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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Oct 17;105(43):16695–16700. doi: 10.1073/pnas.0808993105

Functional recognition of a distinct receptor preferential for leukotriene E4 in mice lacking the cysteinyl leukotriene 1 and 2 receptors

Akiko Maekawa *,†,‡, Yoshihide Kanaoka *,†,‡, Wei Xing *,†, K Frank Austen *,†,§
PMCID: PMC2575482  PMID: 18931305

Abstract

The cysteinyl leukotrienes (cys-LTs) are a family of potent lipid mediators of inflammation derived from arachidonic acid. Activation of certain cell types results in the biosynthesis and export of leukotriene (LT) C4, which then undergoes extracellular metabolism to LTD4 and LTE4. LTE4, the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT1R and CysLT2R, respectively). We had recognized a greater potency for LTE4 than LTC4 or LTD4 in constricting guinea pig trachea in vitro and comparable activity in eliciting a cutaneous wheal and flare response in humans. Thus, we hypothesized that a vascular permeability response to LTE4 in mice lacking both the CysLT1R and CysLT2R could establish the existence of a separate LTE4 receptor. We now report that the intradermal injection of LTE4 into the ear of mice deficient in both CysLT1R and CysLT2R elicits a vascular leak that exceeds the response to intradermal injection of LTC4 or LTD4, and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE4 is ≈64-fold more potent in the CysLT1R/CysLT2R double-deficient mice than in sufficient mice. The administration of a CysLT1R antagonist augmented the permeability response of the CysLT1R/CysLT2R double-deficient mice to LTC4, LTD4, and LTE4. Our findings establish the existence of a third receptor, CysLTER, that responds preferentially to LTE4, the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.

Keywords: inflammation, lipid mediator, knockout mice

To recognize a role for the cysteinyl leukotrienes (cys-LTs) and their receptors in inflammation, we initially developed mice lacking the critical biosynthetic enzyme, leukotriene C4 synthase (LTC4S), which forms leukotriene (LT) C4 by conjugation of reduced glutathione to LTA4. LTA4 is generated from arachidonic acid released from phospholipids of the outer nuclear membrane by cytosolic phospholipase A2α during cell activation. In the presence of 5-lipoxygenase (5-LO) and the 5-LO-activating protein (FLAP), the arachidonic acid is converted sequentially to 5-hydroperoxyeicosatetraenoic acid and LTA4 (1, 2). Both LTC4S and FLAP are integral proteins of the outer nuclear membrane and function as trimers (3–5) in the tightly regulated intracellular synthesis of LTC4. After its export via an energy-dependent step that requires multidrug resistance-associated proteins 1 and 4, LTC4 is metabolized by cleavage removal of glutamic acid and then glycine to provide LTD4 and LTE4, respectively. In a passive cutaneous anaphylaxis model, mice lacking LTC4S (Ltc4s−/−) exhibited a significant reduction in vascular leak after local sensitization of ear mast cells with specific IgE and systemic challenge with antigen (6). These findings revealed a permeability-enhancing function for cys-LTs comparable to that of the amines stored in the mast cell secretory granules. These Ltc4s−/− mice also were significantly protected against bleomycin-induced pulmonary fibrosis and antigen-induced allergic inflammation of the lung (7, 8).

To further analyze the cys-LT-mediated inflammatory pathways, we generated mice deficient in the type 1 cys-LT receptor (CysLT1R) and the type 2 cys-LT receptor (CysLT2R) based on the prior cloning of these seven-transmembrane, G protein-coupled human receptors by others (9, 10). CysLT1R and CysLT2R are 38% homologous in their amino acid sequences. The rank order of affinities of the cys-LTs for the CysLT1R and CysLT2R based on transfected cells is LTD4 > LTC4 > LTE4 and LTC4 = LTD4 > LTE4, respectively (9–12). CysLT1R-deficient (Cysltr1−/−) and CysLT2R-deficient (Cysltr2−/−) mice each showed a significant reduction in vascular permeability in the passive cutaneous anaphylaxis model comparable to that in the Ltc4s−/− mice (13, 14), suggesting either sequential functions in the microvasculature or heterodimerization of these receptors. Bleomycin-induced fibrosis in Cysltr2−/− mice, but not in Cysltr1−/− mice, was as reduced as that in Ltc4s−/− mice, revealing a requirement of CysLT2R for this complex injury. In contrast, neither the Cysltr1−/− nor the Cysltr2−/− mice were protected from antigen-induced pulmonary inflammation, suggesting that additional receptor(s) might account for the protection observed in the Ltc4s−/− mice (unpublished data).

