Of the potent lipid inflammatory mediators comprising the cysteinyl leukotrienes (LTs; LTC4, LTD4, and LTE4), only LTE4 is stable and abundant in vivo. Although LTE4 shows negligible activity at the type 1 and 2 receptors for cys-LTs (CysLT1R and CysLT2R), it is a powerful inducer of mucosal eosinophilia and airway hyperresponsiveness in humans with asthma. We show that the adenosine diphosphate (ADP)–reactive purinergic (P2Y12) receptor is required for LTE4-mediated pulmonary inflammation. P2Y12 receptor expression permits LTE4 -induced activation of extracellular signal-regulated kinase in Chinese hamster ovary cells and permits chemokine and prostaglandin D2 production by LAD2 cells, a human mast cell line. P2Y12 receptor expression by LAD2 cells is required for competition between radiolabeled ADP and unlabeled LTE4 but not for direct binding of LTE4, suggesting that P2Y12 complexes with another receptor to recognize LTE4. Administration of LTE4 to the airways of sensitized mice potentiates eosinophilia, goblet cell metaplasia, and expression of interleukin-13 in response to low-dose aerosolized allergen. These responses persist in mice lacking both CysLT1R and CysLT2R but not in mice lacking P2Y12 receptors. The effects of LTE4 on P2Y12 in the airway were abrogated by platelet depletion. Thus, the P2Y12 receptor is required for proinflammatory actions of the stable abundant mediator LTE4 and is a novel potential therapeutic target for asthma.
Cysteinyl leukotrienes (LTs [cys-LTs]) are lipid inflammatory mediators generated in vivo by mast cells (MCs), eosinophils, myeloid DCs, basophils, and macrophages (Kanaoka and Boyce, 2004). They abound in mucosal inflammation, play a validated role in human asthma (Wenzel et al., 1990; Israel et al., 1996; Liu et al., 1996), and are important mediators in mouse models of pulmonary inflammation, remodeling, and fibrosis (Beller et al., 2004; Henderson et al., 2006; Kim et al., 2006). LTC4, the parent cys-LT, is synthesized from arachidonic acid, which is liberated by calcium-dependent cPLA2 (cytosolic phospholipase A2) from membrane phospholipids (Clark et al., 1991). Arachidonic acid is then converted to LTA4 by 5-lipoxygenase (5-LO) in concert with 5-LO–activating protein (Dixon et al., 1990; Malaviya et al., 1993). LTA4 is conjugated to reduced glutathione by LTC4 synthase, a homotrimeric integral nuclear membrane protein (Ago et al., 2007), forming LTC4. LTC4 is exported to the extracellular space by a multidrug-resistant protein after synthesis (Robbiani et al., 2000), where it is converted to LTD4 by γ-glutamyl leukotrienase-mediated removal of glutamic acid (Shi et al., 2001). LTD4 is then converted to LTE4 by dipeptidase-mediated removal of glycine (Lee et al., 1983). Thus, the cys-LTs comprise three ligands that form in a spatially and temporally distinct fashion. LTC4 is the only intracellular cys-LT, and LTD4 is the most powerful contractile agonist of the airway smooth muscle. The half-life of LTD4 is short (minutes) because of its rapid conversion to LTE4, effectively limiting its duration of action in vivo. LTE4 is stable and excreted in the urine (Sala et al., 1990). The stability of LTE4 accounts for the fact that it is the dominant cys-LT detected in biological fluids. Consequently, LTE4 levels can be monitored in the urine (Drazen et al., 1992), sputum (Lam et al., 1988), and exhaled breath condensate (Csoma et al., 2002) as an index of the cys-LT synthetic pathway activity in human disease states such as asthma, where its concentrations can be markedly elevated.
To date, two G protein–coupled receptors (GPCRs) for cys-LTs, termed type 1 and type 2 cys-LT receptors (CysLT1R and CysLT2R), have been cloned and characterized (Lynch et al., 1999; Heise et al., 2000). These receptors share 38% amino acid identity. Each is 24–32% identical to the purinergic (P2Y) class of GPCRs that regulate cellular responses to extracellular nucleotides (Mellor et al., 2001), suggesting a phylogenetic relationship between these two GPCR classes. The human CysLT1R, encoded by a gene on chromosome Xq21.13, is a high-affinity receptor for LTD4 (Kd ∼1 nM; Lynch et al., 1999), whereas the human CysLT2R is encoded by a gene on chromosome 13q14 and has equal affinity for LTC4 and LTD4 (Kd ∼10 nM; Heise et al., 2000). As neither receptor has significant affinity for LTE4, the existence of an additional cys-LT receptor with a preference for LTE4 has long been suspected. Early studies demonstrated that purified LTE4 is more potent than LTC4 or LTD4 for inducing contraction of guinea pig tracheal rings (Lee et al., 1984), whereas LTC4 and LTD4 were more potent on peripheral lung. Of the three cys-LTs, only LTE4 potentiates the contractile response of guinea pig trachea to histamine, a response which can be blocked by the administration of indomethacin, a nonselective inhibitor of the cyclooxygenase (COX) enzymes (Lee et al., 1984). LTE4 inhalation by asthmatic individuals potentiates airway hyperresponsiveness to subsequent challenges with histamine. This potentiation is also blocked by oral administration of indomethacin (Christie et al., 1992a). Inhalation of LTE4, but not of LTD4, causes eosinophils, basophils, and MCs to accumulate in the bronchial mucosa of asthmatic individuals (Laitinen et al., 1993; Gauvreau et al., 2001). Patients with aspirin-exacerbated respiratory disease (AERD), a syndrome characterized by asthma, nasal polyposis, and marked cys-LT overproduction, exhibit selectively enhanced bronchoconstriction in response to LTE4 relative to LTC4 or to histamine when compared with aspirin-tolerant asthmatic individuals (Christie et al., 1993). Mice lacking both CysLT1R and CysLT2R (Cysltr1/Cysltr2−/− mice) exhibit enhanced skin swelling in response to intracutaneous LTE4 relative to WT controls, indicating that a putative LTE4-reactive GPCR (termed CysLTER) exists in the skin (Maekawa et al. 2008). Thus, the potency of LTE4 as an inducer of inflammatory and physiological effects in vivo is not explained by the pharmacology of the classical GPCRs for cys-LTs, which preferentially bind the metabolic precursors of LTE4. Thus, the three cys-LTs are all potent mediators, and show considerable tissue specificity for their respective actions. Both a 5-LO inhibitor (zileuton) and the drugs that block CysLT1R (Knorr et al., 1998) show clinical efficacy in asthma, despite the negligible activity of LTE4 at CysLT1R and the fact that zileuton blocks only ∼50% of cys-LT generation in vivo (Israel et al., 1996; Liu et al., 1996). Identification of receptors and pathways through which LTE4 exerts its effects may be highly significant in terms of the pathobiology of mucosal inflammation, as well as the treatment of asthma, AERD, and related diseases in which local concentrations of LTE4 are elevated and/or end-organ reactivity to LTE4 is high.
