2 Production in Response to Prostaglandin D for Exaggerated

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Leukotriene E4 Activates Human Th2 Cells for Exaggerated Proinflammatory Cytokine Production in Response to Prostaglandin D2

This information is current as Luzheng Xue, Anna Barrow, Vicki M. Fleming, Michael G. of September 27, 2021. Hunter, Graham Ogg, Paul Klenerman and Roy Pettipher J Immunol 2012; 188:694-702; Prepublished online 14 December 2011; doi: 10.4049/jimmunol.1102474

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Leukotriene E4 Activates Human Th2 Cells for Exaggerated Proinﬂammatory Cytokine Production in Response to Prostaglandin D2

Luzheng Xue,* Anna Barrow,† Vicki M. Fleming,‡ Michael G. Hunter,† Graham Ogg,*,x Paul Klenerman,*,‡ and Roy Pettipher†

PGD2 exerts a number of proinflammatory responses through a high-affinity interaction with chemoattractant receptor- homologous molecule expressed on Th2 cells (CRTH2) and has been detected at high concentrations at sites of allergic inflammation. Because cysteinyl leukotrienes (cysLTs) are also produced during the allergic response, we investigated the possibility that cysLTs may modulate the response of human Th2 cells to PGD2. PGD2 induced concentration-dependent Th2 cytokine production in the absence of TCR stimulation. Leukotrienes D4 and E4 (LTE4) also stimulated the cytokine production but were much less Downloaded from active than PGD2. However, when combined with PGD2, cysLTs caused a greater than additive enhancement of the response, with LTE4 being most effective in activating Th2 cells. LTE4 enhanced calcium mobilization in response to PGD2 in Th2 cells without affecting endogenous PGD2 production or CRTH2 receptor expression. The effect of LTE4 was inhibited by montelukast but not by the P2Y12 antagonist methylthioadenosine 59-monophosphate. The enhancing effect was also evident with endogenous cysLTs produced from immunologically activated mast cells because inhibition of cysLT action by montelukast or cysLT synthesis by

MK886, an inhibitor of 5-lipoxygenase–activating protein, reduced the response of Th2 cells to the levels produced by PGD2 alone. http://www.jimmunol.org/ These findings reveal that cysLTs, in particular LTE4, have a significant proinflammatory impact on T cells and demonstrate their effects on Th2 cells are mediated by a montelukast-sensitive receptor. The Journal of Immunology, 2012, 188: 694–702.

oth PGD2 and cysteinyl leukotrienes (cysLTs) are prod- kine release in transgenic mice overexpressing PGD2 synthase (9). ucts of the oxidative metabolism of arachidonic acid and Two distinct G protein-coupled receptors have been identiﬁed as B have been detected in high concentrations at sites of al- PGD2 receptors: D prostanoid receptor 1 and chemoattractant recep- lergic inﬂammation and play central roles in promoting airway tor-homologous molecule expressed on Th2 cells (CRTH2). In

inflammation and deterioration in lung function, often acting in recent years, increasing evidence suggests that through its action by guest on September 27, 2021 concert (1, 2). on CRTH2, PGD2 elicits many proinflammatory responses in PGD2, produced by the activity of the cyclooxygenase enzymes, leukocytes, including chemotaxis of eosinophils, basophils, and is the major prostanoid released from mast cells during an allergic Th2 cells (10, 11), cytokine production by Th2 cells (12, 13), and response (3, 4), although macrophages (5), dendritic cells (6), and proinflammatory protein expression by eosinophils and Th2 cells + CD4 Th2 lymphocytes (7, 8) may contribute to PGD2 production (12, 14). Our recent studies also demonstrated that activation of in some circumstances. A significant contribution of PGD2 to the CRTH2 suppresses Th2 cell apoptosis (15), a process that is likely development of allergic inflammation has been suggested by the to impede the resolution of allergic inflammation. Allergic re- observations of enhanced eosinophilic lung inflammation and cyto- sponses mediated by IgE, mast cells, Th2 cells, and eosinophils are dramatically reduced in mice in which CRTH2 is genetically *Oxford Biomedical Research Centre, John Radcliffe Hospital, University of Oxford, ablated or by small molecule CRTH2 antagonists (16–19). An- † Oxford OX3 9DU, United Kingdom; Oxagen Ltd., Abingdon OX14 4RY, United tagonism of CRTH2 is currently being considered as a potentially Kingdom; ‡Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford OX1 3SY, United Kingdom; and xMedical Re- useful approach for the treatment of allergic diseases, including search Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, asthma, rhinitis, and atopic dermatitis (20). John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom CysLTs, including cysteinyl leukotrienes C4 (LTC4), D4 (LTD4), Received for publication August 29, 2011. Accepted for publication November 10, and E (LTE ), are derived from the 5-lipoxygenase pathway 2011. 4 4 of the arachidonic acid metabolism (21, 22). LTC is formed by This work was supported in part by a grant from the Oxford Biomedical Research 4 Centre (to L.X., P.K., and G.O.) and the James Trust Grant from the British Medical conjugation of LTA4 with reduced glutathione and after extra- Association (to G.O., P.K., and L.X.), with additional funding from the Wellcome cellular export is converted to LTD4 and then the stable metabolite Trust (Grant WT091663MA to P.K. and V.M.F.), the Oxford Martin School (to P.K.), and the Medical Research Council (to G.O.). LTE4 by sequential enzymatic removal of glutamic acid followed by glycine. Two G protein-coupled receptors for LTs have been Address correspondence and reprint requests to Dr. Luzheng Xue, Oxford Biomed- ical Research Centre, NDM Experimental Medicine, John Radcliffe Hospital, Uni- cloned, characterized, and designated as CysLT1 and CysLT2 (23– versity of Oxford, Oxford OX3 9DU, United Kingdom. E-mail address: luzheng.xue@ 25). LTD4 binds CysLT1 with higher affinity than LTC4, whereas ndm.ox.ac.uk CysLT2 binds these cysLTs with equal affinity. LTE4 has only Abbreviations used in this article: CHO, Chinese hamster ovary; CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells; cysLT, cysteinyl weak activity on either CysLT1 (23, 24) or CysLT2 (25, 26) and leukotriene; LTC4, leukotriene C4;LTD4, leukotriene D4;LTE4, leukotriene E4; has therefore been generally considered to be a stable inactive 2MeS, 2-methylthioadenosine 59-monophosphate; PTX, pertussis toxin; qPCR, breakdown product, although there is accumulating evidence that real-time quantitative PCR. LTE4 can stimulate inflammatory responses through mechanisms Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 independent of CysLT1 or CysLT2 (27–31). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102474 The Journal of Immunology 695

