE 5. Effects of LPA receptor-selective agonists and antagonists on chemokine production by IL-4-primed hMCs. A, Effects of LPA and the LPA2 receptor-selective agonist FAP-12 on the generation of MIP-1{beta} and MCP-1 by IL-4-primed hMCs. Replicate cells were stimulated with LTD4 (100 nM) and anti-IgE as positive controls. B, Effects of the dual-selective LPA1/LPA3 receptor-selective antagonist VPC 32179 and the LPA3 receptor-selective antagonist VPC 32210 (100 nM for both) on MIP-1{beta} and MCP-1 generation. IL-4-primed hMCs were incubated with the indicated antagonists for 30 min before stimulation with LPA (50 µM). C, Effect of PTX (100 ng/ml) on MIP-1{beta} and MCP-1 generation in response to the indicated agonists. Data depicted in A–C are the mean ± SEM of data from three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Strategically situated in perivascular connective tissues and mucosal surfaces, MCs are specialized to initiate inflammation and immune responses to diverse stimuli by generating and releasing soluble mediators. Classical IgE-dependent activation mechanisms underlie the role of MCs in anaphylaxis and contribute substantially to the pathophysiology of allergic asthma and rhinitis in humans (38). Like allergy, mucosal defense in rodents against helminthic worms such as Trichinella spiralis requires contributions from both IL-4 and MCs, suggesting a synergistic interaction between them (39). Although helminth infection strongly elicits IgE synthesis (a signature activity of IL-4 and IL-13), MC-dependent expulsion of adult T. spiralis worms does not depend on IgE (40), implying that uncharacterized mechanisms contribute to MC effector responses in such circumstances. Furthermore, the recognized role of MCs in models of autoimmune disorders (11, 12, 41) as well as their suspected involvement in cardiovascular diseases (42, 43) and nonallergic asthma (44) invoke the importance of nonclassical mechanisms for MC activation in disease and inflammation. Stimulation of MCs in vitro through GPCRs for anaphylatoxins (27), cys-LTs (8, 45), and adenosine (46) induces cytokine and chemokine generation by initiating transcriptional events. The proximity of MCs to the vasculature and their expression of LPA-specific GPCRs (26) probably assure their stimulation by LPA in vivo. Because cytokine and chemokine generation by MCs underlies their ability to initiate inflammatory responses germane to both host defense and disease (7), we determined whether LPA, in addition to its actions as a growth factor, might induce hMCs to produce cytokines and to determine the responsible LPA receptor(s). Moreover, the ability of IL-4 to enhance chemokine generation by MCs activated by diverse ligands prompted an examination of potential mechanisms.

The effects of IL-4 on mouse and/or human MCs in vitro include comitogenesis with SCF (6), apoptosis (47, 48), expression of the biosynthetic enzyme LTC4 synthase (5), and up-regulation of the Fc{epsilon}RI{alpha} subunit (31). The fact that IL-4 also enhances cytokine production by hMCs in response to transmembrane stimuli as diverse as Fc{epsilon}RI cross-linkage (4, 6, 49), Gram-negative bacteria (36), and cys-LTs (8) implies additional effects of IL-4 on fundamental signaling or transcriptional events. To test the ability of LPA to induce cytokine production and to assess the potential effect of IL-4 on this response, we stimulated both IL-4-primed and unprimed hMCs with LPA at 50 µM. This concentration of LPA was chosen so as to exceed potential receptor desensitization due to the maintenance of 5 µM LPA during the culture derivation, to overcome possible inhibition by extracellular lipid phosphate phosphatases (33), and to approximate the LPA doses used previously to elicit receptor-mediated chemotactic responses from human eosinophils (23). IL-4 priming was absolutely required for LPA-mediated MIP-1{beta} generation (Fig. 1A) and for expression of the corresponding mRNA (Fig. 1B), and it resulted as well in the additional generation of MCP-1, IL-8 (Fig. 1C), and TNF-{alpha}. The priming effect required exposure of hMCs to 5–10 ng/ml IL-4 for a minimum of 3 days before activation by either LPA or anti-IgE (Fig. 1, D and E). LPA 18:1 was identical in agonistic activity to LPA 18:2 (the major species found in serum) at concentrations within the range found in normal serum and inflammatory biologic fluids. MIP-1{beta} is a major chemoattractant for CCR5-bearing T cells and dendritic cells in an MC-dependent model of contact hypersensitivity (50), IL-8 is a potent neutrophil chemoattractant, and MCP-1 activates macrophages and plays a major role in a MC-dependent asthma model (51). TNF-{alpha} mediates both neutrophilic inflammation (52) and lymph node hypertrophy (53) in experimentally elicited, MC-dependent anti-microbial responses in vivo. It is thus possible that LPA facilitates the trafficking of blood-borne leukocytes through its actions on MCs in contexts where IL-4 is provided by T lymphocytes (54), NKT cells (55), or basophils (56). Moreover, the lack of LPA-mediated exocytosis of histamine or eicosanoid generation suggests that such responses would occur without histologic evidence of MC degranulation.