MCs respond strongly to cys-LTs and are a useful cell type for modeling cys-LT–induced signaling events and receptor functions. We previously demonstrated that human and mouse MCs express both CysLT1R (Mellor et al., 2001) and CysLT2R (Mellor et al. 2003) and that these receptors constitutively form heterodimers on this cell type (Jiang et al., 2007). Stimulation of MCs with LTD4, the most potent agonist of the CysLT1R, transactivates the kit tyrosine kinase (Jiang et al., 2006), induces calcium flux (Mellor et al., 2001), and phosphorylates extracellular signal-regulated kinase (ERK; Jiang et al., 2006). These signaling events amplify MC proliferation and induce their generation of cytokines and chemokines (Mellor et al., 2002). CysLT1R is required for all of these LTD4-induced responses, whereas CysLT2R inhibits them (Jiang et al., 2007). MCs also express several P2Y receptors (Feng et al., 2004). We recently reported that LTE4 induces ERK activation and COX-2 expression, and causes prostaglandin (PG) D2 and macrophage inflammatory protein 1β (MIP-1β) generation by LAD2 cells, a well-differentiated human MC line (Kirshenbaum et al. 2003; Paruchuri et al., 2008), and to a lesser extent by primary cord blood-derived human MCs (hMCs). LTE4-mediated production of PGD2 by LAD2 cells was unaffected by short hairpin RNA (shRNA)–mediated knockdown of either CysLT1R or CysLT2R (Paruchuri et al., 2008), supporting the presence of a previously unrecognized LTE4-reactive receptor on this cell type. A computer model (Nonaka et al., 2005) had predicted that the P2Y12 receptor, an adenosine diphosphate (ADP)–reactive GPCR which is the target of the thienopyridine anti-thrombotic drugs (Foster et al., 2001), could be an LTE4 receptor. In this study, we demonstrate a potent unique proinflammatory function for LTE4 in the lung, and we demonstrate that P2Y12 receptors are required for the functions of LTE4 in vitro and in the lung in vivo. Our results suggest that P2Y12 receptors may be a novel target for the treatment of asthma.
Recombinant human P2Y12 receptors convey cellular responses to LTE4
To determine if P2Y12 receptors could mediate responses to LTE4, we stably transfected Chinese hamster ovary (CHO) cells (which do not natively express classical CysLTRs [Maekawa et al., 2001] or P2Y12 receptors) with constructs encoding the human P2Y12 receptor protein in forward and reverse orientation. The transfectants were stimulated with exogenous LTC4, LTD4, or LTE4. Fura-2 AM–loaded CHO cells expressing P2Y12 receptors failed to flux calcium in response to LTE4 but did exhibit a response to ADP, reflecting endogenous P2Y1 receptors (Fig. 1 A). However, CHO cells transfected with the P2Y12 receptor construct in forward (but not reverse) orientation responded to LTE4 (and to LTD4) with ERK activation (Fig. 1 B), which occurred at doses as low as 1 nM and reached a plateau at 100 nM (Fig. S1). The cys-LT–induced ERK activation was partially blocked by pretreatment of the cells with 2-methylthioadenosine monophosphate (2-MesAMP), a selective antagonist of the P2Y12 receptor (Fig. 1 C), and was also sensitive to pertussis toxin (PTX; Fig. 1 D) but was resistant to MK571 (Fig. 1 D), an inhibitor which blocks CysLT1R and some P2Y receptors (Mamedova et al., 2005). Although ERK activation in the P2Y12 receptor transfectants was at least as robust in response to LTE4 as to LTD4 (Fig. 1 D), CysLT1R and CysLT2R transfectants reacted to LTD4 in marked preference to LTE4, even at high (500 nM) ligand concentrations. These responses were resistant to PTX and 2-MesAMP and, in the case of the CysLT1R transfectants, susceptible to MK571.