We had observed previously that LTE4 was more potent than LTC4 or LTD4 in contracting the guinea pig trachea in vitro (15, 16) and was comparable to LTC4 or LTD4 in eliciting a dermal wheal and flare response in humans (17). Others noted that inhalation of LTE4 but not LTD4 by patients with asthma elicited an influx of eosinophils and basophils into the bronchial mucosa (18, 19). Furthermore, patients with aspirin-exacerbated respiratory disease, characterized by bronchial asthma, nasal polyposis, and marked overproduction of the cys-LTs as defined by urinary excretion of N-acetyl-LTE4, responded with enhanced bronchoconstriction to inhalation of LTE4 relative to their responses to LTC4 or histamine, as compared with the potency of these agonists in asthmatic patients without aspirin-exacerbated respiratory disease (20). These preferential agonist functions of LTE4 were not explained by the pharmacologic properties of the CysLT1R and CysLT2R.

To seek an additional receptor(s) for the cys-LTs, we generated CysLT1R/CysLT2R double-deficient (Cysltr1/Cysltr2−/−) mice and examined the agonist function of the cys-LTs by intradermal injection into the ear. We found that the Cysltr1/Cysltr2−/− mice expressed a previously unrecognized receptor that mediated a greater vascular leak in response to LTE4 than to LTD4 or LTC4. The increased sensitivity of the Cysltr1/Cysltr2−/− mice compared with WT mice was ≈64-fold by dose-dependent analysis, thereby revealing a profound negative regulation of the novel receptor for LTE4 by the two known receptors with a preference for LTD4 and LTC4. We then found that pretreatment of Cysltr1/Cysltr2−/− mice with a selective CysLT1R antagonist, MK-571, further increased the permeability response of these mice to each of the three cys-LTs. Thus, these studies uncover a distinct receptor with a preference for LTE4, which we term CysLTER, that could be relevant to pathobiology mediated by the most stable cys-LT, LTE4 (21, 22).

Results and Discussion

Enhanced LTE4-Elicited Ear Edema in Cysltr1/Cysltr2−/− Mice Indicates the Presence of a Novel Receptor.

To examine whether any cys-LTs mediate an increase in vascular permeability in the absence of their known receptors, LTC4, LTD4, and LTE4 (0.5 nmol per site) were injected intradermally into the ear of WT, Cysltr1−/−, Cysltr2−/−, and Cysltr1/Cysltr2−/− mice, and tissue edema was assessed by measuring ear thickness at 0, 30, 60, 120, 180, 240, and 300 min after the injection. Ear edema induced by LTC4 and that induced by LTD4 in WT mice were similar, peaking at 30 min and returning to baseline at 300 min (Fig. 1 A and B). LTC4- and LTD4-induced ear edema in Cysltr1−/− mice both were reduced by 50≈60% at the peak compared with levels in WT mice and returned to baseline 60 min earlier. LTC4- and LTD4-induced ear edema in Cysltr2−/− mice showed a delayed peak response at 60 min that was comparable to the earlier peak response in WT mice (Fig. 1 A and B). These results in mice lacking a single receptor indicate that about 50% of LTC4- and LTD4-induced ear edema is mediated by CysLT1R and suggest that CysLT2R may be a negative regulator of the CysLT1R-mediated response. Of particular note, Cysltr1/Cysltr2−/− mice responded to intradermal LTC4 and LTD4, respectively, with a vascular leak similar to that of WT mice (Fig. 1 A and B), revealing an additional receptor that is distinct from CysLT1R and CysLT2R.