We next sought a mechanism to explain the inductive effect of IL-4 on LPA-mediated chemokine production. To identify potential signaling pathways that might be targets for the priming effect of IL-4, we stimulated the primed hMCs with LPA in the presence of various signaling inhibitors. Chemokines are generated de novo by MCs activated through GPCRs involving synergistic functions of calcineurin-dependent NFAT family members as well as MEK-dependent phosphorylation of ERK MAPKs (8, 27), which often occurs through PI3K-dependent mechanisms. ERK induces the activation of several transcription factors involved in chemokine production, including AP-1 and NF for IL-6 (57). Because LPA-dependent chemokine production by primed hMCs was highly susceptible to inhibition by the calcineurin inhibitor CsA (1 µg/ml) and the MEK inhibitor UO126 (5 µg/ml; Fig. 2A) and was partly inhibited by LY294002 (10 µM), we explored whether IL-4 had altered calcium, PI3K, or MEK/ERK signaling. Although LPA-induced calcium fluxes (Fig. 2B) and Akt phosphorylation (an index of PI3K activity; Fig. 2C) occurred regardless of IL-4 priming, LPA-induced ERK phosphorylation (but not p38 phosphorylation) was strikingly IL-4-dependent (Fig. 2, D and E). The fact that IL-4 priming also augmented both baseline and LTD4-mediated ERK phosphorylation also supported a direct effect of IL-4 at (an) upstream regulator(s) of ERK phosphorylation. Several lines of evidence support the conclusion that MEK-1 is a critical target for the priming effect of IL-4. First, MEK-1 was the only MAPK family member that was up-regulated at the transcript level by IL-4 (37). Second, IL-4 clearly up-regulated the MEK-1 protein (Fig. 3) with a dose and time dependency that mirrored its priming effect for LPA-induced MIP-1{beta} generation (Fig. 1, D and E). Third, IL-4 did not alter other potential signaling controls reflected by calcium signaling (Fig. 2B), levels of LPA receptor expression, phosphorylation of p38 MAPK (Fig. 2E; which is not controlled by MEK-1), or PI3K activation (as assessed by Akt phosphorylation; Fig. 2C). Together with the fact that MEK inhibition virtually abrogated LPA-mediated chemokine generation by the primed hMCs (Fig. 2A), these observations are consistent with the critical nature of MEK-1 in LPA-dependent hMC chemokine production and its regulation by IL-4.