P2Y12 receptor requirement for LTE4-mediated activation and binding to LAD2 cells
We had previously reported that LAD2 cells (a well-differentiated human MC sarcoma line; Kirshenbaum et al., 2003) exhibited activation responses to LTE4 > LTD4. To determine whether P2Y12 receptors were responsible for these responses, Fura-2 AM–loaded LAD2 cells were stimulated with LTD4 or LTE4 in the presence or absence of 2-MesAMP or MK571. As reported previously (Paruchuri et al., 2008), MK571 blocked calcium responses of LAD2 cells to both ligands (unpublished data). In contrast, 2-MesAMP treatment failed to attenuate LTD4- or LTE4-mediated calcium flux in LAD2 cells (Fig. 2 A). We determined the effect of MK571 and 2-MesAMP on MIP-1β generation by LAD2 cells in response to stimulation for 6 h with LTD4 and LTE4, using IgE plus anti-IgE as a positive control. LTE4 at doses of 100 and 500 nM induced the generation of large amounts of MIP-1β, exceeding the amounts generated in response to LTD4 and to IgE plus anti-IgE (Fig. 2 B). Pretreatment of the LAD2 cells with 2-MesAMP blocked the LTE4-mediated increment in MIP-1β production by >50% (Fig. 2 B). 2-MesAMP also reduced the response to the higher concentrations of LTD4. MK571 suppressed the response to both ligands and was additive with 2-MesAMP for the suppression of the response to LTD4. Neither antagonist altered the production of MIP-1β in response to IgE plus anti-IgE. To exclude potential off-target effects of the inhibitors, samples of LAD2 cells were transfected with lentiviruses encoding a P2Y12 sequence-specific shRNA, a CysLT1R-specific shRNA, or an empty vector control before stimulation. Knockdown of P2Y12 receptors decreased the receptor messenger RNA (mRNA) expression by ∼90% (Fig. 2 D) and did not alter expression of CysLT1R or CysLT2R proteins (not depicted). P2Y12 receptor knockdown nearly abrogated MIP-1β production, completely eliminated PGD2 production in response to LTE4, and minimally affected the response to LTD4. The knockdown of CysLT1R completely blocked MIP-1β and PGD2 production in response to LTD4 and slightly (but not significantly) decreased the responses to LTE4 (Fig. 2 C, bottom). Neither knockdown altered MIP-1β generation or PGD2 production in response to IgE–anti-IgE (Fig. 2 C). LTE4-mediated MIP-1β generation (Fig. S2 A) and ERK activation (Fig. S2 B) were not altered by treatment of LAD2 cells with apyrase to degrade extracellular ADP.
To determine whether P2Y12 receptors directly mediated LTE4 binding, membranes were prepared from LAD2 cells with and without P2Y12 knockdown. We first performed competitive radioligand binding assays using [3H]ADP (the known natural ligand of P2Y12 receptors) and unlabeled LTs as competitors. Unlabeled LTE4 competed with labeled ADP, blocking 39 ± 9 and 50 ± 9% of specific ADP binding at doses of 0.1 and 1 nM, respectively, and reaching a plateau (60 ± 7%) at 1 µM (mean ± SEM for four separate experiments, as shown for one experiment [Fig. S3 A]). LTE4 was more efficacious than LTD4 (Fig. S3 B). Knockdown of P2Y12 receptors reduced binding of [3H]ADP by 40–60% and completely eliminated competition by LTE4 (Fig. S3, A and B, right). LAD2 cell membranes weakly bound [3H]LTE4 (converted from commercially prepared [3H]LTD4; Fig. S3 C), but specific binding of [3H]LTE4 was not altered by the knockdown of P2Y12 receptor (Fig. S3 C). To determine whether LTE4 could block the ADP binding of P2Y12 receptors expressed in isolation, the human forward and reverse P2Y12 constructs were transiently expressed in COS-7 cells. The membranes from the transfectants expressing the forward construct bound [33P]2-MesADP, a selective P2Y12 receptor agonist. In contrast to ADP binding to LAD2 membranes, this binding was not competed by unlabeled LTE4 or LTD4 (Fig. S3 D). Additionally, these membranes failed to bind [3H]LTE4 (unpublished data).
LTE4 potentiates allergen-induced bronchial inflammation
To determine whether LTE4 could induce or potentiate histological signatures of pulmonary inflammation in mice, we determined the effect of 2.2 nmol of intranasal LTE4 administered on three successive days on the lung histology of naive BALB/c mice. We compared the effect to that of LTD4. The lungs of naive BALB/c mice showed no evidence of cellular influx or goblet cell metaplasia after three doses of either cys-LT alone (unpublished data). Thus, we sought to determine whether either cys-LT amplified pulmonary inflammation induced by the inhalation of low-dose allergen in sensitized mice. 2 wk after sensitization with chicken egg OVA by i.p. injection, BALB/c mice received inhalation challenges on three consecutive days with low-dose OVA (0.1%) for 30 min. 30 min before each challenge, the mice received 2.2 nmol of intranasal LTD4, LTE4, or a buffer control. A cohort of mice treated with 1% OVA were maintained as a positive control. The mice were then euthanized, their bronchoalveolar lavage (BAL) fluid was collected, and their lungs were examined histologically for evidence of inflammation and goblet cell metaplasia. Compared with saline-treated sensitized mice, mice challenged with low-dose OVA demonstrated low-grade BAL fluid eosinophilia (Fig. 3 A). These mice showed very mild pulmonary inflammation, as indicated by the accumulation of lymphocytes, plasma cells, and eosinophils around the bronchovascular bundles (Fig. 3, B and C). The administration of LTD4 did not increase BAL fluid eosinophilia (Fig. 3 A) and slightly potentiated both bronchovascular inflammation (Fig. 3, B and C) and goblet cell metaplasia (Fig. 3, D and E). In contrast, LTE4 significantly enhanced BAL fluid eosinophilia (Fig. 3 A), inflammation (Fig. 3, B and C), and goblet cell metaplasia (Fig. 3, D and E). The extent of the cellularity and goblet cell responses of the LTE4-treated animals approached the levels of these parameters in the mice treated with 1% OVA.
