β2-Adrenoceptor (β2AR) agonists are the most effective class of bronchodilators and a mainstay of asthma management. The first potent β2AR agonist discovered and widely used in reversing the airway constriction associated with asthma exacerbation was the endogenous activator of the β2AR, epinephrine. In this study, we demonstrate that activation of the β2AR by epinephrine is paradoxically required for development of the asthma phenotype. In an antigen-driven model, mice sensitized and challenged with ovalbumin showed marked elevations in three cardinal features of the asthma phenotype: inflammatory cells in their bronchoalveolar lavage fluid, mucin over production, and airway hyperresponsiveness. However, genetic depletion of epinephrine using mice lacking the enzyme to synthesize epinephrine, phenylethanolamine N-methyltransferase, or mice that had undergone pharmacological sympathectomy with reserpine to deplete epinephrine, had complete attenuation of these three cardinal features of the asthma phenotype. Furthermore, administration of the long-acting β2AR agonist, formoterol, a drug currently used in asthma treatment, to phenylethanolamine N-methyltransferase–null mice restored the asthma phenotype. We conclude that β2AR agonist–induced activation is needed for pathogenesis of the asthma phenotype. These findings also rule out constitutive signaling by the β2AR as sufficient to drive the asthma phenotype, and may help explain why chronic administration of β2AR agonists, such as formoterol, have been associated with adverse outcomes in asthma. These data further support the hypothesis that chronic asthma management may be better served by treatment with certain “β-blockers.”
β2-adrenoceptor agonists; formoterol; epinephrine; murine model; asthma
Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.
exocytosis; mast cell; mucin; mucus; Munc18; secretion; AB-PAS, Alcian Blue/periodic acid/Schiff reagent; bHLH, basic helix–loop–helix; CCSP, Clara cell secretory protein; Clca3, chloride channel, calcium-activated, family member 3; CRE, cAMP-response element; DNP, 2,4-dinitrophenol; FBS, fetal bovine serum; FcϵRIα, high-affinity IgE receptor, α subunit; FRT, flippase recognition target; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRE, glucocorticoid-response element; HA, haemagglutinin; HSA, human serum albumin; HRP, horseradish peroxidase; IL-3, interleukin-3; INR, initiator; ISH, in situ hybridization; MC, mast cell; mBMMC, mouse bone-marrow-derived MC; mClca3, mouse Clca3; MFI, mean fluorescent intensity; mtCC, mouse transformed Clara cell; NK, natural killer; OCT, optimal cutting temperature compound; PAFS, periodic acid/fluorescent Schiff reagent; PBST, PBS containing 0.05% Tween 20; PGD2, prostaglandin D2; PGK, phosphoglucokinase; SCF, stem cell factor; SM, Sec1/Munc18; SNAP, soluble N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor; Stxbp2, syntaxin-binding protein 2; TK, thymidine kinase; TNFα, tumour necrosis factor α; WT, wild-type; YFP, yellow fluorescent protein