IL-4Rs induce responses by activating both STAT6 and PI3K (58). Thus, it is thus not surprising that stimulation of hMCs with IL-4 induced Akt phosphorylation (Fig. 3B) with kinetics similar to those described in other cell types (59). This event was probably functionally significant, because treatment of hMCs with LY294002 during their exposure to IL-4 attenuated MEK-1 expression (Fig. 3C) in addition to priming for LPA (but not Fc{epsilon}RI)-induced chemokine generation (Fig. 3D). IL-4 may control MEK-1 expression in part through the insulin/IL-4R motif of the IL-4R{alpha} subunit, which initiates PI3K signaling through insulin receptor substrate-2 (58). This component of IL-4R signaling is linked to IL-4-mediated growth responses (60), whereas JAK1/3/STAT6 signaling is linked dominantly to IL-4-driven gene induction events, such as chromatin remodeling and Th2 cytokine production in T cells (61). Previous studies noted that both IgE-dependent and constitutive production of IL-5 and IL-13 by hMCs hinges critically on IL-4 priming in vitro (4, 6). Taken together, our study is consistent with IL-4 initiating both STAT6-dependent and PI3K-dependent events that regulate the capacity for MCs to generate cytokines and chemokines. In particular, amplified MEK-1 expression downstream of PI3K may be especially crucial for the generation of chemokines by MCs that occurs in response to ligands that use GPCRs (8, 27), such as LPA.

Each LPA receptor exhibits a distinct pattern of G protein-coupling and calcium signaling when expressed heterologously in cell lines (16, 17, 18, 62). However, relatively little is known about the behavior of these receptors in nontransformed cells that naturally express more than one family member. The fact that hMCs expressed LPA1, LPA2, and LPA3 receptor proteins (Fig. 4A) implied the potential contribution of any or all to calcium flux, MAPK phosphorylation, and chemokine generation. The robust calcium flux observed in response to LPA in our study was only partly attenuated by PTX (Fig. 4B), implying the involvement of both Gi-dependent and -independent signaling pathways in this response. The virtually superimposable, partial blockade of LPA-dependent calcium fluxes by both VPC 32210 (which interferes with LPA3 receptors) and VPC 32179 (which blocks both LPA1 and LPA3 receptors) suggested that LPA induces calcium signaling in hMCs through both LPA3 and LPA2 receptors. The respective contributions of these receptors were also supported by the calcium fluxes induced by the LPA2 receptor-selective agonist FAP-12 and the LPA3 receptor-selective agonist 2S-OMPT. The partial blockade of FAP-12-mediated calcium flux by the LPA3 and LPA1/LPA3 receptor antagonists (Fig. 4B) implies some potential cross-over effects of FAP-12 at these receptors at the agonist doses used or functional dimerization between LPA2 and LPA3 receptors. Nonetheless, the fact that both FAP-12- and 2S-OMPT-mediated calcium fluxes were completely abrogated by previous stimulation of hMCs with LPA, but not vice versa (Fig. 4C), indicates that both agonists signaled through their respective target receptors at the concentration ranges tested. Although both receptor-selective agonists induced calcium fluxes and IL-4-dependent ERK phosphorylation (not shown), the fact that FAP-12 was far more robust than 2S-OMPT for IL-4-dependent chemokine generation (Fig. 5) suggests that additional uncharacterized signals that permit chemokine production can be initiated through LPA2, but not LPA3, receptors. The responses to LPA and FAP-12 were similar not only in magnitude, but also in the extent of sensitivity to interference by PTX (100 ng/ml; Fig. 5C), implying the involvement of both Gi family and other G proteins in this response. Combined with the fact that neither the LPA1/LPA3 receptor antagonist VPC 32179 nor the LPA3 receptor antagonist VPC 32210 attenuated the LPA-induced chemokine generation (Fig. 5B), these observations implicate the LPA2 receptor as the inducer of the chemokine response. The inability of dual LPA1/LPA3 blockade to interfere with chemokine generation contrasts sharply with its capacity to completely abrogate proliferative responses of hMCs to LPA in the same culture system (26). Thus, the functions of the different LPA receptor family members on hMCs are strikingly distinct.