LTE4-mediated pulmonary inflammation depends on P2Y12 receptors and is independent of classical CysLTRs
To determine whether P2Y12 receptors accounted for the LTE4-mediated augmentation of pulmonary inflammation, sensitized mice were treated with clopidogrel, an antithrombotic agent that is converted in vivo to an active metabolite that covalently binds to and irreversibly inactivates P2Y12 receptors (Savi et al., 2006). Drug treatment began 2 d before the first administration of LTs to allow conversion of the prodrug. A separate cohort of sensitized mice was maintained without clopidogrel treatment as a control group. Both groups were challenged with low-dose OVA with or without additional LTE4. Treatment with clopidogrel eliminated LTE4-induced potentiation of both inflammation and goblet cell metaplasia (Fig. 4, A and B). To determine the effect of P2Y12 receptor blockade on the induced expression of mRNAs encoding proteins involved in goblet cell metaplasia, real-time PCR was used to analyze the lungs of the mice for the expression of IL-13 and the goblet cell–associated glycoprotein MUC5AC. LTE4 modestly increased the expression of both transcripts, whereas clopidogrel treatment substantially suppressed the expression of both (Fig. 4 C). There was no detectable induction of IL-4 or IL-5 transcripts (unpublished data).
Because pharmacologic antagonists can act in an off-target manner, we sought to determine the receptors required to mediate the effect of LTE4 in the lungs of allergen-sensitized and -challenged mice using a molecular approach. First, we studied the ability of LTE4 to amplify pulmonary inflammation in OVA-sensitized and -challenged C57BL/6 mice lacking P2Y12 receptors (p2ry12−/− mice; Andre et al., 2003) along with age- and sex-matched C57BL/6 controls. Although cellular infiltration into the lung and BAL fluid was less pronounced in the C57BL/6 mice than in the BALB/c mice, LTE4 potentiated BAL fluid eosinophilia (Fig. 4 D), goblet cell metaplasia (Fig. 4 E), and bronchovascular infiltration (Fig. 4 F) in the WT controls, all of which were severely blunted in the p2ry12−/− mice (Fig. 4, D–F). To determine whether classical GPCRs for cys-LTs were also required for the LTE4 effects, BALB/c mice lacking both CysLT1R and CysLT2R (Cysltr1/Cysltr2−/− mice; Maekawa et al., 2008) were also studied. Because of limited numbers of available mice, all animals were sensitized and challenged with low-dose OVA, and half were treated with exogenous LTE4. Strikingly, the potentiation of OVA-induced BAL fluid eosinophilia (Fig. 4 G), goblet cell metaplasia (Fig. 4 H), and inflammation (Fig. 4 I) by LTE4 were completely intact in the Cysltr1/Cysltr2−/− mice, indicating that LTE4 was working independently of the known GPCRs for cys-LTs. Thus, the intrapulmonary actions of LTE4 in vivo require P2Y12 receptors but not classical cys-LT–reactive GPCRs.
Blockade of P2Y12 receptors blunts pulmonary inflammation induced by house dust mite antigen
To determine whether pharmacologic blockade of P2Y12 receptors altered pulmonary inflammatory responses in a more physiological model of pulmonary inflammation, C57BL/6 mice were administered an extract of house dust mite Dermatophagoides farinae (Der f) intranasally twice weekly for 3 wk, with or without clopidogrel treatment. Two different doses of Der f were used to elicit moderate (3 µg) and severe (10 µg) inflammation, respectively. At both antigen doses, the mice treated with clopidogrel showed ∼75% attenuation of BAL fluid eosinophilia (unpublished data), as well as significant reductions in pulmonary inflammation, and goblet cell metaplasia compared with the cohort that did not receive clopidogrel (Fig. 5, A and B).
LTE4/P2Y12 receptor-mediated amplification of pulmonary inflammation requires platelets
Because P2Y12 receptors are essential for normal platelet activation in vivo (Andre et al., 2003), we sought to determine whether platelets were required for the response of sensitized challenged mice to exogenous LTE4. Platelets were depleted in sensitized BALB/c mice by the i.v. injection of a rat mAb against mouse CD42b (GPIbα; Nieswandt et al., 2000) or an isotype-matched control IgG 48 h before to the first administration of LTD4 or LTE4. Treatment with the anti-CD42b antibody depleted platelets almost completely (99% depletion [unpublished data]) at the time of the first dose of LT. Platelet depletion resulted in a complete loss of the LTE4-mediated potentiation of airway eosinophilia, inflammation, and goblet cell metaplasia (Fig. 6, A and B). The effect of platelet depletion was identical to the effect of treatment of the mice with clopidogrel, and the two treatments were not additive (Fig. 6 B). Platelet depletion, like clopidogrel treatment, sharply reduced the LTE4-mediated expression of mRNA encoding IL-13 and MUC5AC (unpublished data). To determine whether LTE4 alone induced platelet activation (leading to ADP release with potential resultant autocrine stimulation of P2Y12 receptors), human blood platelets were stimulated with 1 µM LTE4 or with 100 µM ADP, and degranulation was assessed by cytofluorographic detection of P-selectin (CD62P). As expected, ADP elicited CD62P expression, but no CD62P expression was detected in response to stimulation with LTE4 (Fig. 6 C).
