Procalcitonin (PCT) is expressed in nonthryoidal tissues of humans during severe infections. Serum PCT levels are measured to diagnose and guide therapy, and there is some evidence that PCT may also contribute to the pathogenesis of sepsis. We tested whether disruption of the gene encoding PCT in mice affected the course of sepsis. Mice with exons 2–5 of the gene encoding calcitonin/calcitonin gene–related polypeptide α (Calca) knocked out and congenic C57BL/6J control mice were challenged with aerosolized Streptococcus pneumoniae or Pseudomonas aeruginosa, or injected intraperitoneally with S. pneumoniae. There were no significant differences in the survival of knockout and control mice in the two pneumonia models, and no significant differences in weight loss, splenic bacterial counts, or blood leukocyte levels in the peritoneal sepsis model. To verify disruption of the Calca gene in knockout mice, the absence of calcitonin in the serum of knockout mice and its presence and inducibility in control mice were confirmed. To evaluate PCT expression in nonthyroidal tissues of control mice, transcripts were measured in multiple organs. PCT transcripts were not significantly expressed in liver or spleen of control mice challenged with aerosolized P. aeruginosa or intraperitoneal endotoxin, and were expressed in lung only at low levels, even though serum IL-6 rose 3,548-fold. We conclude that mice are not an ideal loss-of-function model to test the role of PCT in the pathogenesis of sepsis because of low nonendocrine PCT expression during infection and inflammation. Nonetheless, our studies demonstrate that nonendocrine PCT expression is not necessary for adverse outcomes from sepsis.
procalcitonin; calcitonin; sepsis; pneumonia
Respiratory virus infections cause airway hyperreactivity (AHR). Preventative strategies for virus-induced AHR remain limited. Toll-like receptors (TLRs) have been suggested as a therapeutic target because of their central role in triggering antiviral immune responses. Previous studies showed that concurrent treatment with TLR2/6 and TLR9 agonists reduced lethality and the microbial burden in murine models of bacterial and viral pneumonia. This study investigated the effects of TLR2/6 and TLR9 agonist pretreatment on parainfluenza virus pneumonia and virus-induced AHR in guinea pigs in vivo. Synthetic TLR2/6 lipopeptide agonist Pam2CSK4 and Class C oligodeoxynucleotide TLR9 agonist ODN2395, administered in combination 24 hours before virus infection, significantly reduced viral replication in the lung. Despite a fivefold reduction in viral titers, concurrent TLR2/6 and TLR9 agonist pretreatment did not prevent virus-induced AHR or virus-induced inhibitory M2 muscarinic receptor dysfunction. Interestingly, the TLR agonists independently caused non–M2-dependent AHR. These data confirm the therapeutic antiviral potential of TLR agonists, while suggesting that virus inhibition may be insufficient to prevent virus-induced airway pathophysiology. Furthermore, TLR agonists independently cause AHR, albeit through a distinctly different mechanism from that of parainfluenza virus.
Toll-like receptor; airway hyperreactivity; muscarinic receptor; parainfluenza virus
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
Pneumonia is a serious problem worldwide. We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia. To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens. Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus. Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line. Gene expression analysis of lung tissue showed marked activation of NF-κB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways. Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection. Lung-expressed antimicrobial peptides were also highly up-regulated. Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.
innate immunity; pneumonia; immunocompromised host; lung epithelium
Lung cancer is the leading cause of cancer deaths in the United States. In addition to genetic abnormalities induced by cigarette smoke, several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lungs, have an increased risk of lung cancer (1.3- to 4.9-fold) compared to smokers without COPD. This suggests a link between chronic airway inflammation and lung carcinogenesis, independent of tobacco smoke exposure. We studied this association by assaying the inflammatory impact of products of nontypeable Haemophilus influenzae, which colonizes the airways of patients with COPD, on lung cancer promotion in mice with an activated K-ras mutation in their airway epithelium. Two new mouse models of lung cancer were generated by crossing mice harboring the LSL–K-rasG12D allele with mice containing Cre recombinase inserted into the Clara cell secretory protein (CCSP) locus, with or without the neomycin cassette excised (CCSPCre and CCSPCre-Neo, respectively). Lung lesions in CCSPCre-Neo/LSL–K-rasG12D and CCSPCre/LSL–K-rasG12D mice appeared at 4 and 1 month of age, respectively, and were classified as epithelial hyperplasia of the bronchioles, adenoma, and adenocarcinoma. Weekly exposure of CCSPCre/LSL–K-rasG12D mice to aerosolized nontypeable Haemophilus influenzae lysate from age 6–14 weeks resulted in neutrophil/macrophage/CD8 T-cell–associated COPD-like airway inflammation, a 3.2-fold increase in lung surface tumor number (156 ± 9 versus 45 ± 7), and an increase in total lung tumor burden. We conclude that COPD-like airway inflammation promotes lung carcinogenesis in a background of a G12D-activated K-ras allele in airway secretory cells.