Our study suggests a novel mechanism by which IL-4, a pivotal Th2 cytokine, may regulate MEK/ERK signaling and ligand-mediated gene induction. The findings also indicate a function for LPA2 receptors distinct from those of its partners that share its ligand specificity on the same cell type. LPA can directly induce pulmonary inflammation and chemokine generation in experimental systems (22). The ability of LPA to induce chemokine production by MCs may even figure into the sentinel role for these cells in innate immunity; indeed, sphingomyelinase D enzymes from certain pathogenic bacteria convert extracellular lysophosphatidylcholine to LPA as a major mechanism for their virulence (63). Importantly, the ability of IL-4 to up-regulate MEK-1 expression and therefore augment the ERK signaling pathway may provide a mechanism to amplify MC activation and downstream inflammation in mucosal immune responses. Moreover, the same mechanism could amplify cellular and tissue responses to agonists and receptor systems that use this pathway to initiate contraction, gene induction, and cell growth (64, 65). Indeed, systemic administration of IL-4 amplifies responses to challenges with such agonists (66).


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 was supported by National Institutes of Health Grants AI48802, AI52353, AI31599, and HL36110 and grants from the Charles Dana Foundation and the Vinik Family Fund for Research in Allergic Diseases. D.A.L. is the recipient of a Glaxo-SmithKline Allergy Research Fellowship Award. Back

2 Address correspondence and reprint requests to Dr. Joshua A. Boyce, One Jimmy Fund Way, Smith Building Room 626, Boston, MA 02115. E-mail address: jboyce{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: MC, mast cell; CsA, cyclosporin A; cys-LT, cysteinyl leukotriene; FAP-12, fatty alcohol phosphate-12; GPCR, G protein-coupled receptor; h, human; LPA, lysophosphatidic acid; LT, leukotriene; MIP, macrophage inflammatory protein; 2S-OMPT, L-sn-1-O-oleoyl-{gamma}2-O-methyl-glyceryl-3-phosphothionate; PPAR-{gamma}, peroxisome-proliferator activated receptor-{gamma}; PTX, pertussis toxin; SCF, stem cell factor. Back

Received for publication March 8, 2005. Accepted for publication August 8, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Boyce, J. A., K. F. Austen. 2001. Biology of the mast cell. K. F. Austen, and M. M. Frank, and H. P Atkinson, and H. Cantor, eds. Samter’s Immunologic Diseases 6th Ed.253.-269. Lippincott, Williams & Wilkins, Philadelphia.
  2. Rodewald, H. R., M. Dessing, A. M. Dvorak, S. J. Galli. 1996. Identification of a committed precursor for the mast cell lineage. Science 271:818.-822. [Abstract]
  3. Nadler, M. J., J. P. Kinet. 2002. Uncovering new complexities in mast cell signaling. Nat. Immunol. 3:707.-708. [Medline]
  4. Ochi, H., N. H. De Jesus, F. Hsieh, K. F. Austen, J. A. Boyce. 2000. Interleukins 4 and 5 prime human mast cells for different profiles of IgE-dependent cytokine production. Proc. Natl. Acad. Sci. USA 97:10509.-10513. [Abstract/Free Full Text]
  5. Hsieh, F. H., B. K. Lam, J. F. Penrose, K. F. Austen, J. A. Boyce. 2001. T helper cell type 2 cytokines coordinately regulate IgE-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C4 synthase expression by interleukin 4. J. Exp. Med. 193:123.-133. [Abstract/Free Full Text]
