Pulmonary surfactant proteins and lipids are required for lung function after birth. Lung immaturity and resultant surfactant deficiency cause respiratory distress syndrome, a common disorder contributing to morbidity and mortality in preterm infants. Surfactant synthesis increases prior to birth in association with formation of the alveoli that mediate efficient gas exchange. To identify mechanisms controlling perinatal lung maturation, the Calcineurin b1 (Cnb1) gene was deleted in the respiratory epithelium of the fetal mouse. Deletion of Cnb1 caused respiratory failure after birth and inhibited the structural maturation of the peripheral lung. Synthesis of surfactant and a lamellar body–associated protein, ABC transporter A3 (ABCA3), was decreased prior to birth. Nuclear factor of activated T cells (Nfat) calcineurin-dependent 3 (Nfatc3), a transcription factor modulated by calcineurin, was identified as a direct activator of Sftpa, Sftpb, Sftpc, Abca3, Foxa1, and Foxa2 genes. The calcineurin/Nfat pathway controls the morphologic maturation of lungs prior to birth and regulates expression of genes involved in surfactant homeostasis that are critical for adaptation to air breathing.
Vrushank Davé, Tawanna Childs, Yan Xu, Machiko Ikegami, Valérie Besnard, Yutaka Maeda, Susan E. Wert, Joel R. Neilson, Gerald R. Crabtree, Jeffrey A. Whitsett
Kaushik Parthasarathi, Hideo Ichimura, Eiji Monma, Jens Lindert, Sadiqa Quadri, Andrew Issekutz, Jahar Bhattacharya
Acute lung injury (ALI), which is associated with a mortality of 30–40%, is attributable to inflammation that develops rapidly across the lung’s vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 μm from the target site, indicating that Ca2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.
Kaushik Parthasarathi, Hideo Ichimura, Eiji Monma, Jens Lindert, Sadiqa Quadri, Andrew Issekutz, Jahar Bhattacharya
Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase–deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883–treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy.
Chun-Xiao Sun, Hongyan Zhong, Amir Mohsenin, Eva Morschl, Janci L. Chunn, Jose G. Molina, Luiz Belardinelli, Dewan Zeng, Michael R. Blackburn
Bronchial asthma, the most prevalent cause of significant respiratory morbidity in the developed world, typically is a chronic disorder associated with long-term changes in the airways. We developed a mouse model of chronic asthma that results in markedly increased numbers of airway mast cells, enhanced airway responses to methacholine or antigen, chronic inflammation including infiltration with eosinophils and lymphocytes, airway epithelial goblet cell hyperplasia, enhanced expression of the mucin genes Muc5ac and Muc5b, and increased levels of lung collagen. Using mast cell–deficient (KitW-sh/W-sh and/or KitW/W-v) mice engrafted with FcRγ+/+ or FcRγ–/– mast cells, we found that mast cells were required for the full development of each of these features of the model. However, some features also were expressed, although usually at less than wild-type levels, in mice whose mast cells lacked FcRγ and therefore could not be activated by either antigen- and IgE-dependent aggregation of FcεRI or the binding of antigen-IgG1 immune complexes to FcγRIII. These findings demonstrate that mast cells can contribute to the development of multiple features of chronic asthma in mice and identify both FcRγ-dependent and FcRγ-independent pathways of mast cell activation as important for the expression of key features of this asthma model.