CysLT1 mediates bronchoconstriction and also a range of The cells were pretreated with 5 mg/ml purified human myeloma IgE for 4 proinflammatory effects including activation and migration of d, washed, and then sensitized passively with fresh IgE (5 mg/ml) for 2 h. leukocytes (21, 32, 33), whereas CysLT may mediate the vaso- The cells were washed once with medium in absence or presence of 10 mM 2 diclofenac, 10 mM MK886, or both and then continued to be incubated active effects of LTC4 and LTD4. The leukotriene antagonists with medium or challenged with goat anti-human IgE (1 mg/ml) in the approved for use in asthma and allergic rhinitis, most notably presence or absence of diclofenac, MK886, or both for 1 h. The super- montelukast, block the action of cysLTs (predominantly LTD4)on natants of the cells were collected and measured for PGD2 using a PGD2– CysLT but do not inhibit CysLT -mediated effects. Monotherapy MOX enzyme immunoassay kit and LTE4 using a LTE4 enzyme immu- 1 2 noassay kit according to the manufacturer’s instructions. The supernatants with montelukast thereby inhibits the CysLT1-mediated broncho- were aliquoted and stored at 280˚C until used as mast cell supernatants for constrictor element of asthma, but its anti-inflammatory activity the treatment of Th2 cells. and, consequently, clinical efficacy are modest compared with that + + of inhaled steroids (34). However, in patients with asthma not Human CRTH2 CD4 Th2 cell culture and treatment sufficiently controlled with inhaled steroids alone, add-on therapy Human CRTH2+CD4+ Th2 cells were prepared as described in our previous with montelukast to a constant dose of inhaled steroid improves report (13). Briefly, PBMC were isolated from buffy coats (National Blood asthma control (35). Recent data suggest in primary care that Service, Bristol, U.K.) by Lymphoprep density gradient centrifugation, followed by CD4+ cell purification using a CD4+ T cell isolation kit II. leukotriene antagonists may have a more significant role as early After 7 d culture in AIM V medium containing 50 U/ml rIL-2 and 100 ng/ therapy than previously considered because compliance of safe ml rIL-4, CRTH2+ cells were isolated from the CD4+ cultures by positive oral agent is better than with inhaled glucocorticoid (36). selection using an anti-human CRTH2 MicroBead Kit. The harvested CD4+ + We found that cysLTs markedly potentiated proinflammatory CRTH2 cells were treated as Th2 cells and amplified further by stimu- cytokine production from human Th2 cells in response to PGD . lation with the T cell activation/expansion kit and grown in X-VIVO 15 Downloaded from 2 medium containing 10% human serum and 50 U/ml IL-2 before use. The potency of LTE4 in the enhancing effect was significantly The Th2 cells were treated with X-VIVO 15 medium containing 10% higher than that of LTD4 or LTC4, and this enhancing effect of human serum in the presence or absence of PGD2, cysLTs, or other com- LTE4 was inhibited by montelukast. Although the CRTH2 an- pounds or treated with various mast cell supernatants in the presence or tagonist TM30089 alone substantially inhibited IL-13 production absence of antagonist compounds, as indicated in the results at 37˚C and 5% CO2 for different periods of time for qPCR, ELISA, or ELISPOT. In in response to both exogenous and endogenous PGD2 and LTE4, some experiments, Th2 cells were preincubated with pertussis toxin (PTX) a combination of TM30089 and montelukast was required to as indicated in the results. http://www.jimmunol.org/ completely inhibit the response. ELISA These data highlight an important interaction between PGD2 and cysLTs in promoting Th2 cell activation at sites of allergic Th2 cells were treated with different compounds for 6 h, and then the inflammation and further point to the importance of TCR- supernatants were collected. The concentrations of IL-4 and IL-13 in the independent mechanisms of T cell activation in inflammatory supernatants were assayed using enzyme-linked immunoassay kits, according to the manufacturer’s instructions. The results were measured in a responses. Further study of these mechanisms may lead to im- 2 Victor V-1420 multilabel HTS Counter (PerkinElmer Life Sciences). proved therapies for allergic inflammation. ELISPOT cytokine assays by guest on September 27, 2021 Materials and Methods Th2 cells (4 3 103 cell/well) were seeded in a MultiScreen Filter Plate Reagents (Millipore) coated with capture Ab against human IL-4 or IL-13 and treated with PGD2,LTE4 alone, or in combination at 37˚C 5% CO2 for 15 PGD2,LTC4,LTD4, and LTE4 were purchased from Enzo Life Science; h. The plate was washed extensively with PBS containing 0.05% Tween 20 PGD2–MOX enzyme immunoassay kits and LTE4 enzyme immunoassay and then immunoassayed using ELISPOT kits following the manu- kits were purchased from Cayman Chemicals; TM30089 was supplied by + facturer’s instructions. Briefly, the plate was incubated with specific bio- ChemieTek; human CD4 T cell isolation kit II, anti-human CRTH2 tinylated Ab (anti–IL-4 or anti–IL-13) followed by labeling with MicroBead Kits, human CD34 MicroBead kits, and T cell activation/ streptavidin-alkaline phosphatase. Spots were developed by adding sub- expansion kits were from Miltenyi Biotec; human recombinant stem cell strate and then analyzed with an AID ELISPOT Reader System (Auto- factor, human rIL-6, and human IL-4/5/13 immunoassay kits were pur- immunDiagnostika). chased from R&D Systems; IMDM and X-VIVO 15 medium were purchased from Lonza; AIM V medium was from Invitrogen; human my- qPCR eloma IgE and Abs against human tryptase and chymase were purchased from Chemicon International; Lymphoprep was purchased from Axis- Th2 cells were treated for 2.5 h, and the total RNA of the cells was extracted Shield UK; the MultiScreen filter plates for ELISPOT were obtained using an RNeasy Mini kit. cDNA of the samples was prepared from the from Millipore; ELISPOT kits for human IL-4/IL-13 were purchased from same starting amount of RNA using an Omniscript reverse transcription Mabtech; alkaline phosphatase conjugate substrate kit was from Bio-Rad; kit. qPCR was conducted using Master Mix and Probe in a LightCycler 480 RNeasy Mini kit and Omniscript reverse transcription kit were supplied Real-Time PCR System (Roche). GAPDH was used as control gene. The from Qiagen; real-time quantitative PCR (qPCR) Master Mix and probes changes of mRNA level compared with untreated sample were calculated were from Roche; FLIPR Ca2+ 5 dye was purchased from Molecular according to the manufacturer’s instructions. Devices; PathHunter eXpress b-arrestin assay kit for human CRTH2 was supplied by DiscoveRx; and human rIL-2, human rIL-4, goat anti-human IgE, diclofenac, and other chemicals were from Sigma-Aldrich. Human mast cell culture and activation Human mast cells were cultured from CD34+ progenitor cells using a modified method described previously (37). Briefly, CD34+ progenitor cells were isolated from human cord blood (National Blood Service, Ox- ford, U.K.) by using a human CD34 MicroBead kit. The cells were cultured at a density of 1 3 105 cells/ml with IMDM containing 10% human serum, 0·55 mM 2-ME, penicillin/streptomycin, human recombinant stem FIGURE 1. Stimulation of Th2 cells with PGD ,LTD,orLTE evokes cell factor (100 ng/ml), and human rIL-6 (50 ng/ml) in 5% CO at 37˚C for 2 4 4 2 IL-13 production. Th2 cells (1.5 3 106 cell/ml) were treated with various 14 to 15 wk. Half of the culture medium was replaced twice weekly with s n : fresh medium containing the same concentration of cytokines. The ex- concentration of PGD2 ( ), LTE4 ( ), or LTD4 ( ) for 6 h. The cell pression of tryptase and chymase of the cells was tested by immuno- supernatant was collected, and the concentrations of IL-13 were deter- staining using the method described previously (37). The mast cells used in mined by ELISA. Data are expressed mean 6 SEM of two to five inde- this study were tryptase positive (.80%) and chymase negative (,1%). pendent experiments. 696 PROINFLAMMATORY EFFECTS OF LTE4 ON Th2 CELLS Downloaded from http://www.jimmunol.org/

