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Background: Airway mucus hypersecretion is a key pathophysiologic feature in a number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear., Objectives: We sought to characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus., Methods: IL-13 and γ-aminobutyric acid type A receptors (GABA(A)Rs) are implicated in airway mucus. We examined the role of IL-13 and GABA(A)Rs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells and secondhand cigarette smoke-induced, ovalbumin-induced, or both mucus formation in vivo., Results: Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABA(A)R antagonist picrotoxin. Airway epithelial cells express α7-, α9-, and α10-nicotinic acetylcholine receptors (nAChRs), and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen- or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells, murine airways, or both., Conclusions: Nicotine-induced airway mucus formation is independent of IL-13, and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various promucoid agents, including IL-13. In the absence of nicotine, acetylcholine might be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABA(A)Rα2 in the MUC5AC pathway. Acetylcholine and α7-nAChRs might serve as therapeutic targets to control airway mucus., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Sulfur mustard (SM) is a highly toxic chemical warfare agent that remains a threat to human health. The immediate symptoms of pulmonary distress may develop into chronic lung injury characterized by progressive lung fibrosis, the major cause of morbidity among the surviving SM victims. Although SM has been intensely investigated, little is known about the mechanism(s) by which SM induces chronic lung pathology. Increasing evidence suggests that IL-17(+) cells are critical in fibrosis, including lung fibrotic diseases. In this study we exposed F344 rats and cynomolgus monkeys to SM via inhalation and determined the molecular and cellular milieu in their lungs at various times after SM exposure. In rats, SM induced a burst of pro-inflammatory cytokines/chemokines within 72 h, including IL-1β, TNF-α, IL-2, IL-6, CCL2, CCL3, CCL11, and CXCL1 that was associated with neutrophilic infiltration into the lung. At 2 wks and beyond (chronic phase), lymphocytic infiltration and continued elevated expression of cytokines/chemokines were sustained. TGF-β, which was undetectable in the acute phase, was strongly upregulated in the chronic phase; these conditions persisted until the animals were sacrificed. The chronic phase was also associated with myofibroblast proliferation, collagen deposition, and presence of IL-17(+) cells. At ≥30 days, SM inhalation promoted the accumulation of IL-17(+) cells in the inflamed areas of monkey lungs. Thus, SM inhalation causes acute and chronic inflammatory responses; the latter is characterized by the presence of TGF-β, fibrosis, and IL-17(+) cells in the lung. IL-17(+) cells likely play an important role in the pathogenesis of SM-induced lung injury., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Although a number of inflammatory cytokines are increased during sepsis, the clinical trials aimed at down-regulating these mediators have not improved the outcome. These paradoxical results are attributed to loss of the "tolerance" phase that normally follows the proinflammatory response. Chronic nicotine (NT) suppresses both adaptive and innate immune responses, and the effects are partly mediated by the nicotinic acetylcholine receptors in the brain; however, the mechanism of neuroimmune communication is not clear. Here, we present evidence that, in rats and mice, NT initially increases IL-1β in the brain, but the expression is downregulated within 1-2 week of chronic exposure, and the animals become resistant to proinflammatory/pyrogenic stimuli. To examine the relationship between NT, IL-1β, and immunosuppression, we hypothesized that NT induces IL-1β in the brain, and its constant presence produces immunological "tolerance". Indeed, unlike wild-type C57BL/6 mice, chronic NT failed to induce immunosuppression or downregulation of IL-1β expression in IL-1β-receptor knockout mice. Moreover, while acute intracerebroventricular administration of IL-1β in Lewis (LEW) rats activated Fyn and protein tyrosine kinase activities in the spleen, chronic administration of low levels of IL-1β progressively diminished the pyrogenic and T cell proliferative responses of treated animals. Thus, IL-1β may play a critical role in the perception of inflammation by the CNS and the induction of an immunologic "tolerant" state. Moreover, the immunosuppressive effects of NT might be at least partly mediated through its effects on the brain IL-1β. This represents a novel mechanism for neuroimmune communication.

Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8-10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.

Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC(4). Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcepsilonRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using alpha-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) alpha7, alpha9, and alpha10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for > or = 8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A(2) activity and the PI3K/ERK/NF-kappaB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kappaB. The suppressive effect of nicotine on the late-phase response was blocked by the alpha7/alpha9-nAChR antagonists methyllycaconitine and alpha-bungarotoxin, as well as by small interfering RNA knockdown of alpha7-, alpha9-, or alpha10-nAChRs, suggesting a functional interaction between alpha7-, alpha9-, and alpha10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

Sulfur mustard (SM, bis-(2-chloroethyl) sulfide) is a well known chemical warfare agent that may cause long-term debilitating injury. Because of the ease of production and storage, it has a strong potential for chemical terrorism; however, the mechanism by which SM causes chronic tissue damage is essentially unknown. SM is a potent protein alkylating agent, and we tested the possibility that SM modifies cellular antigens, leading to an immunological response to "altered self" and a potential long-term injury. To that end, in this communication, we show that dermal exposure of euthymic hairless guinea pigs induced infiltration of both CD4(+) and CD8(+) T cells into the SM-exposed skin and strong upregulated expression of proinflammatory cytokines and chemokines (TNF-alpha, IFN-gamma, and IL-8) in distal tissues such as the lung and the lymph nodes. Moreover, we present evidence for the first time that SM induces a specific delayed-type hypersensitivity response that is associated with splenomegaly, lymphadenopathy, and proliferation of cells in these tissues. These results clearly suggest that dermal exposure to SM leads to immune activation, infiltration of T cells into the SM-exposed skin, delayed-type hypersensitivity response, and molecular imprints of inflammation in tissues distal from the site of SM exposure. These immunological responses may contribute to the long-term sequelae of SM toxicity., (Copyright 2009 Elsevier B.V. All rights reserved.)

Airway hyperreactivity (AHR), lung inflammation, and atopy are clinical signs of allergic asthma. Gestational exposure to cigarette smoke (CS) markedly increases the risk for childhood allergic asthma. Muscarinic receptors regulate airway smooth muscle tone, and asthmatics exhibit increased AHR to muscarinic agonists. We have previously reported that in a murine model of bronchopulmonary aspergillosis, maternal exposure to mainstream CS increases AHR after acute intratracheal administration of Aspergillus fumigatus extract. However, the mechanism by which gestational CS induces allergic asthma is unclear. We now show for the first time that, compared with controls, mice exposed prenatally to secondhand CS exhibit increased lung inflammation (predominant infiltration by eosinophils and polymorphs), atopy, and airway resistance, and produce proinflammatory cytokines (IL-4, IL-5, IL-6, and IL-13, but not IL-2 or IFN-gamma). These changes, which occur only after an allergen (A. fumigatus extract) treatment, are correlated with marked up-regulated lung expression of M1, M2, and M3 muscarinic receptors and phosphodiesterase (PDE)4D5 isozyme. Interestingly, the PDE4-selective inhibitor rolipram attenuates the increase in AHR, muscarinic receptors, and PDE4D5, but fails to down-regulate lung inflammation, Th2 cytokines, or serum IgE levels. Thus, the fetus is extraordinarily sensitive to CS, inducing allergic asthma after postnatal exposure to allergens. Although the increased AHR might reflect increased PDE4D5 and muscarinic receptor expression, the mechanisms underlying atopy and lung inflammation are unrelated to the PDE4 activity. Thus, PDE4 inhibitors might ease AHR, but are unlikely to attenuate lung inflammation and atopy associated with childhood allergic asthma.

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C(4); and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.

Immunological tolerance during prolonged exposure to allergen is accompanied by a shift in the lymphocyte content and a reduction of goblet cell metaplasia (GCM). Bim initiates negative selection of autoreactive T and B cells and shut down of T cell immune responses in vivo. The present study investigated whether Bim plays a role in the resolution of GCM during prolonged exposure to allergen. Loss of Bim increased T lymphocyte numbers in the bronchoalveolar lavage at 4 and 15 days of allergen exposure. The numbers of pulmonary CD4(+)8(-), CD4(-)8(+), and gammadelta T cells were significantly higher in naive and allergen-challenged bim(-/-) mice compared with wild-type (WT) littermates. When activated, pulmonary bim(-/-) T cells produced increased levels of IFNgamma compared with bim(+/+) T cells. No differences were noted in the total numbers of epithelial cells per millimeter of basal lamina between bim(+/+) and bim(-/-) mice, and the rate of resolution over 15 days of exposure was similar in both groups of mice. However, GCM was significantly enhanced and expression of IL-13Ralpha2 was reduced in bim(-/-) mice compared with WT mice at 4 days. Furthermore, treatment of bronchiolar explant cultures with increasing IFNgamma levels reduced immunostaining for IL-13Ralpha2. Collectively, these studies suggest that, during prolonged exposure to allergen, Bim plays no role in the resolution of GCM, but increased IFNgamma levels in bim(-/-) mice may be responsible for reduced expression of IL-13Ralpha2 and enhanced GCM despite similar levels of IL-13 in bim(+/+) and bim(-/-) mice.