Fig. 1.

Fig. 1.

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Effects of CysLTR deficiency on LTC4-, LTD4-, and LTE4-induced ear edema. WT (closed squares, n = 7), Cysltr1−/− (diamonds, n = 5), Cysltr2−/− (triangles, n = 3), and Cysltr1/Cysltr2−/− (open circles, n = 5) mice received intradermal injections of 0.5 nmol (in 25 μl vehicle) of LTC4 (A), LTD4 (B), or LTE4 (C) in the right ear and 25 μl vehicle in the left ear. Ear thickness was measured with calipers at the indicated times after the injection. Results are expressed as the mean ± SE of the differences between the thickness of the right and left ears at each time point and are combined from two independent experiments. Error bars are too small to be visible compared with the physical size of the symbol. *, P < 0.01 vs. WT mice is presented in the plot for 30, 60 and 240 min.

The ear edema in response to intradermal injection of LTE4 in WT mice with a peak at 30 min and resolution at 300 min was similar to the edema induced by LTC4 and LTD4 at the same 0.5-nmol dose (Fig. 1C). However, unlike the reduced response to LTC4 or LTD4, LTE4-induced ear edema in Cysltr1−/− mice was comparable in magnitude to that in WT mice, indicating a CysLT1R-independent action. The LTE4-induced ear edema in Cysltr2−/− mice showed a peak response similar to that in WT mice, but the resolution was delayed; this finding suggested an inhibitory role directed to a CysLT1R-independent LTE4 receptor. Unexpectedly, Cysltr1/Cysltr2−/− mice showed a significantly enhanced peak response (11.1 ± 0.1 × 10−2 mm, n = 5) to LTE4 injection at 30 min compared with WT mice (8.5 ± 0.32 × 10−2 mm, n = 7), and the response remained evident even at 300 min (5.5 ± 0.32 × 10−2 mm), when the response in WT mice had resolved (Fig. 1C).

To assess whether the 30% increase in permeability in response to LTE4 in the Cysltr1/Cysltr2−/− mice compared with WT mice was limited by being a near-maximum response at the fixed 0.5-nmol dose used for the three ligands, we examined the dose–response to LTE4 (0.5, 0.125, 0.03, and 0.008 nmol per site) in WT and Cysltr1/Cysltr2−/− mice. LTE4 elicited ear edema in both WT and Cysltr1/Cysltr2−/− mice in a dose-dependent manner, with a significantly greater response in the Cysltr1/Cysltr2−/− mice at each dose (Fig. 2). The increased ear swelling at 30 min in the Cysltr1/Cysltr2−/− mice at the dose of 0.008 nmol per site was essentially equal to that in WT mice at the dose of 0.5 nmol per site, indicating that Cysltr1/Cysltr2−/− mice are ≈64-fold more sensitive to LTE4 for ear edema compared with WT mice. The persistence of edema to 300 min in the Cysltr1/Cysltr2−/− mice with each dose, compared with full resolution in the WT mice, indicates that the augmented 30-min peak may be influenced by reduced clearance.

Fig. 2.

Fig. 2.

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Dose dependence of LTE4-induced ear edema in WT and Cysltr1/Cysltr2−/− mice. WT (A) and Cysltr1/Cysltr2−/− (B) mice received intradermal injections of LTE4 in the right ear and vehicle in the left ear (four mice per group). Ear thickness was measured with calipers at the indicated times after the injection. Results are expressed as the mean ± SE of the differences between the thickness of the right and left ears at each time point and are combined from two independent experiments. Some error bars are too small to be visible as compared to the physical size of the symbol. *, P < 0.01 vs. WT mice is presented in the plot for 30, 60, and 240 min.