K-ras; lung cancer; inflammation
Nontypeable Haemophilus influenzae (NTHi) commonly colonizes the lower airways of patients with chronic obstructive pulmonary disease (COPD). Whether it contributes to COPD progression is unknown. Here, we determined which aspects of the COPD phenotype can be induced by repetitive exposure to NTHi products. Mice were exposed weekly to an aerosolized NTHi lysate, and inflammation was evaluated by measurement of cells and cytokines in bronchoalveolar lavage fluid (BALF) and immunohistochemical staining; structural changes were evaluated histochemically by periodic acid fluorescent Schiff's reagent, Masson's trichrome, and Picrosirius red staining; mucin gene expression was measured by quantitative RT-PCR; and the role of TNF-α was examined by transgenic airway overexpression and use of an inhibitory antibody. NTHi lysate induced rapid activation of NF-κB in airway cells and increases of inflammatory cytokines and neutrophils in BALF. Repetitive exposure induced infiltration of macrophages, CD8+ T cells, and B cells around airways and blood vessels, and collagen deposition in airway and alveolar walls, but airway mucin staining and gel-forming mucin transcripts were not increased. Transgenic overexpression of TNF-α caused BALF neutrophilia and inflammatory cell infiltration around airways, but not fibrosis, and TNF-α neutralization did not reduce BALF neutrophilia in response to NTHi lysate. In conclusion, NTHi products elicit airway inflammation in mice with a cellular and cytokine profile similar to that in COPD, and cause airway wall fibrosis but not mucous metaplasia. TNF-α is neither required for inflammatory cell recruitment nor sufficient for airway fibrosis. Colonization by NTHi may contribute to the pathogenesis of small airways disease in patients with COPD.
pulmonary disease, chronic obstructive; Haemophilus influenzae; bronchiolitis; inflammation; fibrosis
Single-dose administration of beta-adrenoceptor agonists produces bronchodilation and inhibits airway hyperresponsiveness (AHR), and is the standard treatment for the acute relief of asthma. However, chronic repetitive administration of beta-adrenoceptor agonists may increase AHR, airway inflammation, and risk of death. Based upon the paradigm shift that occurred with the use of beta-blockers in congestive heart failure, we previously determined that chronic administration of beta-blockers decreased AHR in a murine model of asthma. To elucidate the mechanisms for the beneficial effects of beta-blockers, we examined the effects of chronic administration of several beta-adrenoceptor ligands in a murine model of allergic asthma. Administration of beta-blockers resulted in a reduction in total cell counts, eosinophils, and the cytokines IL-13, IL-10, IL-5, and TGF-β1 in bronchoalveolar lavage, and attenuated epithelial mucin content and morphologic changes. The differences in mucin content also occurred if the beta-blockers were administered only during the ovalbumin challenge phase, but administration of beta-blockers for 7 days was not as effective as administration for 28 days. These results indicate that in a murine model of asthma, chronic administration of beta-blockers reduces inflammation and mucous metaplasia, cardinal features of asthma that may contribute to airflow obstruction and AHR. Similar to heart failure, our results provide a second disease model in which beta-blockers producing an acutely detrimental effect may provide a therapeutically beneficial effect with chronic administration.
beta-blockers; beta-adrenoceptor; asthma; mucin; airway inflammation
Mucus hypersecretion contributes to morbidity and mortality in many obstructive lung diseases. Gel-forming mucins are the chief glycoprotein components of airway mucus, and elevated expression of these during mucous metaplasia precedes the hypersecretory phenotype. Five orthologous genes (MUC2, MUC5AC, MUC5B, MUC6, and MUC19) encode the mammalian gel-forming mucin family, and several have been implicated in asthma, cystic fibrosis, and chronic obstructive pulmonary disease pathologies. However, in the absence of a comprehensive analysis, their relative contributions remain unclear. Here, we assess the expression of the entire gel-forming mucin gene family in allergic mouse airways and show that Muc5ac is the predominant gel-forming mucin induced. We previously showed that the induction of mucous metaplasia in ovalbumin-sensitized and -challenged mouse lungs occurs within bronchial Clara cells. The temporal induction and localization of Muc5ac transcripts correlate with the induced expression and localization of mucin glycoproteins in bronchial airways. To better understand the tight regulation of Muc5ac expression, we analyzed all available 5′-flanking sequences of mammalian MUC5AC orthologs and identified evolutionarily conserved regions within domains proximal to the mRNA coding region. Analysis of luciferase reporter gene activity in a mouse transformed Clara cell line demonstrates that this region possesses strong promoter activity and harbors multiple conserved transcription factor–binding motifs. In particular, SMAD4 and HIF-1α bind to the promoter, and mutation of their recognition motifs abolishes promoter function. In conclusion, Muc5ac expression is the central event in antigen-induced mucous metaplasia, and phylogenetically conserved 5′ noncoding domains control its regulation.
