Leukotriene C₄ Activates Mouse Platelets in Plasma Exclusively Through the Type 2 Cysteinyl Leukotriene Receptor^{^{<a href="#FN3" rid="FN3" class=" fn">1</a>}}

Introduction

Cysteinyl leukotrienes (cys-LTs) play a validated role in asthma (1). After 5-lipoxygenase (5-LO) oxidizes arachidonic acid to LTA₄ (2), eosinophils, basophils, mast cells and monocytes conjugate LTA₄ to reduced glutathione via leukotriene C₄ synthase (LTC₄S)(3), forming LTC₄. After export, LTC₄ is converted to LTD₄ (4), a smooth muscle spasmogen, and then to LTE₄ (5), a stable metabolite. Three G protein coupled receptors, termed the type 1 cys-LT receptor (CysLT₁R) (6,7), type 2 cys-LT receptor (CysLT₂R) (8,9), and GPR99 (10), mediate the effects of cys-LTs. CysLT₁R is a high affinity LTD₄ receptor with lower affinity for LTC₄ (6,7). CysLT₂R binds LTC₄ and LTD₄ with equal affinity (8,9), and GPR99 exhibits a preference for LTE₄ (10). CysLT₁R-selective antagonists are widely prescribed for asthma (11). Although CysLT₂R inhibits dendritic cell priming for T helper type 2 immune responses (12) and GPR99 mediates LTE₄-induced skin edema (10), our understanding of the therapeutic applicability of these receptors is limited. Moreover, since many cell types express more than one cys-LT receptor, assignment of receptor-specific functions through in vitro approaches is challenging.

Platelets play an important role in asthma (13) and vascular inflammation (14). Platelets adhere to granulocytes by a CD62P (P-selectin)-P-selectin glycoprotein-1 (PSGL-1)-dependent mechanism. Adherent platelets upregulate leukocyte integrin avidity (15), and permit transcellular metabolism of arachidonic acid (16). Platelets contain LTC₄S and convert granulocyte-derived LTA₄ to LTC₄ through a transcellular pathway, amplifying the production of cys-LTs (13). Human platelets express both CysLT₁R and CysLT₂R (17). To date, however, no study has definitively addressed whether cys-LTs influence platelet functions, or determined which receptors are most essential.

We now report that LTC₄, but not LTD₄ or LTE₄, activates mouse platelets entirely through CysLT₂R. LTC₄ induces expression of platelet CD62P. This response requires CysLT₂R, but not CysLT₁R or GPR99. LTC₄ induces platelets to release inflammatory mediators, and to augment allergen-induced airway inflammation. CysLT₂R-dependent platelet activation requires amplification from P2Y₁₂ receptors and ADP. LTC₄ may facilitate local activation of platelets in a synapse with leukocytes, in turn amplifying inflammatory responses. This function is distinct from those of its extracellular metabolites. Moreover, CysLT₂R can function as an LTC₄ receptor with high specificity despite its ability to bind LTD₄ in transfected cells (8).

Methods

Animals

Tbxa2r−/− mice were obtained from Dr. Thomas Coffman (Duke University, Durham, NC) (18). P2ry12−/− mice were from Portola Pharmaceuticals (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 ovalbumin (OVA, Sigma, 10 μg) and challenged by inhalation of 0.1% OVA with or without intranasal cys-LTs as described (22). Platelets were depleted by an I.P. injection of an anti-CD41 antibody (clone MWReg30, Biolegend) or an isotype control (23).

Platelet isolation

Blood was obtained by cardiac puncture using a 21G needle into 4% sodium citrate (Sodium Citrate Enzyme Grade, Fisher Scientific, Pittsburgh, PA). Platelet Rich Plasma (PRP) was obtained by slow spin centrifugation of whole blood at 1000 rpm/900xg for 15 minutes. PRP was incubated with CaCl₂ (Fischer) ([final]= 5mM) at 37°C for 10 minutes.