To assess the rank order of agonist action for this novel receptor, we examined the dose–response to intradermal injections of LTC4 (0.5, 0.125, and 0.03 nmol per site) and LTD4 (0.5, 0.125, and 0.03 nmol per site) in WT and Cysltr1/Cysltr2−/− mice. Both WT and Cysltr1/Cysltr2−/− mice showed dose-dependent responses to LTC4 and LTD4, and the peak responses at 30 min for each dose of the ligand in the Cysltr1/Cysltr2−/− mice were essentially similar to the responses in WT mice, although prolonged [supporting information (SI) Fig. S1]. The finding that the peak responses to LTC4 and LTD4 persisted until 60 min in the Cysltr1/Cysltr2−/− mice may reflect conversion to LTE4, since we could detect LTC4 and LTE4 with a 1:3 molar ratio in extracts of ear tissues from two mice 60 min after injection of 400 pmol of LTC4 by reverse-phase HPLC (≈10% of recovery), whereas no cys-LTs were detected in the saline-injected ears. These results strongly indicate that a separate receptor for LTC4, LTD4, and LTE4 can be recognized in the absence of the two known receptors and that the rank order of agonist action for this receptor, assessed by increased vascular permeability, is LTE4 > LTC4 = LTD4. This distinct receptor is termed CysLTER to indicate its functional ligand selectivity.

Histologic Assessment of LTE4-Elicited Ear Edema.

To determine whether the LTE4-elicited ear thickening at the peak and during the resolution phase is due solely to plasma protein extravasation, ear tissues were harvested for histologic analysis 30 and 240 min after LTE4 intradermal injection. LTE4 caused expansion of the extracellular space in an injected side of the ear tissue but no detectable cellular infiltration in either WT or Cysltr1/Cysltr2−/− mice (Fig. 3). The LTE4-elicited ear thickening measured from skin injection site to ear cartilage was 1.7-fold greater in Cysltr1/Cysltr2−/− mice than in WT mice at 30 min (336 ± 3.1 μm vs. 200 ± 6.3 μm, n = 2). At 240 min after injection, Cysltr1/Cysltr2−/− mice still showed tissue edema without cellular infiltration, whereas edema had resolved in the WT mice (Fig. 3).

Fig. 3.

Fig. 3.

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Histology of ear tissues 30 and 240 min after intradermal injection of vehicle or LTE4. WT (Upper) and Cysltr1/Cysltr2−/− (Lower) mice received intradermal injections of LTE4 (0.5 nmol) in the right ear and vehicle in the left ear. Tissues were harvested 30 min and 240 min after injection, and sections were stained by the chloroacetate esterase reaction and counterstained with hematoxylin. (Scale bars: 200 μm.)

Effect of MK-571 on cys-LT-Elicited Ear Edema in CysLTR-Deficient Mice.

To assess whether CysLTER is functionally related to CysLT1R, we examined the effect of the CysLT1R blocker, MK-571 (23), on the response to LTE4 in WT, Cysltr1−/−, Cysltr2−/−, and Cysltr1/Cysltr2−/− mice. MK-571 inhibited the response to LTE4 at 0.5 nmol per site in WT mice by about 50%, implicating a CysLT1R-dependent and comparable independent action via CysLTER (Fig. 4A). MK-571 had no inhibitory effect on the LTE4 response in Cysltr1−/− mice, which was comparable to that of WT mice without MK-571 (Fig. 4B). Thus, the CysLT1R-independent response mediated by CysLTER is resistant to MK-571 and appears to be up-regulated in the absence of CysLT1R. Cysltr2−/− mice showed a significantly enhanced peak response to LTE4 at 30 min in the presence of MK-571 (Fig. 4B), supporting the idea that CysLT1R is a negative regulator of CysLTER. MK-571 treatment did not inhibit the peak response to LTE4 at 30 min at 0.5 nmol per site in Cysltr1/Cysltr2−/− mice but did suppress its persistence (Fig. 4A).

Fig. 4.

Fig. 4.