Mang Yu, Mindy Tsai, See-Ying Tam, Carol Jones, James Zehnder, Stephen J. Galli
Activation of latent TGF-β by the αvβ6 integrin is a critical step in the development of acute lung injury. However, the mechanism by which αvβ6-mediated TGF-β activation is regulated has not been identified. We show that thrombin, and other agonists of protease-activated receptor 1 (PAR1), activate TGF-β in an αvβ6 integrin–specific manner. This effect is PAR1 specific and is mediated by RhoA and Rho kinase. Intratracheal instillation of the PAR1-specific peptide TFLLRN increases lung edema during high-tidal-volume ventilation, and this effect is completely inhibited by a blocking antibody against the αvβ6 integrin. Instillation of TFLLRN during high-tidal-volume ventilation is associated with increased pulmonary TGF-β activation; however, this is not observed in Itgb6–/– mice. Furthermore, Itgb6–/– mice are also protected from ventilator-induced lung edema. We also demonstrate that pulmonary edema and TGF-β activity are similarly reduced in Par1–/– mice following bleomycin-induced lung injury. These results suggest that PAR1-mediated enhancement of αvβ6-dependent TGF-β activation could be one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury, and they identify PAR1 and the αvβ6 integrin as potential therapeutic targets in this condition.
R. Gisli Jenkins, Xiao Su, George Su, Christopher J. Scotton, Eric Camerer, Geoffrey J. Laurent, George E. Davis, Rachel C. Chambers, Michael A. Matthay, Dean Sheppard
Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion-related mortality. To explore the pathogenesis of TRALI, we developed an in vivo mouse model based on the passive transfusion of an MHC class I (MHC I) mAb (H2Kd) to mice with the cognate antigen. Transfusion of the MHC I mAb to BALB/c mice produced acute lung injury with increased excess lung water, increased lung vascular and lung epithelial permeability to protein, and decreased alveolar fluid clearance. There was 50% mortality at a 2-hour time point after Ab administration. Pulmonary histology and immunohistochemistry revealed prominent neutrophil sequestration in the lung microvasculature that occurred concomitantly with acute peripheral blood neutropenia, all within 2 hours of administration of the mAb. Depletion of neutrophils by injection of anti-granulocyte mAb Gr-1 protected mice from lung injury following MHC I mAb challenge. FcRγ–/– mice were resistant to MHC I mAb–induced lung injury, while adoptive transfer of wild-type neutrophils into the FcRγ–/– animals restored lung injury following MHC I mAb challenge. In conclusion, in a clinically relevant in vivo mouse model of TRALI using an MHC I mAb, the mechanism of lung injury was dependent on neutrophils and their Fcγ receptors.
Mark R. Looney, Xiao Su, Jessica A. Van Ziffle, Clifford A. Lowell, Michael A. Matthay
To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic inflammation and mild airway fibrosis and did not shorten survival. In C57BL/6 mice, IL-4 caused profound eosinophilic inflammation, airway fibrosis, emphysematous alveolar destruction, and premature death. These differences could not be accounted for by changes in Th2 or Th1 cytokines, receptor components, STAT6 activation, MMPs, or cathepsins. In contrast, in C57BL/6 mice, alveolar remodeling was associated with decreased levels of tissue inhibitors of metalloproteinase 2, -3, and -4 and α1-antitrypsin, and fibrosis was associated with increased levels of total and bioactive TGF-β1. Impressive differences in adenosine metabolism were also appreciated, with increased tissue adenosine levels and A1, A2B, and A3 adenosine receptor expression and decreased adenosine deaminase (ADA) activity in C57BL/6 animals. Treatment with ADA also reduced the inflammation, fibrosis, and emphysematous destruction and improved the survival of C57BL/6 Tg animals. These studies demonstrate that genetic influences control IL-4 effector pathways in the murine lung. They also demonstrate that IL-4 has different effects on adenosine metabolism in Balb/c and C57BL/6 mice and that these differences contribute to the different responses that IL-4 induces in these inbred animals.
Bing Ma, Michael R. Blackburn, Chun Geun Lee, Robert J. Homer, Wei Liu, Richard A. Flavell, Lynn Boyden, Richard P. Lifton, Chun-Xiao Sun, Hays W. Young, Jack A. Elias
Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment.