FIGURE 2. Enhancement by cysLTs of the Th2 cytokine production in response to PGD2. A, Th2 cells were incubated with 100 nM PGD2,50nMLTD4, 50 nM LTE4 alone, or in combination as indicated for 6 h. The concentrations of IL-13 in the cell supernatants were determined with ELISA. The average background level of IL-13 in the control samples is indicated with a dashed line. B, Th2 cells were incubated with 100 nM PGD2,50nMLTE4 alone, or in combination as indicated for 15 h and then analyzed with ELISPOT for IL-13 or IL-4. Upper panels show representative images. The number (left side in the lower panels) and the average intensity (right side in the lower panels) of the spots were analyzed. C, Th2 cells were treated with various concentration by guest on September 27, 2021 of PGD2 in absence (d) or presence of 50 nM LTD4 (N)or50nMLTE4 (4) for 6 h. The concentrations of IL-13 in the supernatants were measured by ELISA. The IL-13 levels detected in the samples treated with control medium (—), 50 nM LTD4 (s), or 50 nM LTE4 (X) alone are included for comparison. D, Th2 cells were treated with medium alone or medium containing 100 nM PGD2,50nMLTE4, or both for 2.5 h. Total RNA was extracted from the cell pellets. The mRNA levels of IL-13 (upper panel), IL-4 (middle panel), and IL-5 (lower panel) were measured by using qPCR. The mRNA levels in untreated cells were treated as 1 fold. Data are expressed as mean 6 SEM of three to nine independent experiments. p , 0.005 by ANOVA for all graphs. *Signiﬁcant differences between PGD2 +LTE4 or PGD2 +LTD4 and PGD2 alone (p , 0.05 by Newman–Keuls test). Other signiﬁcant differences are not illustrated.

