Pulmonology
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
David Ramos-Barbón, John F. Presley, Qutayba A. Hamid, Elizabeth D. Fixman, James G. Martin
Abstract
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.
Authors
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
Abstract
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.
Authors
Scott S. Wagers, Ryan J. Norton, Lisa M. Rinaldi, Jason H.T. Bates, Burton E. Sobel, Charles G. Irvin
Abstract
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.
Authors
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
Abstract
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.
Authors
Isamu Hokuto, Machiko Ikegami, Mitsuhiro Yoshida, Kiyoshi Takeda, Shizuo Akira, Anne-Karina T. Perl, William M. Hull, Susan E. Wert, Jeffrey A. Whitsett
Abstract
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.
Authors
Dennis W. McGraw, Khalid F. Almoosa, Richard J. Paul, Brian K. Kobilka, Stephen B. Liggett
Abstract
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.
Authors
Robert J. Mason, Tianli Pan, Karen E. Edeen, Larry D. Nielsen, Feijie Zhang, Malinda Longphre, Michael R. Eckart, Steven Neben
Abstract
The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A–null mice and was increased in SP-D–overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D–mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane.
Authors
Huixing Wu, Alexander Kuzmenko, Sijue Wan, Lyndsay Schaffer, Alison Weiss, James H. Fisher, Kwang Sik Kim, Francis X. McCormack
Copyright © 2020 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)