Platelet activation

Aliquots of PRP (50 μl) were stimulated with thrombin (50U/mL, Sigma Aldrich, Saint Louis, MO), LTC₄, LTD₄, or LTE₄ (25–250nM, Cayman Chemical, Ann Arbor, MI) at 37°C for 30 minutes. Samples were stained with PE anti-mouse CD41 (Clone: MWReg30, Biolegend, San Diego, CA) and FITC rat Anti- mouse CD62P (Clone: RB40.34, BD Pharmingen, San Diego, CA) for analysis of CD62P expression on CD41 ^{mouse platelets. PE Rat IgG1κ and FITC Rat IgG1λ were used for isotype controls (BD Pharmingen). Cells were fixed overnight in 1% paraformaldehyde in PBS (Affymetrix®, Cleveland, OH). Some aliquots of PRP were stimulated with at 37°C for 30 minutes for analysis of released thromboxane (Thromboxane B}₂ EIA Kit, Cayman), Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) (eBiosciences, San Diego, CA) and CXCL4 (Sigma) by ELISA, or for ADP (Abcam). Some samples were treated with the CysLT₂R antagonists BayCysLT₂ and HAMI3379 (Cayman Chemical, 300 nM each). In some experiments, supernatants were analyzed for conversion of LTC₄ to LTD₄ and LTE₄ by high performance liquid chromatography (24).

RESULTS AND DISCUSSION

LTC₄ is synthesized by cells that express both 5-LO and LTC₄S (25), or generated through from granulocyte-derived LTA₄ by adherent LTC₄S-expressing platelets (26). Since extracellular enzymes efficiently convert LTC₄ to LTD₄ and LTE₄, LTC₄ most likely functions in a synapse between the cells of origin and adjacent endothelium or platelets. However, apart from its role as a precursor, no unique functions have been attributed to LTC₄. Human platelets express both CysLT₁R and CysLT₂R (17), as is the case for many hematopoietic cells (25). Given that cell recruitment (27), bronchoconstriction (28), airway inflammation (22), and fibrosis (20) all involve both cys-LTs and platelet activation (13,22,29,30), we sought to determine whether platelets might respond directly to cys-LTs.

We first stimulated platelets from WT mice with various concentrations of LTC₄, LTD₄, and LTE₄. Only LTC₄ elicited an increase in surface CD62P expression (Fig. 1), and was active at the lowest dose tested (25 nM). The response to LTC₄ at 250 nM was ~60% of that elicited by thrombin (Fig. 1). PRP did not convert LTC₄ to LTD₄ or LTE₄ (not shown). The induction of CD62P by LTC₄, and the lack of any response to LTD₄ and LTE₄ at physiologic ranges, suggests that LTC₄ has specific functions in the formation of platelet-leukocyte complexes, which depend on induction of CD62P and its interaction with PSGL-1 on the leukocyte surface (26).

Figure 1

Platelet activation by cys-LTs. PRP from WT mice was stimulated with the indicated agonists. CD62P was assessed by flow cytometry. Results are mean ± SD from 5–10 separate experiments using platelets from one mouse/strain.

Given that CysLT₁R and CysLT₂R each bind LTC₄ and LTD₄ at low nM ranges (6,9), the response limited to LTC₄ was unexpected. To identify the responsible receptors, we stimulated PRP obtained from mice lacking CysLT₁R (Cysltr1−/− mice), CysLT₂R (Cysltr2−/− mice), and GPR99 (Gpr99−/− mice). Platelets from Cysltr2−/− mice were unresponsive to LTC₄ (Fig. 2A), whereas platelets from the Cysltr1−/− (Fig. 2B) and Gpr99−/− strains (Fig. 2C) were fully responsive. Platelets from all three strains responded to thrombin, and none reacted to LTD₄ or LTE₄ (Fig. 2A–C). Platelets from WT mice expressed both CysLT₁R and CysLT₂R proteins, as did human platelets (Fig. 2D). Thus, while recombinant CysLT₂R has equal binding affinities for LTC₄ and LTD₄ (8,9), natively expressed CysLT₂R on mouse platelets exhibits a preference for activation by LTC₄. Moreover, despite the presence of CysLT₁R on platelets, CysLT₂R is the dominant effector of responses to LTC₄ in this cell type. In mast cells (31) and dendritic cells (12), CysLT₁R signaling dominates and CysLT₂R serves an inhibitory function. Cell-specific variations in receptor stoichiometry, relative abundances, localization, or G protein-coupling may account for these functional differences.