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Effect of MK-571 on LTC4-, LTD4-, and LTE4-induced ear edema in WT and CysLTR-deficient mice. WT (closed squares, n = 4), Cysltr1−/− (diamonds, n = 4), Cysltr2−/− (triangles, n = 4), and Cysltr1/Cysltr2−/− (open circles, n = 4) mice received intradermal injections of 0.5 nmol (A and B) or 0.008 nmol (C) of LTE4 or 0.5 nmol of LTC4 (D) or LTD4 (E) in the right ear and vehicle in the left ear without (dotted lines) or with (solid lines) i.v. injection of MK-571 (10 mg/kg) 30 min previously. Results are expressed as the mean ± SE of the differences between the thickness of the right and left ears at each time point and are combined from two independent experiments. Some error bars are too small to be visible compared with the physical size of the symbol. Experiments for A and B were carried out at the same time. *, P < 0.01 vs. mice without MK-571 within each individual strain presented for 30, 60, and 240 min.

Because the permeability enhancement might have reached a maximum, we reexamined the effect of MK-571 at a 64-fold lower dose of LTE4. The modest vascular leak elicited by LTE4 at 0.008 nmol per site in WT mice was suppressed by MK-571. The vascular leak elicited by LTE4 at 0.008 nmol per site in Cysltr1/Cysltr2−/− mice was again substantial in peak and duration compared with the WT mice (Fig. 4C). Unexpectedly, the LTE4 response was markedly enhanced by pretreatment of the Cysltr1/Cysltr2−/− mice with MK-571 (15 ± 0.05 × 10−2 mm vs. 7.5 ± 0.0 × 10−2 mm, n = 4), revealing further negative regulation of CysLTER by an MK-571-sensitive component. This finding prompted re-examination of the maximal responses to 0.5 nmol of LTC4 and LTD4 in the presence of MK-571. Whereas the administration of MK-571 reduced the response to LTC4 and LTD4 in WT mice by ≈50%, it enhanced the responses to LTC4 (10.5 ± 0.20 × 10−2 mm vs. 7.0 ± 0.24 × 10−2 mm, n = 4) and LTD4 at their peak of 30 min (11.3 ± 0.14 × 10−2 mm vs. 7.25 ± 0.13 × 10−2 mm, n = 4) in Cysltr1/Cysltr2−/− mice about 1.5- to 2-fold compared with Cysltr1/Cysltr2−/− mice not treated with MK-571 (Fig. 4 D and E). These results indicate that MK-571 up-regulates the sensitivity of CysLTER to each cys-LT.

Effects of Pertussis Toxin (PTX), a Rho Kinase Inhibitor, Indomethacin, and Clopidogrel on LTE4-Elicited Ear Edema.

To examine whether the LTE4-elicited ear edema is mediated through a G protein-coupled receptor, we administered PTX (24, 25) and Y-27632 (26) to inhibit the Gαi and Gα12/13-Rho-Rho kinase pathways (27), respectively. Treatment with PTX and Y-27632 significantly reduced peak LTE4-elicited ear edema at 30 min in WT, Cysltr1−/−, Cysltr2−/−, and Cysltr1/Cysltr2−/− mice compared with no treatment (Fig. 5 A–C). These results suggest that the putative CysLTER is G protein-coupled through Gαi and Gα12/13-Rho-Rho kinase pathways. Indomethacin pretreatment had no effect on the LTE4-elicited response in WT, Cysltr1−/−, and Cysltr2−/− mice but did somewhat suppress the peak and resolution of edema in Cysltr1/Cysltr2−/− mice, thereby implying a small contribution by a prostanoid (Fig. 5D). Recently, in silico screening suggested that LTE4 may be a ligand for an ADP receptor, P2Y12, which is heterologously expressed as a fusion protein with human Gα16 (28). This receptor potently mediates LTE4-stimulated ERK activation in CHO cells and is responsible for a marked potentiating effect of intranasal LTE4 on aerosol antigen-induced airway mucosal eosinophilia and goblet cell hyperplasia in sensitized mice. This effect of LTE4 is inhibited by P2Y12 receptor antagonism with clopidogrel (J. Boyce, personal communication). In contrast, the enhanced response to LTE4 in the Cysltr1/Cysltr2−/− mice was not affected by pretreatment with clopidogrel, indicating that the responsible receptor is not P2Y12 (Fig. 5E).

