Leukotriene E4 elicits respiratory epithelial cell mucin release through the G-protein-coupled receptor, GPR99

Abstract

Cysteinyl leukotrienes (cysLTs), leukotriene C4 (LTC4), LTD4, and LTE4 are proinflammatory lipid mediators with pathobiologic function in asthma. LTE4, the stable cysLT, is a weak agonist for the type 1 and type 2 cysLT receptors (CysLTRs), which constrict airway smooth muscle, but elicits airflow obstruction and pulmonary inflammation in patients with asthma. We recently identified GPR99 as a high-affinity receptor for LTE4 that mediates cutaneous vascular permeability. Here we demonstrate that a single intranasal exposure to extract from the respiratory pathogen Alternaria alternata elicits profound epithelial cell (EpC) mucin release and submucosal swelling in the nasal mucosa of mice that depends on cysLTs, as it is absent in mice deficient in the terminal enzyme for cysLT biosynthesis, LTC4 synthase (LTC4S). These mucosal changes are associated with mast cell (MC) activation and absent in MC-deficient mice, suggesting a role for MCs in control of EpC function. Of the three CysLTRs, only GPR99-deficient mice are fully protected from EpC mucin release and swelling elicited by Alternaria or by intranasal LTE4 GPR99 expression is detected on lung and nasal EpCs, which release mucin to doses of LTE4 one log lower than that required to elicit submucosal swelling. Finally, mice deficient in MCs, LTC4S, or GPR99 have reduced baseline numbers of goblet cells, indicating an additional function in regulating EpC homeostasis. These results demonstrate a novel role for GPR99 among CysLTRs in control of respiratory EpC function and suggest that inhibition of LTE4 and of GPR99 may have therapeutic benefits in asthma.

Keywords: cysteinyl leukotrienes; epithelial cell; lung; mast cells; mucosal immunology.

Fig. 1.

cysLT-dependent submucosal swelling and mucin release elicited by Alternaria. (A) Representative histological coronal section through the incisors of the mouse skull of a PBS-challenged mouse with CAE stain (50× magnification). (B) A 630× magnification of Inset in A, rotated 90°. Black arrowheads point to MCs. (C–E) PAS staining of nasal septum 1 h after i.n. administration of (C) PBS or (D and E) 30 µg of Alternaria (magnification: 630×). (Scale bar, 20 µm.) Black arrows indicate mucin-containing GCs. Red arrows indicate GCs with partial extrusion of mucin. (F and G) WT (black bars) and Ltc4s^−/− (gray bars) mice. (F) Submucosal swelling, indicated by the difference in the submucosal area between PBS (0 µg) and Alternaria-treated (30 µg) mice. (G) Mucin release, indicated by the difference in the total number of PAS⁺ cells detected between PBS and Alternaria-treated mice. Results are means ± SEM pooled from four independent experiments with the number of mice per group indicated on each bar in parentheses. *P < 0.05, **P < 0.01.

Fig. 2.

The innate activation of MCs is required for Alternaria-elicited mucosal responses. (A) Percentage of degranulating MCs in the nasal submucosa 1 h after i.n. administration of PBS (0 µg) or Alternaria (30 µg) to WT (black bars) and Fcer1g^‒/‒ (brown bars) mice. (B) ELISA quantification of PGD₂ in the NL 1 h after i.n. administration of PBS or Alternaria to WT, Fcer1g^‒/‒, and Mcpt5/DTA (white bars) mice. (C) BMMCs from WT, Fcer1g^‒/‒, or Ltc4s^‒/‒ mice were harvested after 5–8 wk of culture and stimulated with Alternaria. The concentrations of cysLTs in the supernatants were measured by enzyme immunoassay at 30 min. Results are means ± SEM pooled from three independent experiments with cultures obtained from 10 WT mice, 3 Ltc4s^‒/‒ mice, and 3 Fcer1g^‒/‒ mice. (D) β-Hexosaminidase release into the supernatant at 30 min as a percentage of total. Results are means ± SEM pooled from three independent BMMC cultures and three independent human CD34⁺ cell-derived MC cultures. (E) Submucosal swelling and (F) mucin release in WT, Mcpt5/DTA, and Fcer1g^‒/‒ mice 1 h after a single dose of PBS or Alternaria. Results are means ± SEM pooled from three (A) or five (B, E, and F) independent experiments with the number of mice per group indicated on each bar in parentheses. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. S1.