Primers and probes used were as follows: IL-4, 59-CACCGAGTTG- TCT-39 with probe 27 generating a 65-bp fragment; and GAPDH, 59- ACCGTAACAG-39 and 59-GCCCTGCAGAAGGTTTCC-39 with probe 16 AGCCACATCGCTCAGACAC-39 and 59-GCCCAATACGACCAAATCC- generating a 72-bp fragment; IL-5, 59-GGTTTGTTGCAGCCAAAGAT-39 39 with probe 60 generating a 66-bp fragment. and 59-TCTTGGCCCTCATTCTCACT-39 with probe 25 generating a 66- Ca2+ mobilization assay bp fragment; IL-13, 59-AGCCCTCAGGGAGCTCAT-39 and 59-CTCC- ATACCATGCTGCCATT-39 with probe 17 generating a 84-bp fragment; Chinese hamster ovary (CHO)/CRTH2 cells (Evotec-OAI) (8 3 104 cells/ CRTH2, 59-CCTGTGCTCCCTCTGTGC-39 and 59-TCTGGAGACGGC- well) were seeded into a poly-D-lysine–coated 96-well plate and incubated TCATCTG-39 with probe 43 generating a 95-bp fragment; CysLT1,59- at 37˚C overnight. The culture medium was changed to HBSS before use. ACTCCAGTGCCAGAAAGAGG-39 and 59-GCGGAAGTCATCAATAG- Th2 cells were washed once with HBSS and then seeded into a poly-D- 5 TGTCA-39 with probe 29 generating a 112-bp fragment; CysLT2,59-CT- lysine–coated 96-well plate at a density of 2 3 10 cells/well. The cells AGAGTCCTGTGGGCTGAAA-39 and 59-GTAGGATCCAATGTGCTTT- were incubated with FLIPR Ca2+ 5 dye in the presence or absence of PTX GC-39 with probe 48 generating a 61-bp fragment; P2Y12,59-TTTGC- at 37˚C 5% CO2 for 1 h. The plate for Th2 cells was centrifuged at 600 CTAACATGATTCTGACC-39 and 59-GGAAAGAGCATTTCTTCACAT- rpm for 10 min before the assay. The changes in ﬂuorescence after test

Table I. IL-13 levels in the supernatants from Th2 cells after various treatments

IL-13 Level (pg/4 3 106 cell/ml)

Treatment Readout Net Increase Control 226.5 6 54.8 (n = 10) 100 nM PGD2 949.1 6 219.6 (n = 11) 682.7 6 202.9 50 nM LTC4 302.4 6 56.8 (n = 2) 35.9 6 28.2 50 nM LTC4 + 100 nM PGD2 1269.3 6 119.9 (n = 2) 1002.9 6 111.3 50 nM LTD4 301.8 6 22.7 (n = 4) 35.4 6 24 50 nM LTD4 + 100 nM PGD2 1226.6 6 174.9 (n = 4) 960.1 6 206.3 50 nM LTE4 554.4 6 181.8 (n = 9) 288 6 121.2 50 nM LTE4 + 100 nM PGD2 2571.2 6 450.5 (n = 9) 2304.8 6 300.3 The Journal of Immunology 697

Table II. Comparison of the additive effect and synergistic effect of PGD2 and LTE4 in Th2 cytokine production

a 100 nM PGD2 50 nM LTE4 Additive PGD2/LTE4 p , 0.05 IL-4 protein (pg/ml) 7.78 6 1.9 5.57 6 2.51 13.36 6 0.61 24.39 6 0.71 Yes IL-13 protein (pg/ml) 662.7 6 143.2 288 6 121.2 970.7 6 250.4 2304.8 6 300.3 Yes IL-4 mRNA (fold) 1.03 6 0.3 0.85 6 0.12 1.88 6 0.42 1.66 6 0.27 No IL-5 mRNA (fold) 2.78 6 0.67 1.23 6 0.07 4.02 6 0.75 7.47 6 1.03 Yes IL-13 mRNA (fold) 3.56 6 0.66 1.72 6 0.28 5.27 6 0.63 8.42 6 1.49 Yes IL-4 ELISPOT intensity 1785.7 6 183.9 91.7 6 55.35 1877.3 6 199.9 2737 6 350.6 Yes IL-13 ELISPOT intensity 726.3 6 68.3 360.2 6 37.3 1086.5 6 84.2 1549.8 6 104 Yes The data are net increase and expressed as mean 6 SEM of two to nine independent experiments. a Signiﬁcant difference between additive (sum of values from the treatments with PGD2 and LTE4 alone) and PGD2/LTE4 (values from combination treatment with PGD2 and LTE4) by Newman–Keuls test. compound loading (50 ml) were monitored over a 75-s time course with To determine the effect of the treatment with the combination compound loading at 17 s, using a Flexstation (Molecular Devices). of PGD2 and cysLTs at the gene transcription level, the levels of b-arrestin recruitment assay mRNA encoding IL-4, IL-5, and IL-13 in Th2 cells were mea- The assay was performed using a PathHunter eXpress b-arrestin kit Downloaded from (DiscoveRx) for human CRTH2 according to the manufacturer’s instructions. Brieﬂy, CHO/CRTH2 EA-arrestin cells were resuspended in OCC 2 media and seeded in a 96-well luminescence plate (white walled, clear bottom) for recovering at 37˚C 5% CO2 for 48 h. Treatment compounds were added for a 90-min incubation at 37˚C 5% CO2, and then detection reagents were added for another 90-min incubation at room temperature. Luminescence was measured using a Victor2 V-1420 multilabel HTS

Counter (PerkinElmer Life Sciences). http://www.jimmunol.org/ Statistics Data were analyzed using one-way ANOVA followed by the Newman– Keuls test. Values of p , 0.05 were considered statistically signiﬁcant.