  6. 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. [Abstract/Free Full Text]
  7. Supajatura, V., H. Ushio, A. Nakao, S. Akira, K. Okumura, C. Ra, H. Ogawa. 2002. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J. Clin. Invest. 109:1351.-1359. [Medline]
  8. Mellor, E. A., K. F. Austen, J. A. Boyce. 2002. Cysteinyl leukotrienes and uridine diphosphate induce cytokine generation by human mast cells through an interleukin 4-regulated pathway that is inhibited by leukotriene receptor antagonists. J. Exp. Med. 195:583.-592. [Abstract/Free Full Text]
  9. Johnson, A. R., T. E. Hugli, H. J. Muller-Eberhard. 1975. Release of histamine from rat mast cells by the complement peptides C3a and C5a. Immunology 28:1067.[Medline]
  10. Supajatura, V., H. Ushio, A. Nakao, K. Okumura, C. Ra, H. Ogawa. 2001. Protective roles of mast cells against enterobacterial infection are mediated by toll-like receptor 4. J. Immunol. 167:2250.-2256. [Abstract/Free Full Text]
  11. Benoist, C., D. Mathis. 2002. Mast cells in autoimmune disease. Nature 420:875.-878. [Medline]
  12. Lee, D. M., D. S. Friend, M. F. Gurish, C. Benoist, D. Mathis, M. B. Brenner. 2002. Mast cells: a cellular link between autoantibodies and inflammatory arthritis. Science 297:1689.-1692. [Abstract/Free Full Text]
  13. Xiao, Y. J., B. Schwartz, M. Washington, A. Kennedy, K. Webster, J. Belinson, Y. Xu. 2001. Electrospray ionization mass spectrometry analysis of lysophospholipids in human ascitic fluids: comparison of the lysophospholipid contents in malignant vs nonmalignant ascitic fluids. Anal. Biochem. 290:302.-313. [Medline]
  14. Goetzl, E. J., H. Lee, H. Dolezalova, K. R. Kalli, C. A. Conover, Y. Hu, T. Azuma, T. P. Stossel, J. S. Karliner, R. B. Jaffe. 2000. Mechanisms of lysolipid phosphate effects on cellular survival and proliferation. Ann. N.Y. Acad. Sci. 905:177.-187. [Abstract/Free Full Text]
  15. Hecht, J. H., J. A. Weiner, S. R. Post, J. Chun. 1996. Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex. J. Cell Biol. 135:1071.-1083. [Abstract/Free Full Text]
  16. An, S., T. Bleu, O. G. Hallmark, E. J. Goetzl. 1998. Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid. J. Biol. Chem. 273:7906.-7910. [Abstract/Free Full Text]
  17. Bandoh, K., J. Aoki, H. Hosono, T. Kobayashi, K. Murakami-Murofushi, M. Tsujimoto, H. Arai, K. I. Inoue. 1999. Molecular cloning and characterization of a novel human G protein-coupled receptor, EDG7, for lysophosphatidic acid. J. Biol. Chem. 274:27776.-27785. [Abstract/Free Full Text]
  18. Noguchi, K., S. Ishii, T. Shimizu. 2003. Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distinct from the Edg family. J. Biol. Chem. 278:25600.-25606. [Abstract/Free Full Text]
  19. McIntyre, T. M., A.V. Pontsler, A. R. Silva, A. St Hilaire, Y. Xu, J. C. Hinshaw, G. A. Zimmerman, K. Hama, J. Aoki, H. Arai, et al 2003. Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPAR {gamma} agonist. Proc. Natl. Acad. Sci. USA 100:131.-136. [Abstract/Free Full Text]
  20. Contos, J. J., N. Fukushima, J. A. Weiner, D. Kaushal, J. Chun. 2000. Requirement for the lpA1 lysophosphatidic acid receptor gene in normal suckling behavior. Proc. Natl. Acad. Sci. USA 97:13384.-13389. [Abstract/Free Full Text]