MCs in the nasal cavity degranulate in response to Alternaria. (A) Representative histological coronal section through the first molar teeth of the mouse skull of a PBS-challenged mouse with CAE stain. MS, maxillary sinuses; NCF, nasal cavity floor; NS, nasal septum (50× magnification). (B) Magnification of Inset from A; CAE-reactive MCs stain red. (C) Degranulating MCs in the nasal cavity floor 1 h after Alternaria administration. Arrows point to degranulating MCs (magnification: 630×). (D) Number of degranulating MCs. (E) Number of intact nondegranulating MCs. (F) Total number of MCs (degranulated + intact MCs) enumerated in seven 200× fields in WT (black bars), Fcer1g^‒/‒ (brown bars), and Mcpt5/DTA (open bars) mice. Data are means ± SEM pooled from three to four independent experiments with the number of mice per group indicated on each bar in parentheses. *P < 0.05, **P < 0.01.

Fig. 3.

GPR99 controls Alternaria-elicited EpC mucin release and baseline EpC composition. (A–D) Alternaria (30 µg) or PBS (0 µg) was administered to WT (black bars), Cysltr1^‒/‒ (blue bars), Cysltr2^‒/‒ (green bars), and Gpr99^‒/‒ (red bars) mice. (A and C) Swelling and (B and D) mucin release were measured in histological samples from the nasal mucosa 1 h after i.n. challenge, as in Fig. 1. Results are means ± SEM pooled from three independent experiments with the numbers of mice per group indicated on each bar in parentheses. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. S2.

MCs in the nasal cavity of Cysltr1^‒/‒ and Gpr99^‒/‒ mice are activated after i.n. Alternaria administration. (A and B) Degranulating MCs in the nasal submucosa of (A) Cysltr1^‒/‒ and (B) Gpr99^‒/‒ mice. Magnification: 400×. Arrows indicate degranulating MCs. (C and D) PGD₂ in the NL of WT (black bars), Cysltr1^‒/‒ (blue bars), and Gpr99^‒/‒ (red bars) mice. Data are means ± SEM from one experiment with the number of mice per group indicated on each bar in parentheses.

Fig. 4.

GPR99 is expressed in respiratory EpCs and mediates LTE₄-elicited submucosal swelling and mucin release. (A) Quantitative RT-PCR analysis of expression of Gpr99 and β-galactosidase (Bgal) genes in the nasal cavity floor (Upper panels) and nasal mucosa of the septum and side wall (Lower panels) compared with 18S rRNA. Results are means ± SEM pooled from two independent experiments with the numbers of mice per group indicated in parentheses in each column. (B and C) X-gal staining in the nasal mucosa of Gpr99^‒/‒ mice (Right panels) and WT mice (Left panels) in the (B) nasal cavity (magnification: 630×) and (C) large bronchi (magnification: 200×). Positive X-gal reactivity produces a blue precipitate. (D and E) LTE₄ (0.01, 0.1, or 1 nmol) or PBS (0 nmol) was administered i.n. to WT (black bars), Cysltr1^‒/‒ (blue bars), and Gpr99^‒/‒ (red bars) mice. (D) Submucosal swelling and (E) mucin release were measured 1 h after LTE₄ administration in histological samples from the nasal mucosa. Data are means ± SEM pooled from three independent experiments with the numbers of mice per group indicated on each bar in parentheses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. S3.

Quantitative RT-PCR analysis of CysLT₁R (blue bars) and CysLT₂R (green bars) expression in the nasal cavity floor (“floor”) and nasal septum (“septum”) of WT mice. Data are means ± SEM from one experiment with number of mice per group indicated on each bar in parentheses.

Fig. S4.

Confocal microscopy of nasal (Upper panels) and bronchial (Lower panels) epithelia stained with X-gal. The X-gal precipitate fluoresces in the far red spectrum. Nuclei stained blue with Hoechst 33342 solution. Magnification: 630×.