Results LTD4 and LTE4 are weak inducers of cytokine production by human Th2 cells compared with PGD2 by guest on September 27, 2021 As previously reported, PGD2 induced production of IL-13 when incubated with Th2 cells (13). Both LTD4 and LTE4 alone also induced IL-13 production in a dose-dependent manner with an EC50 similar to PGD2 (75.6 nM for PGD2, 107.8 nM for LTD4, and 26.2 nM for LTE4) (Fig. 1). However, the maximum responses achieved by LTD4 and LTE4 were only 23 and 41%, respectively, of that achieved by PGD2. CysLTs signiﬁcantly enhance Th2 cytokine production in response to PGD2

To define the effect of the combination of PGD2 and cysLTs on Th2 cytokine production, Th2 cells were treated with PGD2,LTD4,or LTE4 alone (at concentrations close to their relative EC50)orin combination as indicated in Fig. 2 for 6 h. Interestingly, both LTD4 and LTE4 significantly enhanced IL-13 production in response to PGD2 (Fig. 2A, Table I). The enhancement was greater than additive (p , 0.05), and most marked with LTE4 (Table II). LTC4 showed a similar enhancing effect of LTD4 in the combination FIGURE 3. Inhibitory effect of TM30089 and montelukast on IL-13 treatment (Table I). ELISPOT analysis of IL-13 and IL-4 levels production induced by PGD2 and LTE4. Th2 cells were treated with 100 indicated that the enhancement occurred at the level of individual nM PGD2,50nMLTD4 (A, top panel), or 50 nM LTE4 (A, bottom panel) cells and not by more responder cells (Fig. 2B); as compared with alone or in combination in the presence or absence of 1 mM TM30089, the samples treated with PGD2 alone, the combined use of PGD2 1 mM montelukast, or both as indicated or with 100 nM PGD2 and 50 nM LTE (B) in the presence of various concentrations of montelukast (d) for and LTE4 did not change the total spot numbers for both IL-4 and 4 IL-13 significantly but enhanced the average intensities of the 6 h. The levels of IL-13 in the supernatants were determined by ELISA. 4 spots (Table II). The IL-13 levels detected in the samples treated with control medium ( ), 100 nM PGD alone (s), or combined with 50 nM LTE (X) are included To understand the nature of the enhancing effect of cysLTs on 2 4 in B for comparison. Data are expressed as mean 6 SEM of three to seven the Th2 cytokine production, the cells were stimulated with increas- independent experiments. For A, top panel, p = 0.002, and for A, bottom ing concentrations of PGD in the absence or presence of 50 nM 2 panel, p = 0.0002 by ANOVA. The significant differences between PGD2 + cysLTs for 6 h as indicated in Fig. 2C. The addition of cysLTs LTD4 (A, top panel) or PGD2 +LTE4 (A, bottom panel) and all other increased the maximum response to PGD2, but did not signifi- conditions are indicated with an asterisk (*p , 0.05 by Newman–Keuls cantly affect the EC50 of PGD2 (Fig. 2C). test). Other significant differences are not indicated. 698 PROINFLAMMATORY EFFECTS OF LTE4 ON Th2 CELLS

sured by qPCR after different treatments of the cells for 2.5 h (Fig. enhancing effect of LTE4. We also investigated the effect of LTE4 2D). The synergistic effect of PGD2 and LTE4 on the upregulation on the expression of CRTH2 by qPCR (Fig. 4B). LTE4 did not of IL-5 and IL-13 mRNA was apparent, although the effect on IL- show any effect on the level of CRTH2 mRNA, suggesting that the 4 at the level of mRNA was not signiﬁcant (Fig. 2D, Table II). enhancing effect of LTE4 is not mediated by an increase of transcription at the CRTH2 locus. Effect of the CRTH2 antagonist TM30089 and montelukast on cytokine production in response to PGD2 and cysLTs CysLTs do not interact with CRTH2 directly

The effects of the CRTH2 antagonist TM30089 and the CysLT1 To rule out the possibility that cysLTs bind CRTH2, the effects of antagonist montelukast on cytokine production by Th2 cells in a selective CRTH2 antagonist on the cytokine production induced response to combinations of PGD2 with LTD4 (Fig. 3A, LTD4)or by LTE4 alone were examined (Fig. 5A). As expected, 1 mM LTE4 (Fig. 3A, LTE4) were investigated. TM30089 reduced cy- TM30089 completely inhibited responses to PGD2 but not to tokine production to the level produced by the leukotriene alone LTE4; vice versa, 1 mM montelukast blocked the effect of LTE4 to in the case of LTD4 or even below in the case of LTE4.TheLTD4- the background level but had no effect on PGD2. or LTE4-mediated enhancement was inhibited by montelukast It has been established that two signal mechanisms are in- that effectively reduced responses to similar levels to those of volved in the activation of CRTH2, Ca2+ inﬂux, and b-arrestin PGD2 alone. In both cases, the combination of TM30089 and recruitment (10, 38–40). We further analyzed the effect of LTE4 montelukast completely inhibited cytokine production by Th2 cells.