This study establishes that P2Y12 receptors are essential for the actions of LTE4, the only long-lived stable abundant member of the cys-LTs, in inflamed lung. Our findings help to explain long-recognized but unexplained properties of LTE4 in airway biology. The involvement of cys-LTs in the pathobiology of asthma is established by the fact that 5-LO inhibitors (Israel et al., 1996; Liu et al., 1996) and CysLT1R antagonists (Knorr et al., 1998) have clinical efficacy. The cloning and functional characterization of the CysLT1R (Lynch et al., 1999) and CysLT2R (Heise et al., 2000) explained the pharmacology of LTC4 and LTD4 predicted from studies of contractile tissues (Lee et al., 1984). The finding that neither GPCR showed significant binding or reactivity to LTE4 was surprising given the plethora of data in human and animal studies that indicate the unique characteristics of this stable ligand relative to its short-lived precursors (Christie et al., 1992a, 1993; Laitinen et al., 1993; Gauvreau et al., 2001). Because we had previously demonstrated that LTE4 could activate LAD2 cells by a mechanism independent of CysLT1R and CysLT2R (Paruchuri et al., 2008), and because LTE4 was previously identified by an in silico model as a potential surrogate ligand for the P2Y12 receptor (Nonaka et al., 2005), we undertook this study to determine whether the P2Y12 receptor was a bona fide LTE4-reactive receptor and to determine its contribution to LTE4-induced proinflammatory events.
We first established that recombinant human P2Y12 receptor protein conveyed activation responses by CHO cells to LTE4. Unlike most members of the P2Y receptor class, native P2Y12 receptors do not couple to Gαq proteins or activate calcium flux; instead, they induce signaling through PTX-sensitive Gαi2 proteins when stimulated with ADP (Foster et al. 2001; Lova et al., 2002; Woulfe et al., 2002). It was, thus, not surprising that CHO cells expressing P2Y12 receptors failed to flux calcium in response to cys-LTs (Fig. 1 A) or that blockade of P2Y12 receptors on LAD2 cells with 2-MesAMP failed to alter cys-LT–mediated calcium flux (Fig. 2 A), which was totally abrogated by MK571. However, heterologously expressed P2Y12 receptors responded to LTE4 with PTX-sensitive ERK activation (Fig. 1). The dose range for this response (Fig. S1) is similar to the LTE4 dose range required to compete with radiolabeled ADP for binding to LAD2 cell membranes (Fig. S3) and is consistent with that reported by Nonaka et al. (2005) using a P2Y12-Gα16 fusion protein to demonstrate calcium flux, also in CHO cells. The finding that cys-LT–induced ERK activation in the transfectants was resistant to MK571 (Fig. 1 D) implies that P2Y12 receptors could contribute to an element of cys-LT–driven pathobiology that is relatively selective for LTE4 and may resist conventional CysLT1R antagonists. This element may be especially relevant to pathological situations where LTE4 is abundant as a result of its relative stability.
In our previous study, LTE4 had exhibited unanticipated potency for inducing ERK activation and the generation of MIP-1β and COX-2–dependent PGD2 by LAD2 cells (Paruchuri et al., 2008). Because both ERK and calcium-dependent transcriptional events are essential for MC activation, we sought to determine the potential contribution of P2Y12 receptors to the activation responses of LAD2 cells to LTE4 and to contrast these responses to those elicited by LTD4, the most potent CysLT1R ligand. Although ineffective for blocking cys-LT–induced calcium flux, 2-MesAMP effectively blocked the LTE4-mediated increment in MIP-1β production (Fig. 2 B) and also reduced the response to the higher concentrations of LTD4, indicating that LTD4 at high concentrations can also activate native P2Y12 receptors, as supported by its actions as an agonist for ERK activation in the P2Y12 receptor transfectants (Fig. 1). MK571 suppressed MIP-1β generation in response to both ligands (likely reflecting the requirement for calcium flux for chemokine generation) and was additive with 2-MesAMP for the suppression of the response to LTD4. The shRNA-mediated knockdowns of CysLT1R and P2Y12 receptors revealed strong dependence of LTD4-mediated activation on CysLT1R, whereas LTE4-mediated responses were clearly P2Y12 receptor dependent (Fig. 2 C). Although there is segregation of the receptor requirements for these two related ligands on the same cell, the ability of LTE4 to “cross over” and induce some calcium signaling through the CysLT1R may permit some complementarities between the CysLT1R (via Gαq proteins and calcium-induced pathways) and P2Y12 receptors (via Gαi proteins), particularly for chemokine generation. This is analogous to the cooperation by Gαq-linked P2Y1 receptors with P2Y12 receptors in regulating ADP responses of platelets (Lova et al., 2002; Woulfe et al., 2002). The P2Y12-dependent activation of LAD2 cells does not likely reflect autocrine effects of released ADP because it was resistant to treatment of the cells with the ectonucleotidase apyrase (Fig. S2).
Surprisingly, although P2Y12 receptors were essential for competition between LTE4 with ADP for binding to membranes of LAD2 cells (Fig. S3, A and B), they were not involved in the direct low-affinity binding of [3H]LTE4 to these same membranes (Fig. S3 C). Additionally, LTE4 could not compete for binding to P2Y12 receptors expressed in isolation on COS-7 cells (Fig. S3 D). The fact that P2Y12 receptors do not directly bind LTE4 despite their essential nature implies that they are components of a complex with another LTE4-reactive GPCR, perhaps with the putative CysLTER reported in the mouse skin (Maekawa et al., 2008). The facts that P2Y12 receptors are required for LTE4 to activate transfected CHO cells (Fig. 1) and LAD2 cells (Fig. 2), and for competition between LTE4 and ADP for binding to LAD2 cells (Fig. S3 B), and that knockdown of P2Y12 receptors did not reduce direct binding of radiolabeled LTE4 (Fig. S3 C) are all consistent with this thesis. Precedents for such complexes on MCs include CysLT1R and CysLT2R heterodimers (Jiang et al., 2007) and a functional requirement of CysLT1R for the uridine diphosphate-reactive P2Y6 receptor (Jiang et al., 2009). Our data indicate that presence of P2Y12 is required for signaling and activation by LTE4 in a cell-specific context.
