HIF-1α Plays a Critical Role in the Gestational Sidestream Smoke-Induced Bronchopulmonary Dysplasia in Mice.

RATIONALE: Smoking during pregnancy increases the risk of bronchopulmonary dysplasia (BPD) and, in mice, gestational exposure to sidestream cigarette smoke (SS) induces BPD-like condition characterized by alveolar simplification, impaired angiogenesis, and suppressed surfactant protein production. Normal fetal development occurs in a hypoxic environment and nicotinic acetylcholine receptors (nAChRs) regulate the hypoxia-inducible factor (HIF)-1α that controls apoptosis and angiogenesis. To understand SS-induced BPD, we hypothesized that gestational SS affected alveolar development through HIF-1α. METHODS: Pregnant BALB/c mice were exposed to air (control) or SS throughout the gestational period and the 7-day-old lungs of the progeny were examined. RESULTS: Gestational SS increased apoptosis of alveolar and airway epithelial cells. This response was associated with increased alveolar volumes, higher levels of proapoptotic factors (FOXO3a, HIPK2, p53, BIM, BIK, and BAX) and the antiangiogenic factor (GAX), and lower levels of antiapoptotic factors (Akt-PI3K, NF-κB, HIF-1α, and Bcl-2) in the lung. Although gestational SS increased the cells containing the proangiogenic bombesin-like-peptide, it markedly decreased the expression of its receptor GRPR in the lung. The effects of SS on apoptosis were attenuated by the nAChR antagonist mecamylamine. CONCLUSIONS: Gestational SS-induced BPD is potentially regulated by nAChRs and associated with downregulation of HIF-1α, increased apoptosis of epithelial cells, and increased alveolar volumes. Thus, in mice, exposure to sidestream tobacco smoke during pregnancy promotes BPD-like condition that is potentially mediated through the nAChR/HIF-1α pathway.

A critical role of acute bronchoconstriction in the mortality associated with high-dose sarin inhalation: effects of epinephrine and oxygen therapies.

Sarin is an organophosphate nerve agent that is among the most lethal chemical toxins known to mankind. Because of its vaporization properties and ease and low cost of production, sarin is the nerve agent with a strong potential for use by terrorists and rouge nations. The primary route of sarin exposure is through inhalation and, depending on the dose, sarin leads to acute respiratory failure and death. The mechanism(s) of sarin-induced respiratory failure is poorly understood. Sarin irreversibly inhibits acetylcholine esterase, leading to excessive synaptic levels of acetylcholine and, we have previously shown that sarin causes marked ventilatory changes including weakened response to hypoxia. We now show that LD50 sarin inhalation causes severe bronchoconstriction in rats, leading to airway resistance, increased hypoxia-induced factor-1α, and severe lung epithelium injury. Transferring animals into 60% oxygen chambers after sarin exposure improved the survival from about 50% to 75% at 24h; however, many animals died within hours after removal from the oxygen chambers. On the other hand, if LD50 sarin-exposed animals were administered the bronchodilator epinephrine, >90% of the animals survived. Moreover, while both epinephrine and oxygen treatments moderated cardiorespiratory parameters, the proinflammatory cytokine surge, and elevated expression of hypoxia-induced factor-1α, only epinephrine consistently reduced the sarin-induced bronchoconstriction. These data suggest that severe bronchoconstriction is a critical factor in the mortality induced by LD50 sarin inhalation, and epinephrine may limit the ventilatory, inflammatory, and lethal effects of sarin.

HIV gp120 induces mucus formation in human bronchial epithelial cells through CXCR4/α7-nicotinic acetylcholine receptors.

Lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, and lung infections are major causes of morbidity and mortality among HIV-infected patients even in the era of antiretroviral therapy (ART). Many of these diseases are strongly associated with smoking and smoking is more common among HIV-infected than uninfected people; however, HIV is an independent risk factor for chronic bronchitis, COPD, and asthma. The mechanism by which HIV promotes these diseases is unclear. Excessive airway mucus formation is a characteristic of these diseases and contributes to airway obstruction and lung infections. HIV gp120 plays a critical role in several HIV-related pathologies and we investigated whether HIV gp120 promoted airway mucus formation in normal human bronchial epithelial (NHBE) cells. We found that NHBE cells expressed the HIV-coreceptor CXCR4 but not CCR5 and produced mucus in response to CXCR4-tropic gp120. The gp120-induced mucus formation was blocked by the inhibitors of CXCR4, α7-nicotinic acetylcholine receptor (α7-nAChR), and γ-aminobutyric acid (GABA)AR but not the antagonists of CCR5 and epithelial growth factor receptor (EGFR). These results identify two distinct pathways (α7-nAChR-GABAAR and EGFR) for airway mucus formation and demonstrate for the first time that HIV-gp120 induces and regulates mucus formation in the airway epithelial cells through the CXCR4-α7-nAChR-GABAAR pathway. Interestingly, lung sections from HIV ± ART and simian immunodeficiency virus (SIV) ± ART have significantly more mucus and gp120-immunoreactivity than control lung sections from humans and macaques, respectively. Thus, even after ART, lungs from HIV-infected patients contain significant amounts of gp120 and mucus that may contribute to the higher incidence of obstructive pulmonary diseases in this population.