To further understand the potency of montelukast in the inhi- Downloaded from bition of enhancing effect of cysLTs in Th2 cytokine production induced by PGD2, Th2 cells were treated with 100 nM PGD2 and 50 nM LTE4 in the presence of increasing concentrations of montelukast for 6 h (Fig. 3B). The inhibitory effect of montelukast was dose dependent, with IC50 = 0.2 nM. The enhancing effect of

50 nM LTE4 was completely abolished by 10 nM montelukast. http://www.jimmunol.org/

LTE4 does not affect PGD2 production and CRTH2 expression in Th2 cells It has been reported that activated Th2 cells are able to produce PGD2 (7, 8). To understand whether the enhancing effect of cysLTs is mediated by a secondary production of PGD2 by Th2 cells, we examined the effect of diclofenac, a cyclooxygenase inhibitor, on cytokine production induced by LTE4 (Fig. 4A).

Diclofenac (10 mM) did not change the IL-13 production induced by guest on September 27, 2021 by LTE4, demonstrating that PGD2 synthesis is not involved in the

FIGURE 5. Effect of LTE4 on responses mediated by CRTH2. A, Th2 cells were incubated with 100 nM PGD2 or 50 nM LTE4 in the presence or absence of 1 mM TM30089 or 1 mM montelukast for 6 h. The IL-13 levels in the supernatants were measured by ELISA. Calcium mobilization in

response to various concentrations of LTE4 (4) and PGD2 alone (d)orin combination in the absence (N) or presence of 1 mM montelukast (,)in

FIGURE 4. Effect of LTE4 on the expression of PGD2 and CRTH2 in CHO-CRTH2 cells (B) or Th2 cells (D) was measured as described in the Th2 cells. A, Th2 cells were incubated with 50 nM LTE4 in the presence or Materials and Methods. Data are reported in relative fluorescence units absence of 10 mM diclofenac for 6 h. The IL-13 levels in the supernatants (RFU). C, CHO-CRTH2 EA-arrestin cells were treated with various con- were measured by ELISA. B, Th2 cells were stimulated with 50 nM LTE4 centrations of PGD2 (d)orLTE4 (4) for 90 min. b-arrestin recruitment in for 2.5 h. Total RNA was extracted from the cell pellets. The mRNA levels the cells was measured with a PathHunter eXpress b-arrestin kit (Dis- of CRTH2 were measured by using qPCR. The mRNA levels in untreated coveRx). Data are presented in relative light units (RLU). Data are cells were treated as 1 fold. Data are expressed as mean 6 SEM of two (A) expressed as mean 6 SEM of two to eight independent experiments. For A, or three (B) independent experiments. For A, p = 0.01 and for B, p , p , 0.01 by ANOVA. Only significant difference between PGD2 and PGD2 + 0.0001 by ANOVA. *Significant difference between control and other TM30089 is indicated with an asterisk (*p , 0.05 by Newman–Keuls test). conditions in A (p , 0.05 by Newman–Keuls test). Other significant values are not indicated. The Journal of Immunology 699 on the downstream signaling of CRTH2 (Fig. 5B, C). In CHO cells activated with IgE/anti-IgE on Th2 cytokine production was 6 cells overexpressing human rCRTH2, PGD2, but not LTE4,in- investigated (Fig. 7). Only low levels of PGD2 (,0.1 ng/2 3 10 2+ 6 duced Ca mobilization (Fig. 5B)andb-arrestin recruitment cell/ml) and LTE4 (,10 ng/2 3 10 cell/ml) were detectable in the (Fig. 5C) in a dose-dependent manner. Addition of LTE4 did supernatant from the resting mast cells (supernatant 1 in upper 2+ not enhance the Ca mobilization induced by PGD2 in the panel of Fig. 7A). After 1 h activation with IgE/anti-IgE, mast 6 recombinant cell system (Fig. 5B). However, in Th2 cells, both cells produced high levels of both PGD2 (.12 ng/2 3 10 cell/ml) 2+ 6 PGD2 and LTE4 induced Ca mobilization in a dose-dependent and LTE4 (.85 ng/2 3 10 cell/ml) (supernatant 2). Cotreatment manner, although the efficacy of LTE4 was much weaker (Fig. of IgE/anti-IgE–activated mast cells with diclofenac (10 mM) 2+ 5D). Addition of LTE4 enhanced the Ca mobilization induced during the period of anti-IgE stimulation abolished production of 6 by PGD2 significantly, an effect that was completely inhibited by PGD2 but enhanced production of LTE4 (.135 ng/2 3 10 cell/ montelukast. ml) (supernatant 3). Cotreatment of the mast cells with MK886 (10 mM), an inhibitor of FLAP, during the activation blocked The enhancing effect of LTE4 was not mediated by P2Y12 production of LTE4 but increased the level of PGD2 (.15 ng/2 3 receptor but by a receptor that was partially sensitive to PTX 106 cell/ml) (supernatant 4). Cotreatment of the mast cells with It has also been reported that LTE4 can act as an agonist at the both diclofenac and MK886 abolished production of both PGD2 P2Y12 receptor (41, 42) and that montelukast can antagonize some and LTE4 (supernatant 5). Only very low levels of IL-13 (,200 P2Y receptors (43), although the effect of montelukast has not pg/2 3 106 cell/ml) could be detected in any of these mast cell been tested on P2Y12. The expression of P2Y12 can be detected in supernatants (Fig. 7A, lower panel). human Th2 cells, but the level of this receptor is much lower than Using these supernatants to treat human Th2 cells revealed, Downloaded from that seen with CysLT1 (Fig. 6A). To address the role of P2Y12 as expected, different capacities to stimulate IL-13 production in an LTE4-induced enhancing effect, the effect of 2-methyl- depending on the levels of PGD2 and LTE4 contained in the thioadenosine 59-monophosphate (2MeS), an inhibitor of P2Y12 supernatants (Fig. 7B). Supernatant 2 containing both PGD2 and (42), was investigated. 2MeS (100 mM) had no significant effect LTE4 possessed the highest stimulatory capacity to induce IL-13 on the IL-13 production induced by PGD2 or LTE4 alone or in production. Treatment with diclofenac alone (supernatant 3) combination, suggesting that the P2Y12 receptor is not involved in caused substantial (∼76%) reduction of Th2 stimulatory activity http://www.jimmunol.org/ mediating the effects of LTE4 in this system (Fig. 6B). as reported previously (37), and MK886 (supernatant 4) partially To further understand the potential receptor mediating the effect (∼45%) reduced activity. Treatment of the mast cells with both of LTE4 in Th2 cells, we examined the effects of PTX, an inhibi- diclofenac and MK886 (supernatant 5) reduced the level of tor of the Gai pathway, on Ca2+ mobilization (Fig. 6C) and IL-13 stimulatory activity to the level produced by unactivated mast cell production (Fig. 6D) induced by LTE4 in Th2 cells. Both re- supernatant (supernatant 1). sponses to LTE4 were partially reduced by 1 mg/ml PTX but not by The stimulatory activity of the mast cell supernatant contain- 100 ng/ml PTX. ing both PGD2 and LTE4 (supernatant 2) on the Th2 cytokine production was reduced by treatment of Th2 cells with TM30089 Effect of mast cell supernatants containing endogenous PGD2 (1 mM) or to a lesser extent by montelukast (1 mM) (Fig. 8). by guest on September 27, 2021 and LTE4 on cytokine production by Th2 cells Treatment of the Th2 cells with both TM30089 and montelukast To analyze the impact of endogenously synthesized eicosanoids on reduced the level of stimulatory activity of supernatant 2 to that of Th2 function, the effect of supernatants collected from human mast the supernatant from unactivated mast cells (supernatant 1).