The fact that LTE4, but not LTD4, induces bronchial eosinophilia when administered by inhalation to the airways of individuals with asthma (Gauvreau et al., 2001) argued for the presence of an LTE4-reactive receptor in inflamed lung. In our model, which was designed to study potentiation of submaximal bronchial inflammation in sensitized mice, we found that LTE4 exceeded the potency of LTD4 for potentiating BAL fluid eosinophilia (Fig. 3 A), cellular infiltration of the bronchovascular bundles (Fig. 3, B and C), and goblet cell metaplasia (Fig. 3, D and E). Thus, LTE4 exceeds the efficacy of LTD4 for potentiating bronchial inflammation in both mouse and man, a pattern not explicable by the known properties of CysLT1R or CysLT2R. Several pieces of evidence link these LTE4-mediated responses in mice to the P2Y12 receptor. First, treatment of the mice with a highly potent selective antagonist of the P2Y12 receptor, clopidogrel, completely eliminated the histological response to LTE4 (Fig. 4, A and B), which is associated with blockade of LTE4-potentiated expression of IL-13, the Th2 cytokine most closely linked to the development of goblet cell metaplasia (Zhu et al., 1999), and of MUC5AC, a major mucus glycoprotein which is controlled by IL-13 (Fig. 4 C). Second, LTE4 fully amplified pulmonary inflammation in Cysltr1/Cysltr2−/− mice (Fig. 4, G–I). Lastly, the ability of LTE4 to potentiate mucosal inflammation and goblet cell metaplasia was nearly completely abrogated in the absence of P2Y12 receptors (Fig. 4, D–F). Thus, LTE4 requires P2Y12 receptors, but not the classical CysLTRs, to amplify the histological signatures of allergen-induced pulmonary inflammation. Because LTD4 is converted in vivo to LTE4, it was somewhat surprising that the ligands do not behave interchangeably in potentiating bronchial eosinophilia in mouse or man. In addition to P2Y12-dependent effects of LTE4, LTD4 may initiate inhibitory signals through CysLT2R (Jiang et al., 2007) or other yet-to-be-identified GPCRs that recognize LTD4 but not LTE4. The effects of clopidogrel in the model of airway disease induced by the natural allergen Der f without the use of exogenous LTE4 (Fig. 5) support the importance of the P2Y12 pathway in the integrated biology of pulmonary inflammation (although this does not discriminate between effects mediated by LTE4 from ADP). The blockade of the response by clopidogrel distinguishes the P2Y12 receptor-dependent response of the lung to LTE4 from the clopidogrel-resistant LTE4 response in the skin (Maekawa et al., 2008). Thus, it is likely that different receptors or receptor complexes mediate response to the stable ligand LTE4 in distinct anatomical distributions.
Platelets accumulate in the lungs of individuals with asthma (Jeffery et al., 1989) and also are recruited to the lungs of OVA-sensitized and -challenged mice by an IgE-dependent mechanism (Pitchford et al., 2008). Activated platelets generate mediators (serotonin and thromboxane) that can potentiate airway inflammation. The essential nature of P2Y12 receptors for normal platelet function (Andre et al., 2003) led us to examine the effect of platelet depletion on the response of sensitized challenged mice to exogenous LTE4. Indeed, the response to LTE4 was totally abrogated by platelet depletion (Fig. 6, A and B). Whether the requirement for platelets is direct or indirect is less clear. The fact that LTE4 did not induce platelet expression of CD62P (an activation marker used as a surrogate for ADP release; Abrams and Shattil, 1991; Fig. 6 C) argues that LTE4 does not trigger an ADP-dependent autocrine loop initiated by LTE4-mediated activation of another receptor. Because platelet activation involves complementary signaling in both Gi and Gq protein–mediated pathways (Jin et al., 2002; Lova et al., 2002; Woulfe et al., 2002) and P2Y12 provides only the Gi-linked component, LTE4 (and P2Y12) likely synergizes in vivo with a second agonist to facilitate platelet functions in the lung. It is noteworthy that LTE4-mediated potentiation of the contractile responses of both guinea pig and human airway smooth muscle is COX dependent (Christie et al., 1992a) and was attributed to secondary generation of thromboxane, a major platelet-derived eicosanoid (Jacques et al., 1991). In retrospect, this finding may have reflected P2Y12 receptor-dependent signaling on platelets or other cell types. The fact that LTE4 potentiated inflammation only in sensitized challenged mice may reflect the previously described platelet-mediated pathway for leukocyte recruitment that depends on sensitization and IgE (Pitchford et al., 2008). The complete lack of LTE4 reactivity in the face of platelet depletion argues against a role for MCs and other P2Y12-bearing cell types, at least in this model.
Our findings suggest a potential therapeutic application for P2Y12 receptor antagonists that may be especially relevant to AERD, which is associated with both high levels of LTE4 in the urine (Christie et al., 1992b) and selective hyperresponsiveness to LTE4 (Christie et al., 1993). In AERD, both 5-LO antagonists and CysLT1R antagonists provide disease control and attenuate the consequences of aspirin challenge (Israel et al., 1993; Dahlén et al., 1998, 2002; White et al., 2006), in which the reaction is characterized by a marked surge in LTE4 generation. The fact that the effects of LTE4 persist in the absence of CysLT1R and CysLT2R implies that this pathway would be resistant to the available cys-LT receptor antagonists, all of which selectively block CysLT1R. We speculate that simultaneous interference with the bronchoconstrictive effects of LTD4 (via CysLT1R) and with proinflammatory effects of LTE4 (by P2Y12), or more complete blockade of 5-LO, might improve clinical efficacy. Our study furthermore highlights the biological significance of the structural and functional relationships between the P2Y and cys-LT–reactive classes of GPCRs.