Jörg Köhl, Ralf Baelder, Ian P. Lewkowich, Manoj K. Pandey, Heiko Hawlisch, Lihua Wang, Jennifer Best, Nancy S. Herman, Alyssa A. Sproles, Jörg Zwirner, Jeffrey A. Whitsett, Craig Gerard, Georgia Sfyroera, John D. Lambris, Marsha Wills-Karp
In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2–/– mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2–/– BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2–/– mice reconstituted with CXCR2–/– BM showed no PMN recruitment. Conversely, CXCR2–/– mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.
Jörg Reutershan, Margaret A. Morris, Tracy L. Burcin, David F. Smith, Daniel Chang, Mary S. Saprito, Klaus Ley
Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2–) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2– also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.
Sang Sun Yoon, Ray Coakley, Gee W. Lau, Sergei V. Lymar, Benjamin Gaston, Ahmet C. Karabulut, Robert F. Hennigan, Sung-Hei Hwang, Garry Buettner, Michael J. Schurr, Joel E. Mortensen, Jane L. Burns, David Speert, Richard C. Boucher, Daniel J. Hassett
Epithelial hyperplasia and metaplasia are common features of inflammatory and neoplastic disease, but the basis for the altered epithelial phenotype is often uncertain. Here we show that long-term ciliated cell hyperplasia coincides with mucous (goblet) cell metaplasia after respiratory viral clearance in mouse airways. This chronic switch in epithelial behavior exhibits genetic susceptibility and depends on persistent activation of EGFR signaling to PI3K that prevents apoptosis of ciliated cells and on IL-13 signaling that promotes transdifferentiation of ciliated to goblet cells. Thus, EGFR blockade (using an irreversible EGFR kinase inhibitor designated EKB-569) prevents virus-induced increases in ciliated and goblet cells whereas IL-13 blockade (using s-IL-13Rα2-Fc) exacerbates ciliated cell hyperplasia but still inhibits goblet cell metaplasia. The distinct effects of EGFR and IL-13 inhibitors after viral reprogramming suggest that these combined therapeutic strategies may also correct epithelial architecture in the setting of airway inflammatory disorders characterized by a similar pattern of chronic EGFR activation, IL-13 expression, and ciliated-to-goblet cell metaplasia.
Jeffrey W. Tyner, Edy Y. Kim, Kyotaro Ide, Mark R. Pelletier, William T. Roswit, Jeffrey D. Morton, John T. Battaile, Anand C. Patel, G. Alexander Patterson, Mario Castro, Melanie S. Spoor, Yingjian You, Steven L. Brody, Michael J. Holtzman
Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.
Allison D. Fryer, Louis H. Stein, Zhenying Nie, Damian E. Curtis, Christopher M. Evans, Simon T. Hodgson, Peter J. Jose, Kristen E. Belmonte, Erin Fitch, David B. Jacoby
Airway smooth muscle (ASM) growth contributes to the mechanism of airway hyperresponsiveness in asthma. Here we demonstrate that CD4+ T cells, central to chronic airway inflammation, drive ASM remodeling in experimental asthma. Adoptive transfer of CD4+ T cells from sensitized rats induced an increase in proliferation and inhibition of apoptosis of airway myocytes in naive recipients upon repeated antigen challenge, which resulted in an increase in ASM mass. Genetically modified CD4+ T cells expressing enhanced GFP (EGFP) were localized by confocal microscopy in juxtaposition to ASM cells, which suggests that CD4+ T cells may modulate ASM cell function through direct cell-cell interaction in vivo. Coculture of antigen-stimulated CD4+ T cells with cell cycle–arrested ASM cells induced myocyte proliferation, dependent on T cell activation and direct T cell–myocyte contact. Reciprocally, direct cell contact prevented postactivation T cell apoptosis, which suggests receptor-mediated T cell–myocyte crosstalk. Overall, our data demonstrate that activated CD4+ T cells drive ASM remodeling in experimental asthma and suggest that a direct cell-cell interaction participates in CD4+ T cell regulation of myocyte turnover and induction of remodeling.