FIGURE 6. Effects of 2MeS and PTX on the responses induced by LTE4. A, Total RNA was extracted from untreated Th2 cells. The mRNA levels of CysLT1,CysLT2, and P2Y12 were measured by using qPCR. The mRNA levels of GAPDH were treated as 1 fold. B, Th2 cells were incubated with 100 nM PGD2 and 50 nM LTE4 alone or in combination in the presence or absence of 100 mM 2MeS for 6 h. The levels of IL-13 in the supernatants were determined by ELISA. Th2 cells were preincubated with 1 mg/ml or 100 ng/ml PTX for 10 min (C)or30min(D) and then stimulated with 1 mMLTE4 for calcium mobilization assay (C) or treated with 100 nM LTE4 in the presence of PTX for 6 h for IL-13 ELISA (D). Data in B–D are expressed as mean 6 SEM of two to four independent experiments. For B, p , 0.002, for C, p , 0.0001, and for D, p , 0.01 by ANOVA. Only signiﬁcant differences between control and

LTE4 +1mg/ml PTX, LTE4,andLTE4 +1mg/ml PTX are indicated with an asterisk (*p , 0.05 by Newman–Keuls test). RFU, relative ﬂuorescence units. 700 PROINFLAMMATORY EFFECTS OF LTE4 ON Th2 CELLS

activation of CRTH2 (12, 13). In this study, we have revealed a previously unrecognized role of cysLTs in potentiating cytokine production by human Th2 cells in response to PGD2. CysLTs alone are weak stimulators of Th2 cytokine production but sig- niﬁcantly enhanced cytokine production in response to PGD2. The combined effects of PGD2 and cysLTs at pathophysiological concentrations (similar to the concentrations produced by activated mast cells) were greater than additive (Table II), and interestingly, LTE4 was more potent than that of LTC4 and LTD4 in mediating this enhancing effect. The enhancing effect of cysLTs was inhibited by montelukast. A combination of TM30089 and montelukast was required to completely inhibit the synergistic effect of PGD2 and cysLTs. CysLTs are produced by mast cells, eosinophils, and other cells during the allergic response and have been detected in high concentrations in airways of asthmatics challenged with allergen (47), and LTE4 is present in the urine of patients during spontaneous asthmatic exacerbations (48). CysLTs are the most potent known

bronchoconstrictors (49, 50) and promote airway hyperrespon- Downloaded from siveness to histamine and PGD2 when they are administered by inhalation to human subjects (1, 2, 51). At the cellular level, they induce chemotaxis of eosinophils and neutrophils and prolong eosinophil survival (52, 53). The involvement of cysLTs in Th2 cytokine production is suggested by the observations that Th2

cytokine production and consequent airway inﬂammation are re- http://www.jimmunol.org/ FIGURE 7. Effect of mast cell supernatants on the cytokine production by Th2 cells. A, Human mast cells were activated with IgE/anti-IgE as duced in LTC4 synthase null mice (33), and cysLTs contribute to described in the Materials and Methods in the presence or absence of 10 cytokine production by allergen-speciﬁc Th2 cells stimulated with mM diclofenac, MK886, or both for 1 h as indicated, and the supernatants GM-CSF–primed macrophages (54). The present study provides were collected and assigned as mast cell supernatants 1 to 5. The levels of direct evidence that cysLTs are able to elicit cytokine production