Fig. 5.

Fig. 5.

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Effects of PTX, a Rho kinase inhibitor, indomethacin, and clopidogrel on LTE4-induced ear edema in CysLTR-deficient mice. (A) WT (filled squares), Cysltr1−/− (diamonds), Cysltr2−/− (triangles), and Cysltr1/Cysltr2−/− (open circles) mice received intradermal injections of 0.5 nmol (in 25 μl) LTE4 in the right ear and 25 μl vehicle in the left ear in the presence or absence of various inhibitors. Mice were injected intravenously with PTX (30 μg/kg) 6 h before or with Y-27632 (10 mg/kg) or indomethacin (10 mg/kg) 1 h before intradermal injection of LTE4. Clopidogrel was administered in drinking water for 2 days before the LTE4 injection. Results are expressed as the mean ± SD of the differences between the thickness of the right and left ears at each time point and are combined from two independent experiments. Some error bars are too small to be visible compared with the physical size of the symbol. (two mice per group for B and E; three mice per group for C; four mice per group for A and D). Results are combined from two independent experiments. *, P < 0.01 vs. mice of the same strain with no inhibitor treatment, A is presented for 30, 60, and 240 min.

Our finding of a functional CysLTER in the ear of Cysltr1/Cysltr2−/− mice that mediates an increase in vascular permeability to each cys-LT with a preference for LTE4 highlights the complexity of the cys-LT system in vivo. The permeability increase in WT mice at the dose of 0.5 nmol per site was comparable for the three cys-LTs, but the response to LTE4 was not inhibited in the Cysltr1−/− mice, implying recognition by CysLTER (Fig. 1). The vascular leak in the Cysltr2−/− mice was prolonged but not increased in peak for each of the three cys-LTs, suggesting that CysLT2R is a negative regulator of CysLT1R and of CysLTER. The role of CysLT2R as a negative regulator of CysLT1R has been previously observed in proliferation assays of culture-derived human and mouse mast cells (29). The finding that the peak response to LTE4 at 30 min was enhanced in Cysltr1/Cysltr2−/− mice, but not in either Cysltr1−/− or Cysltr2−/− mice, indicates that these receptors may each be intrinsic negative regulators of CysLTER function.

The introduction of the prototype “lukast” inhibitor uncovered yet another level of complexity. Although MK-571 reduced the permeability effect of LTE4 in WT mice, it did not inhibit the effect in Cysltr1−/− mice, and it increased the permeability response to LTE4 in Cysltr2−/− mice to the level of Cysltr1/Cysltr2−/− mice (Fig. 4 A and B). Importantly, the enhanced permeability response in Cysltr1/Cysltr2−/− mice not only to LTE4 but also to LTC4 and LTD4 in the presence of MK-571 indicates the presence of a class-related negative regulation independent of expression of CysLT1R and CysLT2R (Fig. 4 C–E). Such an array of intrinsic and induced negative regulation may have evolved because LTE4, the most stable cys-LT, persists in inflammatory responses in human and mouse tissues in contrast to its labile precursors, LTC4 and LTD4 (21, 30, 31). In view of the comparable vascular potency of LTE4 to that of LTC4 and LTD4 in mouse ear skin (Fig. 1) and human forearm skin (17), it is possible that constitutive expression of CysLTER may be harmful to the host homeostasis, and hence highly regulated. However, the fact that CysLT1R, and probably CysLT2R as well, is rapidly internalized in an agonist-dependent manner (32) could provide settings that favor the presence of CysLTER in acute and chronic inflammation.