  21. Zhang, C., D. L. Baker, S. Yasuda, N. Makarova, L. Balazs, L. R. Johnson, G. K. Marathe, T. M. McIntyre, Y. Xu, G. D. Prestwich, et al 2004. Lysophosphatidic acid induces neointima formation through PPAR-{gamma} activation. J. Exp. Med. 199:763.-774. [Abstract/Free Full Text]
  22. Cummings, R., Y. Zhao, D. Jacoby, E. W. Spannhake, M. Ohba, J. G. Garcia, T. Watkins, D. He, B. Saatian, V. Natarajan. 2004. Protein kinase C-{delta} mediates lysophosphatidic acid-induced NF-{kappa}B activation and interleukin-8 secretion in human bronchial epithelial cells. J. Biol. Chem. 279:41085.-41094. [Abstract/Free Full Text]
  23. Idzko, M., M. Laut, E. Panther, S. Sorichter, T. Durk, J. W. Fluhr, Y. Herouy, M. Mockenhaupt, D. Myrtek, P. Elsner, et al 2004. Lysophosphatidic acid induces chemotaxis, oxygen radical production, CD11b up-regulation, Ca2+ mobilization, and actin reorganization in human eosinophils via pertussis toxin-sensitive G proteins. J. Immunol. 172:4480.-4485. [Abstract/Free Full Text]
  24. Goetzl, E. J., Y. Kong, J. K. Voice. 2000. Differential constitutive expression of functional receptors for lysophosphatidic acid by human blood lymphocytes. J. Immunol. 164:4996.-4999. [Abstract/Free Full Text]
  25. Hornuss, C., R. Hammermann, M. Fuhrmann, U. R. Juergens, K. Racke. 2001. Human and rat alveolar macrophages express multiple EDG receptors. Eur. J. Pharmacol. 429:303.-308. [Medline]
  26. Bagga, S., K. S. Price, D. A. Lin, D. S. Friend, K. F. Austen, J. A. Boyce. 2004. Lysophosphatidic acid accelerates the development of human mast cells. Blood 104:4080.-4087. [Abstract/Free Full Text]
  27. Ali, H., J. Ahamed, C. Hernandez-Munain, J. L. Baron, M. S. Krangel, D. D. Patel. 2000. Chemokine production by G protein-coupled receptor activation in a human mast cell line: roles of extracellular signal-regulated kinase and NFAT. J. Immunol. 165:7215.-7223. [Abstract/Free Full Text]
  28. Qian, L., Y. Xu, Y. Hasegawa, J. Aoki, G. B. Mills, G. D. Prestwich. 2003. Enantioselective responses to a phosphorothioate analogue of lysophosphatidic acid with LPA3 receptor-selective agonist activity. J. Med. Chem. 46:5575.-5578. [Medline]
  29. Heasley, B. H., R. Jarosz, K.R. Lynch, T.L. MacDonald. 2004. Initial structure-activity relationships of lysophosphatidic acid receptor antagonists: discovery of a high-affinity LPA1/LPA3 receptor antagonist. Bioorg. Med. Chem. Lett. 14:2735.-2740. [Medline]
  30. Virag, T., D. B. Elrod, K. Liliom, V. M. Sardar, A. L. Parrill, K. Yokoyama, G. Durgam, W. Deng, D. D. Miller, G. Tigyi. 2003. Fatty alcohol phosphates are subtype-selective agonists and antagonists of lysophosphatidic acid receptors. Mol. Pharmacol. 63:1032.-1042. [Abstract/Free Full Text]
  31. Toru, H., C. Ra, S. Nonoyama, K. Suzuki, J. Yata, T. Nakahata. 1996. Induction of the high-affinity IgE receptor (Fc{epsilon}RI) on human mast cells by IL-4. Int. Immunol. 8:1367.-1373. [Abstract/Free Full Text]
  32. Ochi, H., W. M. Hirani, Q. Yuan, D. Friend, K. F. Austen, J. A. Boyce. 1999. T helper type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro. J. Exp. Med. 190:267.-280. [Abstract/Free Full Text]
  33. Smyth, S. S., V. A. Sciorra, Y. J. Sigal, Z. Pamuklar, Z. Wang, Y. Xu, G. D. Prestwich, A. J. Morris. 2003. Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity. J. Biol. Chem. 278:43214.-43223. [Abstract/Free Full Text]