MATERIALS AND METHODS
All animal protocols were approved by the Dana Farber Cancer Institute Animal Care and Utilization Committee. BALB/c mice lacking both CysLT1R and CysLT2R (Cysltr1/Cysltr2−/− mice) and their WT littermate controls were derived as described elsewhere (Maekawa et al., 2008). Mice lacking P2Y12 receptors (p2ry12−/− mice) were derived as described elsewhere (Andre et al., 2003) on a mixed C57BL/6-129 background and backcrossed for 10 generations with C57BL/6 mice. WT BALB/c and C57BL/6 mice were purchased from Taconic.
The LAD2 line (Kirshenbaum et al., 2003) isolated from the bone marrow of a patient with MC leukemia was a gift from A. Kirshenbaum (National Institutes of Health). These cells were cultured in StemPro-34 (Invitrogen) supplemented with 2 mM l-glutamine (Invitrogen), 100 IU/ml Pen-strep (Invitrogen), and 100 ng/ml stem cell factor (SCF; Thermo Fisher Scientific). Cell culture medium was hemidepleted every week with fresh medium and 100 ng/ml SCF. CHO cells were grown in DMEM/F-12 with 10% FBS and 100 IU/ml Pen-strep (Invitrogen). Primary cord blood MCs were derived in vitro in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM l-glutamine, 0.1 mM nonessential amino acids, 0.2 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 µg/ml gentamycin supplemented with SCF, IL-6, and IL-10 (R&D Systems) as previously described (Ochi et al., 1999).
0.5–1 × 106 cells/sample were washed and labeled with Fura 2-AM for 30 min at 37°C. Cells were stimulated with the indicated concentrations of LTD4 or LTE4, and changes in intracellular calcium concentration were measured using excitation at 340 and 380 nm in a fluorescence spectrophotometer (F-4500; Hitachi; Paruchuri et al., 2008). The relative ratios of fluorescence emitted at 510 nm were recorded and displayed as a reflection of intracellular calcium concentration. In some experiments, cells were preincubated with the 1 µM of the CysLT1R antagonist MK571 or with 100 µM of the P2Y12 receptor antagonist 2-MesAMP for 5 min before the stimulation.
Real-time quantitative PCR.
The expressions of human P2Y12 receptor mRNA and mouse IL-13 and MUC5AC RNAs were determined with real-time PCR performed on an ABI PRISM 7700 Sequence detection system (Applied Biosystems). RNA was isolated with an RNeasy mini kit (QIAGEN) and was treated with RNase-free DNase (Invitrogen) according to the manufacturer’s protocol. cDNA was synthesized from 1 µg RNA with Superscript II RNase H-RT (Invitrogen). RT was performed using TaqMan RT reagents. Primers and FAM-labeled PCR mix were purchased from SABiosciences.
shRNA constructs targeting human CysLT1R and P2Y12 receptors were purchased from Thermo Fisher Scientific. The constructs were cloned into a lentiviral vector (pLKo1; Thermo Fisher Scientific) and used to generate infectious particles with a lentiviral packaging mix (Virapower; Invitrogen) according to the manufacturer’s protocol. The transfections were performed as described previously (Jiang et al., 2007). FACS analysis was used to confirm the knockdowns of CysLT1R, whereas quantitative PCR was used to verify the knockdown of the P2Y12 receptor.
Generation of stable P2Y12 receptor transfectants.
A human P2Y12 receptor cDNA was amplified by 30 cycles of PCR from RT total RNA extracted from primary hMCs. The primer sequences were 5′-CAACAAGAAATGCAAGCCGTCGA-3′ and 5′-TACATTGGAGTCTCTTCATTTGG-3′. The fragment was cloned into a TA vector (Invitrogen). After verifying the nucleotide sequence, the fragment was subcloned into the multiple cloning site of the expression vector pEF1/His B, encoding a C-terminal HIS tag. A plasmid expressing the P2Y12 receptor construct in the forward orientation was transfected into CHO cells using Fugene HD reagent according to manufacturer’s protocol. A construct in the reverse sequence was transfected in parallel as a negative control. Stably expressing clones were selected in the medium containing 1,000 µg/ml G418 (Invitrogen), and expression of the construct was confirmed by FACS analysis of permeabilized cells with a mAb against the HIS tag.
LAD2 cells were stimulated with the indicated concentrations of LTD4 or LTE4 (Cayman Chemical) in the presence or absence of MK571 (Cayman Chemical) or 2-MesAMP (Sigma-Aldrich) or were passively sensitized with 2 µg/ml of human myeloma IgE (Millipore) overnight and stimulated with 1 µg/ml of rabbit anti–human anti-IgE (Millipore) as detailed elsewhere (Paruchuri et al., 2008). In some experiments, LAD2 cells were incubated with 10 µM apyrase (Sigma-Aldrich) to degrade extracellular nucleotides during the activation. The concentration of MIP-1β was measured by an ELISA (Thermo Fisher Scientific). PGD2 was quantitated by a PGD2-methoxylamine hydrochloride assay (Cayman Chemical).
SDS PAGE immunoblotting.
After stimulation with the respective agonists, 0.5 × 106 LAD2 cells and CHO cells were lysed with lysis buffer (BD) supplemented with protease inhibitor cocktail (Roche) and 1 mM sodium vanadate. Lysates were subjected to 4–12% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with antibodies against phospho and total ERK (Cell Signaling Technology) in PBS, 5% dry milk, and 0.1% Tween-20 (1:1,000) overnight at 4°C on shaker and then with secondary antibody (peroxidase-conjugated anti–rabbit or anti–mouse). Bands were visualized with enhanced chemiluminescence (Thermo Fisher Scientific).