PGD2,LTE4 (upper panel of A), and IL-13 (lower panel of A) in the by Th2 cells in the absence of Ag or any other costimulation and supernatants were measured by ELISA. B, Th2 cells were incubated with highlights the possibility that PGD2 and cysLTs act in concert to 1:1.5 diluted mast cell supernatants as indicated for 6 h. The IL-13 levels promote Th2 cytokine production. in the supernatants after the incubation were determined by ELISA. Data Our previous report demonstrated the critical role of PGD2 and

6 by guest on September 27, 2021 are expressed as mean SEM of three to four independent experiments. its receptor CRTH2 in mediating cytokine production from Th2 For upper panel in A, p , 0.0005, for lower panel in A, p , 0.05, and for cells in response to activated mast cells (37). The current study B, p , 0.001 by ANOVA. The significant differences in B are indicated with an asterisk (*p , 0.05 by Newman–Keuls test). indicates that cysLTs, particularly LTE4, can activate Th2 cells for exaggerated cytokine production in response to PGD2. Both PGD2 and LTE4 were detected in high levels in supernatants from im- Discussion munologically activated mast cells. Although the effect of the mast Cytokine production from Th2 cells plays an important role in cell supernatants is predominantly through interaction of PGD2 orchestrating allergic responses. High local concentrations of IL-4, with CRTH2, a contribution of cysLTs to this response was dem- IL-5, and IL-13 are detected in both airway and bronchial alveolar onstrated by selectively inhibiting mast cell cysLT production with lavage fluids from patients with asthma and are present at increased the FLAP inhibitor MK886 or inhibiting cysLT action with mon- levels following allergen challenge (44–46). It has been well telukast. Removal of the cysLT-dependent component of the re- established that PGD induces Th2 cytokine production through 2 sponse to that produced by PGD2 alone illustrates that endogenous cysLTs can activate Th2 cells for a heightened response to PGD2. The enhancing effect of cysLTs was not mediated by direct interaction with CRTH2, because in CHO cells overexpressing 2+ rCRTH2, LTE4 had no effect on Ca influx and b-arrestin recruitment that are downstream signaling events following CRTH2 activation (10, 39, 40). However, in human Th2 cells, LTE4 elicits Ca2+ mobilization in a dose-dependent manner and enhances Ca2+ mobilization in response to PGD2 markedly, indicating that cysLTs enhance signaling pathways downstream of CRTH2 receptor activation. It has been reported that IFN-g and TNF-a potentiated PGD2-induced eosinophil chemotaxis through upregulation of CRTH2 (55). However, the enhancing effect of cysLTs FIGURE 8. Inhibitory effect of TM30089 and montelukast on IL-13 on Th2 cytokine production was not mediated by enhanced pro- production induced by mast cell supernatants. Th2 cells were incubated duction of PGD or increased CRTH2 receptor expression as the with 1:1.5 diluted mast cell supernatants 1 or 2 in the presence or absence 2 of 1 mM TM30089, 1 mM montelukast, or both for 6 h. The IL-13 levels in inhibition of PGD2 synthesis with diclofenac did not change the the supernatants after the incubation were measured by ELISA. Data are enhancing effect, and LTE4 did not change the mRNA level of expressed as mean 6 SEM of four independent experiments. p = 0.0002 by CRTH2 in Th2 cells. ANOVA. *The values between supernatant 2 treatment and all other The receptor mediating the enhancing effect of cysLTs is un- treatments are significantly different (p , 0.05 by Newman–Keuls test). clear. On the one hand, the potent activity of LTE4 suggests that The Journal of Immunology 701

the effect is not mediated by either CysLT1 or CysLT2, although 2. Sampson, S. E., A. P. Sampson, and J. F. Costello. 1997. Effect of inhaled both receptors are expressed in human Th2 cells (Fig. 6A), be- prostaglandin D2 in normal and atopic subjects, and of pretreatment with leukotriene D4. Thorax 52: 513–518. cause LTE4 binds these receptors with only low affinity (23–25), 3. Lewis, R. A., N. A. Soter, P. T. Diamond, K. F. Austen, J. A. Oates, and L. J. Roberts, II. 1982. Prostaglandin D generation after activation of rat and and LTE4 is a weak bronchoconstrictor in human airways com- 2 human mast cells with anti-IgE. J. Immunol. 129: 1627–1631. pared with LTD4 (56). On the other hand, although LTE4 has only 4. Schleimer, R. P., C. C. Fox, R. M. Naclerio, M. Plaut, P. S. Creticos, weak affinity for CysLT1, the enhancing effect of LTE4 was A. G. Togias, J. A. Warner, A. Kagey-Sobotka, and L. M. Lichtenstein. 1985. 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However, treatment of Th2 cells with 2MeS, a selective tiocytes, dendritic, and Kupffer cells in various rat tissues. J. Immunol. 143: 2982–2989. antagonist of P2Y12, did not reduce significantly the enhancing 7. Tanaka, K., K. Ogawa, K. Sugamura, M. Nakamura, S. Takano, and K. Nagata. 2000. Cutting edge: differential production of prostaglandin D2 by human helper effect of LTE4, indicating that P2Y12 does not mediate the effect T cell subsets. J. Immunol. 164: 2277–2280. of LTE4. However, it is clear that LTE4 can stimulate inflamma- 8. Vinall, S. L., E. R. Townsend, and R. Pettipher. 2007. A paracrine role for tory responses in mice deficient in both CysLT1 and CysLT2 (57), chemoattractant receptor-homologous molecule expressed on T helper type 2 + + so it is possible that the enhancing effect observed is mediated cells (CRTH2) in mediating chemotactic activation of CRTH2 CD4 T helper type 2 lymphocytes. 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