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

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) C(4), which then undergoes extracellular metabolism to LTD(4) and LTE(4). LTE(4), the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT(1)R and CysLT(2)R, respectively). We had recognized a greater potency for LTE(4) than LTC(4) or LTD(4) 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 LTE(4) in mice lacking both the CysLT(1)R and CysLT(2)R could establish the existence of a separate LTE(4) receptor. We now report that the intradermal injection of LTE(4) into the ear of mice deficient in both CysLT(1)R and CysLT(2)R elicits a vascular leak that exceeds the response to intradermal injection of LTC(4) or LTD(4), and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE(4) is approximately 64-fold more potent in the CysLT(1)R/CysLT(2)R double-deficient mice than in sufficient mice. The administration of a CysLT(1)R antagonist augmented the permeability response of the CysLT(1)R/CysLT(2)R double-deficient mice to LTC(4), LTD(4), and LTE(4). Our findings establish the existence of a third receptor, CysLT(E)R, that responds preferentially to LTE(4), the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.)

Fig. 4.

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.

Fig. 5.

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.