David Ramos-Barbón, John F. Presley, Qutayba A. Hamid, Elizabeth D. Fixman, James G. Martin
Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11–induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11–induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11– and VEGF-induced cytoprotection.
Chuan Hua He, Aaron B. Waxman, Chun Geun Lee, Holger Link, Morgan E. Rabach, Bing Ma, Qingsheng Chen, Zhou Zhu, Mei Zhong, Keiko Nakayama, Keiichi I. Nakayama, Robert Homer, Jack A. Elias
Mechanisms underlying airway hyperresponsiveness are not yet fully elucidated. One of the manifestations of airway inflammation is leakage of diverse plasma proteins into the airway lumen. They include fibrinogen and thrombin. Thrombin cleaves fibrinogen to form fibrin, a major component of thrombi. Fibrin inactivates surfactant. Surfactant on the airway surface maintains airway patency by lowering surface tension. In this study, immunohistochemically detected fibrin was seen along the luminal surface of distal airways in a patient who died of status asthmaticus and in mice with induced allergic airway inflammation. In addition, we observed altered airway fibrinolytic system protein balance consistent with promotion of fibrin deposition in mice with allergic airway inflammation. The airways of mice were exposed to aerosolized fibrinogen, thrombin, or to fibrinogen followed by thrombin. Only fibrinogen followed by thrombin resulted in airway hyperresponsiveness compared with controls. An aerosolized fibrinolytic agent, tissue-type plasminogen activator, significantly diminished airway hyperresponsiveness in mice with allergic airway inflammation. These results are consistent with the hypothesis that leakage of fibrinogen and thrombin and their accumulation on the airway surface can contribute to the pathogenesis of airway hyperresponsiveness.
Scott S. Wagers, Ryan J. Norton, Lisa M. Rinaldi, Jason H.T. Bates, Burton E. Sobel, Charles G. Irvin
To determine the role of IL-5 in airway remodeling, IL-5–deficient and WT mice were sensitized to OVA and challenged by repetitive administration of OVA for 3 months. IL-5–deficient mice had significantly less peribronchial fibrosis (total lung collagen content, peribronchial collagens III and V) and significantly less peribronchial smooth muscle (thickness of peribronchial smooth muscle layer, α-smooth muscle actin immunostaining) compared with WT mice challenged with OVA. WT mice had a significant increase in the number of peribronchial cells staining positive for major basic protein and TGF-β. In contrast, IL-5–deficient mice had a significant reduction in the number of peribronchial cells staining positive for major basic protein, which was paralleled by a similar reduction in the number of cells staining positive for TGF-β, suggesting that eosinophils are a significant source of TGF-β in the remodeled airway. OVA challenge induced significantly higher levels of airway epithelial αVβ6 integrin expression, as well as significantly higher levels of bioactive lung TGF-β in WT compared with IL-5–deficient mice. Increased airway epithelial expression of αVβ6 integrin may contribute to the increased activation of latent TGF-β. These results suggest an important role for IL-5, eosinophils, αVβ6, and TGF-β in airway remodeling.
Jae Youn Cho, Marina Miller, Kwang Je Baek, Ji Won Han, Jyothi Nayar, Sook Young Lee, Kirsti McElwain, Shauna McElwain, Stephanie Friedman, David H. Broide
Acute lung injury syndromes remain common causes of morbidity and mortality in adults and children. Cellular and physiologic mechanisms maintaining pulmonary homeostasis during lung injury remain poorly understood. In the present study, the Stat-3 gene was selectively deleted in respiratory epithelial cells by conditional expression of Cre-recombinase under control of the surfactant protein C gene promoter. Cell-selective deletion of Stat-3 in respiratory epithelial cells did not alter prenatal lung morphogenesis or postnatal lung function. However, exposure of adult Stat-3–deleted mice to 95% oxygen caused a more rapidly progressive lung injury associated with alveolar capillary leak and acute respiratory distress. Epithelial cell injury and inflammatory responses were increased in the Stat-3–deleted mice. Surfactant proteins and lipids were decreased or absent in alveolar lavage material. Intratracheal treatment with exogenous surfactant protein B improved survival and lung histology in Stat-3–deleted mice during hyperoxia. Expression of Stat-3 in respiratory epithelial cells is not required for lung formation, but plays a critical role in maintenance of surfactant homeostasis and lung function during oxygen injury.