  34. Feng, C. L., A. G. Mery, E. M. Beller, C. L. Favot, J. A. Boyce. 2004. Adenine nucleotides inhibit cytokine generation by human mast cells through a Gs-coupled receptor. J. Immunol. 173:7539.-7547. [Abstract/Free Full Text]
  35. Price, K. S., D. S. Friend, E. A. Mellor, N. De Jesus, G. F. Watts, J. A. Boyce. 2003. CC chemokine receptor 3 mobilizes to the surface of human mast cells and potentiates immunoglobulin E-dependent generation of interleukin 13. Am. J. Respir. Cell Mol. Biol. 28:420.-427. [Abstract/Free Full Text]
  36. 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. [Medline]
  37. Lora, J. M., A. Al-Garawi, M. D. Pickard, K. S. Price, S. Bagga, J. Sicoli, M. R. Hodge, J.-C. Gutiérrez-Ramos, M. Briskin, J. A. Boyce. 2003. Fc{epsilon}RI-dependent gene expression in human mast cells is differentially controlled by T helper type 2 cytokines. J. Allergy Clin. Immunol. 112:1119.-1126. [Medline]
  38. Milgrom, H., R. B. Fick, Jr, J.Q. Su, J. D. Reimann, R. K. Bush, M. L. Watrous, W. J. Metzger. 1999. Treatment of allergic asthma with monoclonal anti-IgE antibody: rhuMAb-E25 Study Group. N. Engl. J. Med. 341:1966.-1973. [Abstract/Free Full Text]
  39. Urban, J. F., Jr, L. Schopf, S. C. Morris, T. Orekhova, K. B. Madden, C. J. Betts, H. R. Gamble, C. Byrd, D. Donaldson, K. Else, et al 2000. Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism. J. Immunol. 164:2046.-2052. [Abstract/Free Full Text]
  40. Gurish, M. F., P. J. Bryce, H. Tao, A. B. Kisselgof, E. M. Thornton, H. R. Miller, D. S. Friend, H. C. Oettgen. 2004. IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis. J. Immunol. 172:1139.-1145. [Abstract/Free Full Text]
  41. Chen, R., G. Ning, M. L. Zhao, M. G. Fleming, L. A. Diaz, Z. Werb, Z. Liu. 2001. Mast cells play a key role in neutrophil recruitment in experimental bullous pemphigoid. J. Clin. Invest. 108:1151.-1158. [Medline]
  42. Clejan, S., S. Japa, C. Clemetson, S. S. Hasabnis, O. David, J. V. Talano. 2002. Blood histamine is associated with coronary artery disease, cardiac events and severity of inflammation and atherosclerosis. J. Cell. Mol. Med. 6:583.-592. [Medline]
  43. Filipiak, K. J., B. Tarchalska-Krynska, G. Opolski, A. Rdzanek, J. Kochman, D. A. Kosior, A. Czlonkowski. 2003. Tryptase levels in patients after acute coronary syndromes: the potential new marker of an unstable plaque. Clin. Cardiol. 26:366.-372. [Medline]
  44. Humbert, M., J. A. Grant, L. Taborda-Barata, S. R. Durham, R. Pfister, G. Menz, J. Barkans, S. Ying, A. B. Kay. 1996. High-affinity IgE receptor (Fc{epsilon}RI)-bearing cells in bronchial biopsies from atopic and nonatopic asthma. Am. J. Respir. Crit. Care Med. 153:1931.-1937. [Abstract]
  45. Mellor, E. A., N. Frank, D. Soler, J. M. Jora, M. R. Hodge, K. F. Austen, J. A. Boyce. 2003. Expression of the type 2 cysteinyl leukotriene receptor by human mast cells; demonstration of function distinct from that of CysLTR1. Proc. Natl. Acad. Sci. USA 100:11589.-11593. [Abstract/Free Full Text]
  46. Ryzhov, S., A. E. Goldstein, A. Matafonov, D. Zeng, I. Biaggioni, I. Feoktistov. 2004. Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma. J. Immunol. 172:7726.-7733. [Abstract/Free Full Text]