Gestational exposure of mice to secondhand cigarette smoke causes bronchopulmonary dysplasia blocked by the nicotinic receptor antagonist mecamylamine.

BACKGROUND: Cigarette smoke (CS) exposure during gestation may increase the risk of bronchopulmonary dysplasia (BPD)-a developmental lung condition primarily seen in neonates that is characterized by hypoalveolarization, decreased angiogenesis, and diminished surfactant protein production and may increase the risk of chronic obstructive pulmonary disease. OBJECTIVE: We investigated whether gestational exposure to secondhand CS (SS) induced BPD and sought to ascertain the role of nicotinic acetylcholine receptors (nAChRs) in this response. METHODS: We exposed BALB/c and C57BL/6 mice to filtered air (control) or SS throughout the gestation period or postnatally up to 10 weeks. Lungs were examined at 7 days, 10 weeks, and 8 months after birth. RESULTS: Gestational but not postnatal exposure to SS caused a typical BPD-like condition: suppressed angiogenesis [decreased vascular endothelial growth factor (VEGF), VEGF receptor, and CD34/CD31 (hematopoietic progenitor cell marker/endothelial cell marker)], irreversible hypoalveolarization, and significantly decreased levels of Clara cells, Clara cell secretory protein, and surfactant proteins B and C, without affecting airway ciliated cells. Importantly, concomitant exposure to SS and the nAChR antagonist mecamylamine during gestation blocked the development of BPD. CONCLUSIONS: Gestational exposure to SS irreversibly disrupts lung development leading to a BPD-like condition with hypoalveolarization, decreased angiogenesis, and diminished lung secretory function. Nicotinic receptors are critical in the induction of gestational SS-induced BPD, and the use of nAChR antagonists during pregnancy may block CS-induced BPD.

Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo.

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.

Inhalation of sulfur mustard causes long-term T cell-dependent inflammation: possible role of Th17 cells in chronic lung pathology.

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-1ß, 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-ß, 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-ß, fibrosis, and IL-17(+) cells in the lung. IL-17(+) cells likely play an important role in the pathogenesis of SM-induced lung injury.

Prenatal secondhand cigarette smoke promotes Th2 polarization and impairs goblet cell differentiation and airway mucus formation.

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.

The role of IL-1ß in nicotine-induced immunosuppression and neuroimmune communication.

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-1ß 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-1ß, and immunosuppression, we hypothesized that NT induces IL-1ß 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-1ß expression in IL-1ß-receptor knockout mice. Moreover, while acute intracerebroventricular administration of IL-1ß in Lewis (LEW) rats activated Fyn and protein tyrosine kinase activities in the spleen, chronic administration of low levels of IL-1ß progressively diminished the pyrogenic and T cell proliferative responses of treated animals. Thus, IL-1ß 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-1ß. This represents a novel mechanism for neuroimmune communication.

Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors.

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.

Fate and effects of adult bone marrow cells in lungs of normoxic and hyperoxic newborn mice.

Cell-based therapy in adult lung injury models is associated with highly variable donor cell engraftment and epithelial reconstitution. The role of marrow-derived cell therapy in neonatal lung injury is largely unknown. In this study, we determined the fate and effects of adult bone marrow cells in a model of neonatal lung injury. Wild-type mice placed in a normoxic or hyperoxic (95% O(2)) environment received bone marrow cells from animals expressing green fluorescent protein (GFP) at Postnatal Day (P)5. Controls received vehicle buffer. Lungs were analyzed between Post-Transplantation (TPX) Day 2 and Week 8. The volume of GFP-immunoreactive donor cells, monitored by stereologic volumetry, remained constant between Post-TPX Weeks 1 and 8 and was similar in normoxic and hyperoxia-exposed recipients. Virtually all marrow-derived cells showed colocalization of GFP and the pan-macrophage marker, F4/80, by double immunofluorescence studies. Epithelial transdifferentiation was not seen. Marrow cell administration had adverse effects on somatic growth and alveolarization in normoxic mice, while no effects were discerned in hyperoxia-exposed recipients. Reexposure of marrow-treated animals to hyperoxia at P66 resulted in significant expansion of the donor-derived macrophage population. In conclusion, intranasal administration of unfractionated bone marrow cells to newborn mice does not achieve epithelial reconstitution, but establishes persistent alveolar macrophage chimerism. The predominantly adverse effects of marrow treatment in newborn lungs are likely due to macrophage-associated paracrine effects. While this model and route of cell therapy may not achieve epithelial reconstitution, the role of selected stem cell populations and/or alternate routes of administration for cell-based therapy in injured newborn lungs deserve further investigation.

The Fas system confers protection against alveolar disruption in hyperoxia-exposed newborn mice.