Antagonists selective for CysLT1R alone or for both CysLT1R and CysLT2R have been used to implicate a separate receptor refractory to these inhibitors for contractions of pulmonary artery from humans (33, 34) and pigs (35). In the latter study, both LTC4 and LTD4 contracted intact and rubbed rings from porcine pulmonary artery in a dose-dependent manner that was resistant to either a CysLT1R or a dual-receptor antagonist. That LTE4 had a negligible agonist effect in this assay even at the highest dose of 1 μM indicates that functional CysLTER was not involved. Recently, Paruchuri et al. reported that LTE4 can stimulate human mast cells to produce prostaglandin D2 through the nuclear receptor peroxisome proliferator-activated receptor-γ with more potency than LTD4, and that this response was intact after lentiviral knockdown of the CysLT1R but blocked by MK-571 (36). Lentiviral knockdown of the P2Y12 receptor blocked this LTE4 response (J. Boyce, personal communication), thereby distinguishing this receptor from CysLTER.

Our finding in mice of the presence of a novel LT receptor with a preference for LTE4 relative to LTC4 or LTD4 raises the question of whether such a putative receptor has been recognized in human studies. Aerosolized LTE4 elicited impaired pulmonary air flow with mean 39-fold and 14-fold greater potency than histamine in five normal and six asthmatic individuals, respectively, thereby revealing a possible role for LTE4 in asthma (37). Of particular note is the finding that LTE4 was much more potent than LTC4 in causing a fall in specific conductance in asthmatic patients intolerant to aspirin but not in aspirin-tolerant patients with asthma (20, 38). This finding could mean up-regulation of CysLTER in the aspirin-intolerant group. Furthermore, the inhalation of LTE4 but not LTD4 by humans with asthma induced the subsequent recruitment of granulocytes, particularly eosinophils, into the airway mucosa (18). Thus, both the inherent contractile and proinflammatory activities of LTE4 are apparent in the setting of bronchial asthma. Here, we show that an LTE4-mediated increase in vascular permeability is intrinsically counterregulated by the two known cys-LT receptors and, when uncovered by their absence, is further regulated by an MK-571-susceptible mechanism. It is reasonable to suggest that CysLT1R blockade may contribute to the heterogeneity of its clinical efficacy in bronchial asthma, particularly in a circumstance in which the CysLTER is functionally up-regulated.

Materials and Methods

Reagents.

LTC4, LTD4, LTE4, and MK-571 were purchased from Cayman Chemical. The cys-LTs were reconstituted in DMSO at a concentration of 200 μg/ml after ethanol evaporation under nitrogen gas. Indomethacin, PTX, and a Rho kinase inhibitor, Y-27632, were from Sigma–Aldrich. A P2Y12 receptor blocker, clopidogrel bisulfate (Plavix), was from Sanofi-Aventis.

Animals and Treatment Procedures.

Cysltr1−/− and Cysltr2−/− mice were generated on a C57BL/6 background as reported previously (13, 14) and were backcrossed for 10 generations to a BALB/c background. Cysltr1/Cysltr2−/− mice were generated by crossbreeding BALB/c Cysltr1−/− and Cysltr2−/− mice. Two- to four-month-old, sex-matched Cysltr1−/−, Cysltr2−/−, and Cysltr1/Cysltr2−/− mice were used. Lack of expression of mRNAs for CysLT1R and CysLT2R in the lung of Cysltr1/Cysltr2−/− mice was confirmed by RT-PCR (Fig. S2). WT littermates from the intercrossing of Cysltr1+/− or Cysltr2+/− mice or age-matched BALB/c mice (Charles River Laboratories) were used as controls.

For pharmacologic studies, MK-571 (10 mg/kg, i.v.) and indomethacin (10 mg/kg, i.v.) were each dissolved in DMSO, diluted in PBS, and administered 30 min before intradermal injection of LTE4, LTD4, or LTC4. Clopidogrel was administered in drinking water for 2 days before the injection of LTE4. Mice were treated with saline dilutions of PTX (30 μg/kg, i.v.) 6 h before or with Y-27632 (10 mg/kg, i.v.) 1 h before the intradermal injection of LTE4. All animal studies were approved by the Animal Care and Use Committee of the Dana–Farber Cancer Institute.