mucin; metaplasia; airway; lung; epithelium
Salmeterol is a long-acting β2-adrenergic receptor (β2AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other β-agonists in that it has a very low intrinisic efficacy, especially when compared with the other available long-acting β-agonist, formoterol. Receptor desensitization and down-regulation has been described with the chronic use of β-agonists. This effect may not be the same with all β-agonists and may be related to their stabilization of altered receptor states. The extreme hydrophobicity and high-affinity quasi-irreversible binding of salmeterol have rendered studies examining the mechanisms by which it mediates receptor desensitization, down-regulation, and internalization difficult. We determined the capacity of salmeterol to induce β2AR endocytosis, G protein–coupled receptor kinase (GRK)-site phosphorylation, degradation, and β-arrestin2 translocation in HEK293 cells as compared with other agonists of varying intrinsic efficacies. Despite stimulating GRK-mediated phosphorylation of Ser355,356 after 30 min and 18 h to an extent similar to that observed with agonists of high intrinsic efficacy, such as epinephrine and formoterol, salmeterol did not induce significant β2AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-β–arrestin2 chimera (EGFP-β–arrestin2) to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP-β–arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit β-arrestin or undergo significant endocytosis or degradation despite stimulating considerable GRK-site phosphorylation. Defects in these components of salmeterol-induced receptor desensitization may be important determinants of its sustained bronchodilation with chronic use.
β2-adrenergic receptor; salmeterol; phosphorylation; internalization; desensitization
Mucin hypersecretion is a prominent feature of obstructive airway diseases such as asthma. Clara cells conditionally produce mucin in response to inflammatory signals in a process termed mucous metaplasia. This can be followed by mucin secretion stimulated by various signaling molecules. The cellular and molecular mechanisms that regulate mucin production and secretion are not well understood. Adenosine is a signaling nucleoside that has been implicated in airway diseases in which mucus obstruction is prominent. Furthermore, the A3 adenosine receptor (A3AR) is upregulated in mucin-producing goblet cells of the airway, thereby implicating it in processes involved in mucous cell biology. Here we use genetic approaches to investigate the contribution of A3AR signaling to mucus production and secretion in a mouse model of allergen-induced pulmonary disease. We found that the degree of mucin production in response to allergen is similar in wild-type and A3AR-deficient mice, and that overexpression of this receptor in Clara cells neither induces mucin production itself, nor enhances mucin production in response to allergen challenge. Collectively, these experiments demonstrate that the A3AR is neither necessary nor sufficient for mucous cell metaplasia. In contrast to the lack of effect on mucin production, agonist-induced mucin secretion was increased in goblet cells overexpressing the A3AR, and was absent in A3AR-deficient mice. Thus, the A3AR contributes to mucin secretion in allergen-induced metaplasia. Signaling through this receptor may contribute to mucus airway obstruction seen in pulmonary disorders in which adenosine levels are elevated.
mucin; mucous cell metaplasia; secretion; adenosine receptors; allergic lung disease
Mucus hypersecretion is a phenotype associated with multiple obstructive lung diseases. However, in spite of its nefarious reputation under pathologic conditions, there are significant benefits to having low levels of mucus present in the airways at baseline, such as the ability to trap and eliminate inhaled particles and to prevent desiccation of airway surfaces. Mucins are high–molecular-weight glycoproteins that are the chief components that render viscoelastic and gel-forming properties to mucus. Recent advances in animal models and in vitro systems have provided a wealth of information regarding the identification of the mucin genes that are expressed in the lungs, the signal transduction pathways that regulate the expression of these mucins, and the secretory pathways that mediate their release into the airways. In addition, the clinical and pathologic literature has corroborated many of the basic laboratory findings. As a result, mucin overproduction and hypersecretion are moving away from being markers of disease and toward being testable as functional components of lung disease processes.
epithelium; lung; metaplasia; mucin; secretion