The functional significance of the Fas/Fas-ligand (FasL) system in hyperoxia-induced lung injury and alveolar disruption in newborn lungs in vivo remains undetermined. To assess the role of the Fas/FasL system, we compared the effects of hyperoxia (95% O2 from birth to Postnatal Day [P]7) in Fas-deficient lpr mice and wild-type mice. Alveolar disruption was more severe in hyperoxic lpr mice than in wild-type mice. In addition, a transient alveolarization defect was noted in normoxic lpr mice. Hyperoxia induced marked up-regulation of pulmonary Fas expression in wild-type mice, as well as elevated mRNA levels of pro-apoptotic Bax, Bad, and Bak. Pulmonary apoptotic activity was similar in hyperoxic wild-type and lpr mice. In contrast, lung growth and proliferation, assessed by stereologic volumetry and Ki67 proliferation studies, were significantly higher in hyperoxic wild-type mice compared with lpr mice, suggesting the Fas/FasL system has a pro-proliferative role in hyperoxic conditions. Levels of the prosurvival MAPkinase, pERK1/2, were significantly higher in hyperoxic wild-type mice compared with lpr mice, while pAkt levels were similar. These data suggest that the primary role of the Fas/FasL system in hyperoxic newborn lungs is pro-proliferative, rather than pro-apoptotic, and likely mediated through a Fas-ERK1/2 pathway. Fas-induced proliferation and lung growth in hyperoxic newborn lungs may counteract, in part, the detrimental effects of apoptosis mediated by non-Fas pathways, such as pro-apoptotic Bax/Bcl-2 family members. The capacity of the Fas/FasL signaling pathway to mediate protective rather than destructive functions in hyperoxic newborn lungs highlights the versatility of this complex pathway.

Fas-ligand-induced apoptosis of respiratory epithelial cells causes disruption of postcanalicular alveolar development.

Premature infants are at risk for bronchopulmonary dysplasia, a complex condition characterized by impaired alveolar development and increased alveolar epithelial apoptosis. The functional involvement of pulmonary apoptosis in bronchopulmonary dysplasia- associated alveolar disruption remains undetermined. The aims of this study were to generate conditional lung-specific Fas-ligand (FasL) transgenic mice and to determine the effects of FasL-induced respiratory epithelial apoptosis on alveolar remodeling in postcanalicular lungs. Transgenic (TetOp)(7)-FasL responder mice, generated by pronuclear microinjection, were bred with Clara cell secretory protein (CCSP)-rtTA activator mice. Doxycycline (Dox) was administered from embryonal day 14 to postnatal day 7, and lungs were studied between embryonal day 19 and postnatal day 21. Dox administration induced marked respiratory epithelium-specific FasL mRNA and protein up-regulation in double-transgenic CCSP-rtTA(+)/(TetOp)(7)-FasL(+) mice compared with single-transgenic CCSP-rtTA(+) littermates. The Dox-induced FasL up-regulation was associated with dramatically increased apoptosis of alveolar type II cells and Clara cells, disrupted alveolar development, decreased vascular density, and increased postnatal lethality. These data demonstrate that FasL-induced alveolar epithelial apoptosis during postcanalicular lung remodeling is sufficient to disrupt alveolar development after birth. The availability of inducible lung-specific FasL transgenic mice will facilitate studies of the role of apoptosis in normal and disrupted alveologenesis and may lead to novel therapeutic approaches for perinatal and adult pulmonary diseases characterized by dysregulated apoptosis.

Endoglin (CD105) up-regulation in pulmonary microvasculature of ventilated preterm infants.

RATIONALE: Preterm infants exposed to mechanical ventilation and oxygen are at risk for bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disorder characterized by arrested alveolar development. Studies have described disruption of microvascular development in BPD, characterized by primitive angioarchitectural patterns reminiscent of the canalicular/saccular stages of lung development. The molecular regulation of this BPD-associated dysangiogenesis remains undetermined. OBJECTIVES: Endoglin (CD105), a hypoxia-inducible transforming growth factor-beta coreceptor, has been implicated as an important regulator of angiogenesis in various neoplastic and nonneoplastic conditions. The aim of this study was to investigate the expression of endoglin and other angiogenesis-related factors in ventilated preterm human lungs. METHODS: We have studied endoglin protein and mRNA expression in postmortem lungs of short-term and long-term ventilated preterm infants. Control subjects were age-matched infants who had lived for less than 1 hour. MEASUREMENTS AND MAIN RESULTS: Lungs of short-term ventilated preterm infants showed significant upregulation of endoglin mRNA and protein levels, immunolocalized to the microvasculature. Similar but more variable endoglin upregulation was noted in lungs of long-term ventilated infants with BPD. The mRNA levels of vascular endothelial growth factor, angiopoietin-1, and their respective receptors were significantly lower in ventilated lungs than in age-matched nonventilated control lungs. CONCLUSIONS: BPD is associated with a shift from traditional angiogenic growth factors (vascular endothelial growth factor, angiopoietin-1) to alternative regulators such as endoglin, which may contribute to BPD-associated microvascular dysangiogenesis.