RT-PCR.

Total RNA from the tissue of WT, Cysltr1−/−, Cysltr2−/−, and Cysltr1/Cysltr2−/− mice was isolated with TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. Total RNA (1 μg) was reverse transcribed with SuperScript III (Invitrogen), and RT-PCR was performed with primers specific for mouse CysLT1R (5′-CTTGAACGTACTCTGACACTACAA-3′ and 5′-GAGATGTCGTCAGATTTT-3′), mouse CysLT2R (5′-AGATAAGATGTCCATCATGT-3′ and 5′-ACAGTTCCTGTTACTATAAC-3′), and mouse GAPDH (5′-CATGACCACAGTCCATGCCATCACT-3′ and 5′-TGAGGTCCACCACCCTGTTGCTGTA-3′) under the following condition: 98°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 40 cycles.

Intradermal Injection of Leukotrienes and Assessment of Ear Edema.

Mice received intradermal injections of various concentration of LTC4, LTD4, or LTE4 in 25 μl saline/DMSO in the right ear and 25 μl saline/DMSO (vehicle) in the left ear. At 0, 30, 60, 120, 240, and 300 min after the intradermal injection, ear thickness was measured with calipers (Dyer Company). The results are presented as the difference of ear thickness between right ear and left ear.

Histology.

Mice were killed by inhalation of isoflurane ether 30 or 240 min after intradermal LTE4 injection. Ear tissues were fixed in 4% paraformaldehyde and embedded in glycolmethacrylate as described previously (39). Sections 2 μm thick were stained by the chloroacetate esterase reaction to identify mast cells, and neutrophils and were counterstained with hematoxylin.

Measurement of cys-LTs from Ear Tissues After LTC4 Injection.

Mice received intradermal injections of 400 pmol LTC4 in 20 μl saline/DMSO in the right ear and 20 μl saline/DMSO (vehicle) in the left ear. At 60 min after the intradermal injection, ear tissues were isolated and homogenated with a Tissue-Tearor homogenizer (Biospec Products) in 1 ml of 50 mM Hepes buffer, pH 7.9, containing 50% methanol and 200 ng prostaglandin B2. The tissue homogenates were centrifuged at 1,000 × g for 5 min at 4°C, and the supernatants were collected for measurements of cys-LTs by reverse-phase HPLC as previously described (40). Briefly, samples were applied to a C18 Ultrasphere RP column (Beckman Instruments) equilibrated with a solvent of methanol/acetonitrile/water/acetic acid (10:15:100:0.2, vol/vol), pH 6.0 (solvent A). After injection of the sample, the column was eluted at a flow rate of 1 ml/min with a programmed concave gradient to 55% of the equilibrated solvent A and 45% solvent B (100% methanol) over 2.5 min. After 5 min, solvent B was increased linearly to 75% over 15 min and was maintained at this level for an additional 15 min. The UV absorbance at 280 nm and the UV spectra were recorded simultaneously. The retention times for prostaglandin B2, LTC4, LTD4, and LTE4 were 20.4, 21.5, 23.2, and 24.5 min, respectively. LTC4 and LTE4 were quantitated by calculating the ratio of each peak area to the peak area of the internal standard prostaglandin B2.

Statistics.

Student's t test was used for the statistical analysis in cases in which the variance was homogeneous, and Welch's test was used when the variance was heterogeneous. For simplicity, statistical analyses were performed at 30 and 60 min for the peak and 240 min for resolution. A value of P < 0.05 was considered significant.

Supplementary Material

Supporting Information
supp_105_43_16695__index.html (681B, html)

Acknowledgments.

We thank Juying Lai for technical assistance, Dr. Bing Lam for HPLC analysis of cys-LTs, and Drs. Joshua Boyce and Jonathan Arm for critical reading of the manuscript. This work was supported by National Institutes of Health grants AI-31599, AI-52353, HL-36110, and HL-82695.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808993105/DCSupplemental.

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