Isamu Hokuto, Machiko Ikegami, Mitsuhiro Yoshida, Kiyoshi Takeda, Shizuo Akira, Anne-Karina T. Perl, William M. Hull, Susan E. Wert, Jeffrey A. Whitsett
β-adrenergic receptors (βARs) relax airway smooth muscle and bronchodilate, but chronic β-agonist treatment in asthma causes increased sensitivity to airway constriction (hyperreactivity) and is associated with exacerbations. This paradox was explored using mice with ablated βAR genes (βAR–/–) and transgenic mice overexpressing airway smooth muscle β2AR (β2AR-OE) representing two extremes: absence and persistent activity of airway βAR. Unexpectedly, βAR–/– mice, lacking these bronchodilating receptors, had markedly decreased bronchoconstrictive responses to methacholine and other Gq-coupled receptor agonists. In contrast, β2AR-OE mice had enhanced constrictive responses. Contraction to permeabilization with β-escin was unaltered by gene ablation or overexpression. Inositol phosphate accumulation by Gq-coupled M3-muscarinic, thromboxane-A2, and 5-HT2 receptors was desensitized in airway smooth muscle cells from βAR–/– mice and sensitized in cells from β2AR-OE mice. Thus, βAR antithetically regulates constrictive signals, affecting bronchomotor tone/reactivity by additional means other than direct dilatation. Studies of signaling elements in these pathways revealed the nodal point of this cross talk as phospholipase C-β1, whose expression was altered by βAR in a direction and magnitude consistent with the physiologic and cellular responses. These results establish a mechanism of the β-agonist paradox and identify a potential asthma modifier gene (phospholipase C-β1), which may also be a therapeutic target in asthma when chronic β-agonists are required.
Dennis W. McGraw, Khalid F. Almoosa, Richard J. Paul, Brian K. Kobilka, Stephen B. Liggett
Strategies to stimulate endogenous surfactant production require a detailed understanding of the regulation of lipogenesis in alveolar type II cells. We developed culture conditions in which keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis. KGF stimulated acetate incorporation into phosphatidylcholine, disaturated phosphatidylcholin, and phosphatidylglycerol more than 5% rat serum alone. To determine the mRNA levels of lipogenic enzymes and transport proteins, we analyzed gene expression by oligonucleotide microarrays. KGF increased the mRNA levels for fatty acid synthase, stearoyl-CoA desaturase-1 (SCD-1), and epidermal fatty acid–binding protein more than rat serum alone. In addition, KGF increased the mRNA levels of the transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPδ as well as SREBP-1c (ADD-1), but not PPARγ. These changes in C/EBPα and C/EBPδ were confirmed by in situ hybridization. SCD-1 was also found to be highly expressed in alveolar type II cells in vivo. Furthermore, KGF increased protein levels of fatty acid synthase, C/EBPα, C/EBPδ, SREBP-1, epidermal fatty acid–binding protein, and SCD. Finally, the liver X receptor agonist T0901317 increased acetate incorporation and SREBP-1 but not SREBP-2 protein levels. In summary, KGF stimulates lipogenesis in type II cells by a coordinated expression of lipogenic enzymes and transport proteins regulated by C/EBP isoforms and SREBP-1c.
Robert J. Mason, Tianli Pan, Karen E. Edeen, Larry D. Nielsen, Feijie Zhang, Malinda Longphre, Michael R. Eckart, Steven Neben