Leukotriene C4 Activates Mouse Platelets in Plasma Exclusively Through the Type 2 Cysteinyl Leukotriene Receptor This Information Is Current As of September 29, 2021

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Cutting Edge: Leukotriene C4 Activates Mouse Platelets in Plasma Exclusively through the Type 2 Cysteinyl Leukotriene Receptor This information is current as of September 29, 2021. Hannah E. Cummings, Tao Liu, Chunli Feng, Tanya M. Laidlaw, Pamela B. Conley, Yoshihide Kanaoka and Joshua A. Boyce J Immunol 2013; 191:5807-5810; Prepublished online 15 November 2013; Downloaded from doi: 10.4049/jimmunol.1302187 http://www.jimmunol.org/content/191/12/5807 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/11/18/jimmunol.130218 Material 7.DC1 References This article cites 36 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/191/12/5807.full#ref-list-1

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Cutting Edge: Leukotriene C4 Activates Mouse Platelets in Plasma Exclusively through the Type 2 Cysteinyl Leukotriene Receptor ,†,‡,1 ,†,‡,1 ,† ,†,‡ Hannah E. Cummings,*x Tao Liu,* Chunli Feng,* Tanya M.{ Laidlaw,* Pamela B. Conley, Yoshihide Kanaoka,*,†,‡ and Joshua A. Boyce*,†,‡,

Leukotriene C4 (LTC4) and its extracellular metabo- diate the effects of cys-LTs. CysLT1Risahigh-affinityLTD4 lites, LTD4 and LTE4, mediate airway inflammation. receptor with lower affinity for LTC4 (6, 7). CysLT2Rbinds They signal through three specific receptors (type 1 LTC4 and LTD4 with equal affinity (8, 9), and GPR99 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) exhibits a preference for LTE4 (10). CysLT1R-selective an-

with overlapping ligand preferences. In this article, we tagonists are widely prescribed for asthma (11). Although Downloaded from CysLT R inhibits dendritic cell priming for Th2 immune demonstrate that LTC4, but not LTD4 or LTE4, acti- 2 responses(12)andGPR99mediatesLTE-induced skin vates mouse platelets exclusively through CysLT2R. 4 edema (10), our understanding of the therapeutic applica- Platelets expressed CysLT1R and CysLT2R proteins. bility of these receptors is limited. Moreover, because many LTC4 induced surface expression of CD62P by wild- type mouse platelets in platelet-rich plasma (PRP) and cell types express more than one cys-LT receptor, assignment caused their secretion of thromboxane A and CXCL4. of receptor-specific functions through in vitro approaches is http://www.jimmunol.org/ 2 challenging. LTC was fully active on PRP from mice lacking either 4 Platelets play an important role in asthma (13) and vascular CysLT1R or GPR99, but completely inactive on PRP 2/2 inflammation (14). Platelets adhere to granulocytes by a from CysLT2R-null (Cysltr2 )mice.LTC4/CysLT2R CD62P (P-selectin)-P-selectin glycoprotein-1–dependent signaling required an autocrine ADP-mediated response mechanism. Adherent platelets upregulate leukocyte integrin through P2Y12 receptors. LTC4 potentiated airway in- avidity (15) and permit transcellular metabolism of arachidonic flammationinaplatelet-andCysLTR-dependent man- 2 acid (16). Platelets contain LTC4S and convert granulocyte- ner. Thus, CysLT2R on platelets recognizes LTC4 with derived LTA4 to LTC4 through a transcellular pathway, am- by guest on September 29, 2021 unexpected selectivity. Nascent LTC4 may activate pla- plifying the production of cys-LTs (13). Human platelets telets at a synapse with granulocytes before it is con- express both CysLT1R and CysLT2R (17). To date, however, verted to LTD4, promoting mediator generation and no study has definitively addressed whether cys-LTs influence the formation of leukocyte–platelet complexes that fa- platelet functions or determined which receptors are most cilitate inflammation. The Journal of Immunology, essential. 2013, 191: 5807–5810. We report that LTC4,butnotLTD4 or LTE4, activates mouse platelets entirely through CysLT2R. LTC4 induces expression of platelet CD62P. This response requires CysLT2R, ysteinyl leukotrienes (cys-LTs) play a validated role but not CysLT1RorGPR99.LTC4 induces platelets to release in asthma (1). After 5-lipoxygenase oxidizes arach- inflammatory mediators, and to augment allergen-induced C idonic acid to LTA4 (2), eosinophils, basophils, airway inflammation. CysLT2R-dependent platelet activation mast cells, and monocytes conjugate LTA4 to reduced glu- requires amplification from P2Y12 receptors and ADP. LTC4 tathione via leukotriene (LT) C4 synthase (LTC4S) (3), may facilitate local activation of platelets in a synapse with forming LTC4.Afterexport,LTC4 is converted to LTD4 leukocytes, in turn amplifying inflammatory responses. This (4), a smooth muscle spasmogen, and then to LTE4 (5), function is distinct from those of its extracellular metabolites. a stable metabolite. Three G protein–coupled receptors, Moreover, CysLT2R can function as an LTC4 receptor with termed the type 1 cys-LT receptor (CysLT1R) (6, 7), type 2 high specificity despite its ability to bind LTD4 in transfected cys-LT receptor (CysLT2R) (8, 9), and GPR99 (10), me- cells (8).

*Jeff and Penny Vinik Center for Allergic Disease Research, Boston, MA 02115; †Division Address correspondence and reprint requests to Dr. Joshua A. Boyce, Brigham and of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA Women’s Hospital, 1 Jimmy Fund Way, Smith Building, Boston, MA 02115. E-mail x 02115; ‡Department of Medicine, Harvard Medical School, Boston, MA 02115; Depart- address: [email protected] { ment of Pediatrics, Harvard Medical School, Boston, MA 02115; and Portola Pharma- The online version of this article contains supplemental material. ceuticals, San Francisco, CA 94080 Abbreviations used in this article: BAL, bronchoalveolar lavage; cys-LT, cysteinyl leuko- 1H.C. and T.L. contributed equally to these studies. triene; CysLT1R, type 1 cys-LT receptor; LT, leukotriene; LTC4, leukotriene C4; Received for publication August 19, 2013. Accepted for publication October 21, 2013. LTC4S, leukotriene C4 synthase; PRP, platelet-rich plasma; TXA2, thromboxane A2; WT, wild-type. This work was supported by the National Institutes of Health (Grants AI078908, AI095219, AT002782, AI082369, HL111113, HL117945, and HL36110) and the Ó Vinik family. Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302187 5808 CUTTING EDGE: LTC4 ACTIVATES PLATELETS

Materials and Methods Animals 2 2 Tbxa2r / mice were obtained from Dr. Thomas Coffman (Duke Univer- 2 2 sity, Durham, NC) (18). P2ry12 / mice were from Portola Pharmaceuticals 2 2 2 2 2 2 (San Francisco, CA) (19). Cysltr1 / , Cysltr2 / , and Gpr99 / mice were generated in our institution (10, 20, 21). Mice were sensitized i.p. on days 0 and 7 with Alum-precipitated chicken egg OVA (10 mg; Sigma) and challenged by inhalation of 0.1% OVA with or without intranasal cys-LTs as described previously (22). Platelets were depleted by an i.p. injection of an anti-CD41 Ab (clone MWReg30; Biolegend, San Diego, CA) or an isotype control (23).

Platelet isolation

Blood was obtained by cardiac puncture using a 21G needle into 4% sodium FIGURE 1. Platelet activation by cys-LTs. PRP from WT mice was stimu- citrate (Sodium Citrate Enzyme Grade; Fisher Scientific, Pittsburgh, PA). lated with the indicated agonists. CD62P was assessed by flow cytometry. Platelet-rich plasma (PRP) was obtained by slow-spin centrifugation of whole Results are mean 6 SD from 5–10 separate experiments using platelets blood at 1000 rpm/900 3 g for 15 min. PRP was incubated with CaCl 2 from 1 mouse/strain. (Fisher) ([final]= 5 mM) at 37˚C for 10 min.

Platelet activation

LTA4 by adherent LTC4S-expressing platelets (25). Because Downloaded from Aliquots of PRP (50 ml) were stimulated with thrombin (50 U/ml; Sigma extracellular enzymes efficiently convert LTC4 to LTD4 and Aldrich, St. Louis, MO), LTC , LTD , or LTE (25–250 nM; Cayman 4 4 4 LTE4, LTC4 most likely functions in a synapse between the Chemical, Ann Arbor, MI) at 37˚C for 30 min. Samples were stained with PE anti-mouse CD41 (clone MWReg30; Biolegend) and FITC rat anti-mouse cells of origin and adjacent endothelium or platelets. How- CD62P (clone RB40.34; BD Pharmingen, San Diego, CA) for analysis of ever, apart from its role as a precursor, no unique functions + k CD62P expression on CD41 mouse platelets. PE rat IgG1 and FITC rat have been attributed to LTC4. Human platelets express both IgG1l were used for isotype controls (BD Pharmingen). Cells were fixed CysLT R and CysLT R (17), as is the case for many hema- http://www.jimmunol.org/ overnight in 1% paraformaldehyde in PBS (Affymetrix, Cleveland, OH). 1 2 Some aliquots of PRP were stimulated with at 37˚C for 30 min for analysis of topoietic cells (24). Given that cell recruitment (26), bron- released thromboxane (Thromboxane B2 EIA Kit; Cayman), RANTES choconstriction (27), airway inflammation (22), and fibrosis (eBioscience, San Diego, CA) and CXCL4 (Sigma) by ELISA, or for ADP (20) all involve both cys-LTs and platelet activation (13, 22, (Abcam). Some samples were treated with the CysLT2R antagonists Bay- 28, 29), we sought to determine whether platelets might re- CysLT2 and HAMI3379 (300 nM each; Cayman Chemical). In some experiments, supernatants were analyzed for conversion of LTC4 to LTD4 spond directly to cys-LTs. and LTE4 by high-performance liquid chromatography (3). We first stimulated platelets from wild-type (WT) mice with various concentrations of LTC4,LTD4,andLTE4.Only LTC4 elicited an increase in surface CD62P expression (Fig.

Results and Discussion by guest on September 29, 2021 LTC4 is synthesized by cells that express both 5-lipoxygenase 1) and was active at the lowest dose tested (25 nM). The ∼ and LTC4S (24), or generated through granulocyte-derived response to LTC4 at 250 nM was 60% of that elicited by

FIGURE 2. Cys-LT receptors involved in LTC4-induced platelet activation. PRP from mice of the indicated genotypes was stimulated with various con- A B C D centrations of cys-LTs, or with thrombin as a positive control. ( ) Effect of CysLT2R deletion. ( ) Effect of CysLT1R deletion. ( ) Effect of GPR99 deletion. ( ) Western blot of proteins from human and WT mouse platelets showing bands corresponding to the anticipated molecular sizes of CysLT1R and CysLT2R. Results in (A)–(C) are mean 6 SD from three to ﬁve separate experiments. The Journal of Immunology 5809

thrombin (Fig. 1). PRP did not convert LTC4 to LTD4 or platelets exhibits a preference for activation by LTC4.More- LTE4 (not shown). The induction of CD62P by LTC4, and over, despite the presence of CysLT1R on platelets, CysLT2R the lack of any response to LTD4 and LTE4 at physiologic is the dominant effector of responses to LTC4 in this cell type. ranges, suggests that LTC4 has speciﬁc functions in the for- In mast cells (30) and dendritic cells (12), CysLT1R signaling mation of platelet–leukocyte complexes, which depend on dominates and CysLT2R serves an inhibitory function. Cell- induction of CD62P and its interaction with (P-selectin)-P- speciﬁc variations in receptor stoichiometry, relative abundan- selectin glycoprotein-1 on the leukocyte surface (25). ces, localization, or G protein coupling may account for these Given that CysLT1R and CysLT2R each bind LTC4 and functional differences. LTD4 at low nanomolar ranges (6, 9), the response limited to Endogenous ADP can amplify platelet activation through LTC4 was unexpected. To identify the responsible receptors, P2Y1 and P2Y12 receptors (31). P2Y12 receptors are impli- we stimulated PRP obtained from mice lacking CysLT1R cated in cellular responses to cys-LTs (particularly LTE4) (22, 2/2 2/2 (Cysltr1 mice), CysLT2R(Cysltr2 mice), and GPR99 32), but do not bind cys-LTs (22), suggesting an indirect 2/2 2/2 (Gpr99 mice). Platelets from Cysltr2 mice were unre- functional relationship to cys-LT receptors. LTC4-mediated 2/2 sponsive to LTC4 (Fig. 2A), whereas platelets from the induction of CD62P was markedly impaired in P2ry12 2 2 2 2 Cysltr1 / (Fig. 2B) and Gpr99 / strains(Fig.2C)were platelets (Fig. 3A). Treatment of WT platelets with apyrase fully responsive. Platelets from all three strains responded to attenuated their responses to LTC4 (Fig. 3B) while depleting thrombin, and none reacted to LTD4 or LTE4 (Fig. 2A–C). extracellular ADP (Fig. 3C). Although the doses of LTE4 used

Platelets from WT mice expressed both CysLT1R and CysLT2R in this study may exceed those required to demonstrate ac- Downloaded from proteins, as did human platelets (Fig. 2D). Thus, whereas tivity at P2Y12,onlyLTC4 caused platelets to release ADP; recombinant CysLT2R has equal binding afﬁnities for LTC4 this response required CysLT2R(Fig.3C).P2Y12-targeted and LTD4 (8, 9), natively expressed CysLT2Ronmouse thienopyridine drugs, which prevent cardiovascular events (33), may interfere with the LTC4/CysLT2R-dependent pathway of platelet activation in vivo.

Activated platelets generate thromboxane A2 (TXA2), a po- http://www.jimmunol.org/ tent inﬂammatory mediator, and secrete chemokines (34). Human platelets released RANTES when stimulated with cys- LTs in a prior study (17). In our study, LTC4 induced mouse platelets to release large quantities of TXA2, as well as CXCL4 and, to a lesser extent, RANTES (Supplemental Fig. 1A–C), in a CysLT2R- and P2Y12 receptor–dependent manner. Two by guest on September 29, 2021

FIGURE 4. LTC4 amplifies allergen-induced pulmonary inflammation in FIGURE 3. Involvement of P2Y12 receptors and extracellular nucleotides in a platelet, CysLT2R, and P2Y12-dependent manner. Mice were sensitized i.p. A 2/2 3 CysLT2R- mediated platelet activation. ( ) Platelets from WT or P2ry12 with OVA/Alum and challenged 3 with 0.1% OVA with or without in- A mice were stimulated with the indicated concentrations of cys-LTs or tranasal LTC4 (2 nmol). ( ) BAL fluid total cell counts (top panel) and eo- thrombin. CD62P induction was assessed by flow cytometry. (B) WT plate- sinophil counts (bottom panel) from mice of the indicated genotypes. (B) lets were stimulated with cys-LTs or thrombin in the absence or presence of Effect of platelet depletion (using anti-CD41 versus an isotype control) of 2/2 C 6 apyrase. PRP from P2ry12 mice was included as a control. ( ) Release of WT mice challenged with OVA LTC4 on BAL fluid cell counts and eo- ADP by stimulated platelets and effects of apyrase and genotypes. Results are sinophil counts. Results are mean 6 SEM from a single experiment using mean 6 SD from three separate experiments. 5–10 mice/group. Results from a second experiment were similar. 5810 CUTTING EDGE: LTC4 ACTIVATES PLATELETS

duction in aspirin-exacerbated respiratory disease is driven by platelet-adherent CysLT2R antagonists, BayCysLT2 and HAMI3379 (300 nM leukocytes. Blood 119: 3790–3798. each), suppressed TXA2 release by WT platelets (Supple- 14. Sarma, J., C. A. Laan, S. Alam, A. Jha, K. A. Fox, and I. Dransfield. 2002. Increased 2/2 mental Fig. 1D). Studies using platelets from Tbxa2r mice platelet binding to circulating monocytes in acute coronary syndromes. Circulation 105: 2166–2171. revealed that TXA2 was not necessary for LTC4-induced ac- 15. Pitchford, S. C., S. Momi, S. Giannini, L. Casali, D. Spina, C. P. Page, and tivation, although there was a trend toward less activation at P. Gresele. 2005. Platelet P-selectin is required for pulmonary eosinophil and the lowest LTC doses (Supplemental Fig. 2). lymphocyte recruitment in a murine model of allergic inflammation. Blood 105: 4 2074–2081. Intrapulmonary administration of LTE4 to sensitized mice 16. Antoine, C., R. C. Murphy, P. M. Henson, and J. Maclouf. 1992. Time-dependent challenged with low-dose OVA potentiates eosinophil recruit- utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations. Biochim. Biophys. Acta ment in a platelet- and P2Y12-dependent manner (35). We 1128: 139–146. treated sensitized mice intranasally with LTC4 (2 nmol) on 3 17. Hasegawa, S., T. Ichiyama, K. Hashimoto, Y. Suzuki, R. Hirano, R. Fukano, and S. Furukawa. 2010. Functional expression of cysteinyl leukotriene receptors on consecutive days before low-dose (0.1%) OVA challenges. human platelets. Platelets 21: 253–259. LTC4 markedly potentiated the recruitment of eosinophils to 18. Thomas, D. W., and T. M. Coffman. 1998. A genetic approach for studying the role of thromboxane A2 in the kidney. Kidney Int. Suppl. 67: S84–S87. the bronchoalveolar lavage (BAL) fluid. This response depen- 19. Hollopeter, G., H. M. Jantzen, D. Vincent, G. Li, L. England, V. Ramakrishnan, ded on CysLT2R, P2Y12 (Fig. 4A), and platelets (Fig. 4B). R. B. Yang, P. Nurden, A. Nurden, D. Julius, and P. B. Conley. 2001. Identifi- LTC may therefore contribute to platelet activation in asthma, cation of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409: 4 202–207. aspirin-exacerbated respiratory disease (13), myocardial infarc- 20. Beller, T. C., A. Maekawa, D. S. Friend, K. F. Austen, and Y. Kanaoka. 2004. tion (36), and stroke (37). Moreover, this pathway likely resists Targeted gene disruption reveals the role of the cysteinyl leukotriene 2 receptor in increased vascular permeability and in bleomycin-induced pulmonary fibrosis in blockade by the available antagonists, which do not target mice. J. Biol. Chem. 279: 46129–46134. Downloaded from CysLT2R, but may be sensitive to P2Y12 receptor–active drugs. 21. Maekawa, A., Y. Kanaoka, B. K. Lam, and K. F. Austen. 2001. Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing. Proc. Natl. Acad. Sci. USA 98: 2256–2261. 22. Paruchuri, S., H. Tashimo, C. Feng, A. Maekawa, W. Xing, Y. Jiang, Y. Kanaoka, Disclosures P. Conley, and J. A. Boyce. 2009. Leukotriene E4-induced pulmonary inflamma- The authors have no financial conflicts of interest. tion is mediated by the P2Y12 receptor. J. Exp. Med. 206: 2543–2555. 23. Liu, T., T. M. Laidlaw, H. R. Katz, and J. A. Boyce. 2013. Prostaglandin E2 de-

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Y. Acad. Sci. 1207: 11–17. Supplemental Figure 1. Receptor requirements for cys-LT-induced mediator generation by platelets. PRP From mice of the indicated genotypes was stimulated for

30 min with the indicated cys-LTs (100 nM each). A. TXB2 concentrations measured in the supernatants. B. Levels of CXCL4 and C. RANTES from the same samples as in A.

Result are from three separate experiments. D. Effects of CysLT2R antagonists

BayCysLT2 and HAMI3379 (300 nM each) on TXA2 release. Results are mean ± SD from five mice/group. Supplemental Figure 2. Effect of TP receptor deletion on cys-LT-mediated induction of

CD62P. Results are mean ± SD from 5 mice/group.