[3H] LTD4 ([14,15,19,20-3H(N)]; specific activity 100–240 Ci/mmol; PerkinElmer) was converted to [3H]LTE4 by the dipeptidases present in the serum. In brief, 100 µl (80 nM) [3H]LTD4 was incubated with 100 µl of 10% serum for 2 h at room temperature. The converted product was extracted into 400 µl of methanol, evaporated in the presence of nitrogen, and diluted to the required concentration with the binding buffer for the binding assay. The conversion was confirmed by running an aliquot on high-performance reverse-phase liquid chromatography. The fraction eluting with the LTE4 peak accounted for >99% of the radioactivity used in the assays. The specific activity of the resultant LTE4 was consistently 80–100% that of the LTD4 from which it was converted. [3H]ADP and [3H]LTE4 binding assays were performed using membrane proteins as described earlier (Maekawa et al., 2001). In brief, cells were washed in PBS, resuspended in PBS supplemented with protease inhibitor cocktail, and lysed by sonication for 5 min. The lysed cells were centrifuged at 100,000 g for 45 min and the microsomal pellet was resuspended in 1 ml PBS. Protein concentration was determined using a Protein Assay kit (Bio-Rad Laboratories). 50 µg of membrane protein was incubated for 1 h at room temperature in 250 µl of 10 mM Hepes/KOH, pH 7.4, with various concentrations of radiolabeled ligand and cold competitor. Bound [3H] was separated from free [3H] by filtration through GF/C filters (GE Healthcare) and washed twice with 10 mM Hepes/KOH, pH 7.4, containing 0.01% BSA. The residual membrane-associated [3H] on the filter was determined in 2 ml scintillation fluid by β counter (PerkinElmer). For [3H]ADP, specific binding was determined by subtracting the amount of [3H] bound in the presence of 100 µM 2-MesADP from total binding. For [3H]LTE4, nonspecific binding was calculated as the residual radioactivity bound in the presence of 10 µM of unlabeled LTE4.
Induction of pulmonary inflammation.
Male BALB/c mice (6–8 wk old) received i.p. injections of 10 µg of reagent-grade chicken egg OVA precipitated with 2.25 µg aluminum hydroxide on days 0 and 7. On days 14–16, the mice received single intranasal doses of 2.2 nmol LTD4, LTE4, or buffer. 30 min after each dose, the mice were exposed to an aerosol of 0.1% OVA for 30 min delivered by an ultrasonic nebulizer. As a positive control group, some mice received 1% OVA without LTD4 or LTE4 pretreatment. The mice were euthanized 24 h after their third and final OVA aerosol challenge. In some experiments, the mice received 500 µg/ml clopidogrel in drinking water for 3 d before the first intranasal dose of LTs. The treatment was continued throughout the procedure. For Der f–mediated pulmonary inflammation, 6–8-wk-old C57BL/6 mice were lightly anaesthetized and received either 3 or 10 µg Der f extract (Greer Laboratories) on days 0, 3, 7, 10, 14, and 17. Some mice received clopidogrel throughout the duration of the experiment. Mice were euthanized 24 h after the last intranasal instillation.
Sensitized mice were treated i.v. with 50 µg (∼2 µg/g body weight) of a monoclonal rat IgG directed against mouse CD42b (GPIbα) or an equal amount of isotype control (both from Emfret Analytics; Nieswandt et al., 2000). Each antibody was diluted in 50 µl of sterile saline. The depletion of platelets was confirmed by automated counting.
Histological assessment .
The left lungs were fixed for at least 8 h in 4% paraformaldehyde and embedded in glycol methacrylate, as described previously (Kim et al., 2006). Then, 2.5-µm-thick glycol methacrylate sections were stained with H&E for general morphological examination. For histological study of the mucus-secreting cells of the epithelium (goblet cells) of the airways, lung sections were stained with PAS. The extent of cellular infiltration in the bronchovascular bundles was evaluated without knowledge of the particular treatment and was categorized arbitrarily into the following grades: 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, severe inflammation. 15 bronchovascular bundles of each mouse were evaluated. The stained goblet cells were enumerated in at least four independent bronchovascular bundles from the lung sections obtained for each animal in the different experimental groups. The length of basal lamina of corresponding bronchus was measured by ImageJ image analysis software (National Institutes of Health). Only the comparable large-caliber preterminal bronchi (diameter 200–220 µm) were examined because minimal changes occur in terminal bronchioles. The data were expressed as the mean of goblet cell counts stained in each bronchus in each section per millimeter of bronchial basal lamina.
Data are expressed as mean ± SEM from at least three experiments except where otherwise indicated. Significance was determined with the Welch’s test for samples of unequal variance. Analysis of variance was used to test differences between multiple groups.
Online supplemental material.
Fig. S1 shows the dose-dependent effects of LTE4 and LTD4 on ERK activation in CHO cells stably transfected with the human P2Y12 construct. Fig. S2 shows the effect of apyrase treatment on LT-mediated activation of LAD2 cells. Fig. S3 shows competitive ligand binding assays.
We thank Arlene Simon for her careful editing of this manuscript and K. Frank Austen and Jilly Evans for helpful comments and suggestions. We thank Gretchen Bain for her assistance with the binding assays.
This work was supported by grants from the National Institutes of Health and by a generous contribution from the Vinik Family.
Pamela Conley is employed by Portola Pharmaceuticals. The other authors have no financial conflicts of interest to report.
aspirin-exacerbated respiratory disease
Chinese hamster ovary
- Der f
extracellular signal-regulated kinase
G protein–coupled receptor
macrophage inflammatory protein 1β
short hairpin RNA