Figure 2

Cys-LT receptors involved in LTC₄-induced platelet activation. PRP from mice of the indicated genotypes was stimulated with various concentrations of cys-LTs, or with thrombin as a positive control. A. Effect of CysLT₂R deletion. B. Effect of CysLT₁R deletion. C. Effect of GPR99 deletion. D. 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 ± SD from 3–5 separate experiments.

Endogenous ADP can amplify platelet activation through P2Y₁ and P2Y₁₂ receptors (32). P2Y₁₂ receptors are implicated in cellular responses to cys-LTs (particularly LTE₄) (22,33), but do not bind cys-LTs (22), suggesting an indirect functional relationship to cys-LT receptors. LTC₄-mediated induction of CD62P was markedly impaired in P2ry12−/− platelets (Fig. 3A). Treatment of WT platelets with apyrase attenuated their responses to LTC₄ (Fig. 3B) while depleting extracellular ADP (Fig. 3C). While the doses of LTE₄ used in this study may exceed those required to demonstrate activity at P2Y₁₂, only LTC₄ caused platelets to release ADP; this response required CysLT₂R (Fig. 3C). P2Y₁₂-targeted thienopyridine drugs, which prevent cardiovascular events (34), may interfere with the LTC₄/CysLT₂R-dependent pathway of platelet activation in vivo.

Figure 3

Involvement of P2Y₁₂ receptors and extracellular nucleotides in CysLT₂R-mediated platelet activation. A. Platelets from WT or P2ry12−/− micewere stimulated with the indicated concentrations of cys-LTs or thrombin. CD62P induction was assessed by flow cytometry. B. WT platelets were stimulated with cys-LTs or thrombin in the absence or presence of apyrase. PRP from P2ry12−/− mice was included as a control. C. Release of ADP by stimulated platelets and effects of apyrase and genotypes. Results mean ± SD from 3 separate experiments.

Activated platelets generate TXA₂, a potent inflammatory mediator, and secrete chemokines (35). 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 CysLT₂R antagonists, BayCysLT₂ and HAMI3379 (300 nM each) suppressed TXA₂ release by WT platelets (Supplemental Fig. 1D). Studies using platelets from Tbxa2r−/− mice revealed that TXA₂ was not necessary for LTC₄-induced activation, although there was a trend toward less activation at the lowest LTC₄ doses (Supplemental Fig. 2).

Intrapulmonary administration of LTE₄ to sensitized mice challenged with low-dose OVA potentiates eosinophil recruitment in a platelet- and P2Y₁₂-dependent manner (36). We treated sensitized mice intranasally with LTC₄ (2 nmol) on three consecutive days before low-dose (0.1%) OVA challenges. LTC₄ markedly potentiated the recruitment of eosinophils to the BAL fluid. This response depended on CysLT₂R, P2Y₁₂ (Fig. 4A), and platelets (Fig. 4B). LTC₄ may therefore contribute to platelet activation in asthma, aspirin exacerbated respiratory disease (13), myocardial infarction (37), and stroke (38). Moreover, this pathway likely resists blockade by the available antagonists, which do not target CysLT₂R, but may be sensitive to P2Y₁₂ receptor-active drugs.

Figure 4

LTC₄ amplifies allergen-induced pulmonary inflammation in a platelet, CysLT₂R and P2Y₁₂-dependent manner. Mice were sensitized intraperitoneally with OVA/Alum and challenged 3x with 0.1% OVA with or without intranasal LTC₄ (2 nmol). A. BAL fluid total cell counts (top) and eosinophil counts (bottom) from mice of the indicated genotypes. B. Effect of platelet depletion (using anti-CD41 vs. an isotype control) of WT mice challenged with OVA ± LTC₄ on BAL fluid cell counts and eosinophil counts. Results are mean ± SEM from a single experiment using 5–10 mice/group. Results from a second experiment were similar.

Supplementary Material

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