  47. Yeatman, C. F., Jr, S. M. Jacobs-Helber, P. Mirmonsef, S. R. Gillespie, L. A. Bouton, H. A. Collins, S. T. Sawyer, C. P. Shelburne, J. J. Ryan. 2000. Combined stimulation with the T helper cell type 2 cytokines interleukin (IL)-4 and IL-10 induces mouse mast cell apoptosis. J. Exp. Med. 192:1093.-1103. [Abstract/Free Full Text]
  48. Oskeritzian, C. A., Z. Wang, J. P. Kochan, M. Grimes, Z. Du, H. W. Chang, S. Grant, L. B. Schwartz. 1999. Recombinant human (rh) IL-4-mediated apoptosis and recombinant human IL-6-mediated protection of recombinant human stem cell factor-dependent human mast cells derived from cord blood mononuclear cell progenitors. J. Immunol. 163:5105.-5115. [Abstract/Free Full Text]
  49. Toru, H., R. Pawankar, C. Ra, J. Yata, T. Nakahata. 1998. Human mast cells produce IL-13 by high-affinity IgE receptor cross-linking: enhanced IL-13 production by IL-4-primed human mast cells. J. Allergy Clin. Immunol. 102:491.-502. [Medline]
  50. Wang, H. W., N. Tedla, A. R. Lloyd, D. Wakefield, P. H. McNeil. 1998. Mast cell activation and migration to lymph nodes during induction of an immune response in mice. J. Clin. Invest. 102:1617.-1626. [Medline]
  51. Campbell, E. M., I. F. Charo, S. L. Kunkel, R. M. Strieter, L. Boring, J. Gosling, N. W. Lukacs. 1999. Monocyte chemoattractant protein-1 mediates cockroach allergen-induced bronchial hyperreactivity in normal but not CCR2–/– mice: the role of mast cells. J. Immunol. 163:2160.-2167. [Abstract/Free Full Text]
  52. Malaviya, R., T. Ikeda, E. Ross, S. N. Abraham. 1996. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-{alpha}. Nature 381:77.-80. [Medline]
  53. McLachlan, J. B., J. P. Hart, S. V. Pizzo, C. P. Shelburne, H. F. Staats, M. D. Gunn, S. N. Abraham. 2003. Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat. Immunol. 4:1199.-1205. [Medline]
  54. Stevens, T. L., A. Bossie, V. M. Sanders, R. Fernandez-Botran, R. L. Coffman, T. R. Mosmann, E. S. Vitetta. 1988. Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334:255.-258. [Medline]
  55. Akbari, O., P. Stock, E. Meyer, M. Kronenberg, S. Sidobre, T. Nakayama, M. Taniguchi, M. J. Grusby, R. H. DeKruyff, D. T. Umetsu. 2003. Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nat. Med. 9:582.-588. [Medline]
  56. Min, B., M. Prout, J. Hu-Li, J. Zhu, D. Jankovic, E. S. Morgan, J. F. Urban, Jr, A. M. Dvorak, F. D. Finkelman, G. LeGros, et al 2004. Basophils produce IL-4 and accumulate in tissues after infection with a Th2-inducing parasite. J. Exp. Med. 200:507.-517. [Abstract/Free Full Text]
  57. Page, K., V. S. Hughes, K. K. Odoms, K. E. Dunsmore, M. B. Hershenson. 2005. German cockroach proteases regulate interleukin-8 expression via nuclear factor for interleukin-6 in human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 32:225.-231. [Abstract/Free Full Text]
  58. Blaeser, F., P. J. Bryce, N. Ho, V. Raman, F. Dedeoglu, D. D. Donaldson, R. S. Geha, H. C. Oettgen, T. A. Chatila. 2003. Targeted inactivation of the IL-4 receptor {alpha} chain I4R motif promotes allergic airway inflammation. J. Exp. Med. 198:1189.-1200. [Abstract/Free Full Text]
  59. Ahmad, F., G. Gao, L. M. Wan, T. R. Landstrom, E. Degerman, J. H. Pierce, V. C. Manganiello. 1999. IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells. J. Immunol. 162:4864.-4875. [Abstract/Free Full Text]
  60. Keegan, A. D., K. Nelms, M. White, L. M. Wang, J. H. Pierce, W. E. Paul. 1994. An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated