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A role for neutrophils in asthma?

      The prevalence of asthma is increasing, and it continues to be one of the leading diagnoses in emergency rooms. Analysis of the cellular and biochemical characteristics of airway secretions can provide valuable information about asthma pathogenesis, and sputum induction has recently emerged as a useful noninvasive method for collecting airway secretions. Sputum induction relies on the inhalation of hypertonic saline that causes cough and sputum production, even in patients free of airway disease. This observation has been leveraged in clinical medicine for many decades in which sputum induction and analysis of induced sputum have been used to diagnose lung cancer and lung infections. Despite this proven utility as a diagnostic test, it was only in the early 1990s that clinical researchers began to use sputum induction for research purposes in airway disease (
      • Pin I.
      • Gibson P.G.
      • Kolendowicz R.
      • et al.
      Use of induced sputum cell counts to investigate airway inflammation in asthma.
      ,
      • Fahy J.V.
      • Liu J.
      • Wong H.
      • Boushey H.A.
      Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects.
      ). Standardized protocols were developed, and cytologic analyses evolved beyond sputum smears to more rigorous processing methods involving cytocentrifugation and quantitative total and differential cell counts. The fluid phase of induced sputum was also analyzed for various inflammatory mediators using specific immunoassays or enzymatic assays. In direct comparisons with bronchoalveolar lavage, sputum induction held up well; induced sputum samples are more concentrated and more reflective of airway secretions.
      A major advantage of sputum induction over bronchoscopy is that it can be applied to larger groups of patients. This advantage has facilitated relatively large studies on airway inflammation and airway function in asthma. One such study by Little et al. (
      • Little S.A.
      • MacLeod K.J.
      • Chalmers G.W.
      • et al.
      Association of forced expiratory volume with disease duration and sputum neutrophils in chronic asthma.
      ) is presented in this issue of the Journal. They analyze the relation between best achievable forced expiratory volume in 1 second (FEV1) and the cellular constituents of induced sputum in 59 patients with asthma. Best achievable FEV1 was determined by treatment for 2 weeks with oral or high-dose inhaled corticosteroids. The average baseline FEV1 rose from 67% to 76% predicted. The best achievable FEV1 was inversely associated with the number of neutrophils in sputum, measures of neutrophil activation, and the duration of asthma. The authors concluded that long disease duration predisposes to the development of irreversible airway obstruction in chronic asthma and that neutrophils may play a role in the pathophysiology of this airway obstruction.
      Although current concepts of asthma pathogenesis focus on the role of CD4+ T cells and eosinophilic inflammation in asthma (
      • Woodruff P.G.
      • Fahy J.V.
      Asthma prevalence, pathogenesis, and prospects for novel therapies.
      ), Little et al. are not alone in finding that neutrophilic inflammation may be important in the pathophysiology of asthma. About 15 years ago, Seltzer and colleagues (
      • Seltzer J.
      • Bigby B.G.
      • Stulbarg M.
      • et al.
      O3 induced change in bronchial reactivity to methacholine and airway inflammation in humans.
      ) reported that nonasthmatic subjects who had been exposed acutely to ozone developed airway hyperresponsiveness in association with airway neutrophilia. Other clinical studies have since found, as have Little et al., that measures of chronic asthma severity, such as FEV1, correlate with the degree of neutrophilia in sputum or bronchial biopsy specimens (
      • Wenzel S.E.
      • Szefler S.J.
      • Leung D.Y.M.
      • et al.
      Bronchoscopic evaluation of severe asthma.
      ,
      • Jatakanon A.
      • Uasuf C.
      • Maziak W.
      • et al.
      Neutrophilic inflammation in severe persistent asthma.
      ,
      • Louis R.
      • Lau L.C.K.
      • Bron A.O.
      • et al.
      The relationship between airway inflammation and asthma severity.
      ,
      • Woodruff P.G.
      • Khashayar R.
      • Lazarus S.C.
      • et al.
      Relationship between airway inflammation, hyperresponsiveness, and obstruction in asthma.
      ). Neutrophilic inflammation in the airway is also increasingly recognized in acute exacerbations of asthma and in status asthmaticus (
      • Sur S.
      • Crotty T.B.
      • Kephart G.M.
      • et al.
      Sudden onset fatal asthma a distinct clinical entity with few eosinophils and relatively more neutrophils in the airway submucosa?.
      ,
      • Ordonez C.L.
      • Shaughnessy T.E.
      • Matthay M.A.
      • Fahy J.V.
      Increased neutrophil numbers and IL-8 levels in airway secretions in acute severe asthma clinical and biologic significance.
      ).
      Are neutrophils therefore causally related to acute or chronic airway obstruction in asthma? How can a role for these cells be reconciled with the dominant hypothesis that asthma is a T-cell–mediated disease characterized by Th2 cytokines, immunoglobulin E (IgE), and eosinophils? The answers to these questions cannot be definitive at this time, but several points are worth making. First, the consistency of the finding that neutrophils are present in airway secretions and tissues from patients with more severe forms of chronic asthma cannot be ignored or disregarded as nonspecific. The effects of smoking and concomitant acute infections do not explain this observation. Second, analysis of induced sputum from nonselected patients with asthma estimates that up to 60% of patients have noneosinophilic airway inflammation (
      • Gibson P.G.
      • Simpson J.L.
      • Saltos N.
      Heterogeneity of airway inflammation in persistent asthma.
      ). In addition, treatment of asthma with anti-IgE has little effect on measures of airway obstruction (
      • Milgrom H.
      • Fick Jr, R.B.
      • Su J.Q.
      • et al.
      Treatment of allergic asthma with monoclonal anti-IgE antibody. The rhuMAb-E25 Study Group.
      ). These suggest that other mechanisms, such as neutrophils, contribute to the pathogenesis of chronic airway narrowing in asthma. Third, it is biologically plausible that neutrophils can cause acute or chronic airway obstruction in asthma. Neutrophils secrete a variety of inflammatory mediators, including proteases, cytokines (e.g., tumor necrosis factor α, transforming growth factor α), and reactive oxygen species, which can cause airway epithelial injury and mucus hypersecretion. Finally, although most animal models of asthma seek to model allergic T-cell and eosinophilic airway inflammation, some show that airway neutrophilia and airway hyperresponsiveness can occur together. These include murine models of asthma in which airway infection with mycoplasma or sendai virus cause neutrophilia and hyperresponsiveness (
      • Martin R.J.
      • Chu H.W.
      • Honour J.M.
      • Harbeck R.J.
      Airway inflammation and bronchial hyperresponsiveness after mycoplasma pneumonia infection in a murine model.
      ,
      • Castleman W.L.
      • Sorkness R.L.
      • Lemanske R.F.
      • McAllister P.K.
      Viral bronchiolitis during early life induces increased numbers of bronchiolar mast cells and airway hyperresponsiveness.
      ). These animals develop airway hyperresponsiveness that persists long after the resolution of the acute viral infection. In this respect, these models mimic severe forms of human asthma better than do models that are based on allergen sensitization.
      We can only hypothesize about the mechanisms of airway neutrophilia in chronic severe asthma. This subgroup of asthmatic patients may have had chronic airway infection with viruses or atypical bacteria, such as mycoplasma or chlamydia, which is in keeping with the recent emergence of chronic infections as plausible causes for disease in some patients with peptic ulcer disease or coronary artery disease (
      • Leinonen M.
      Chlamydia pneumoniae and other risk factors for atherosclerosis.
      ,
      • Cohen H.
      Peptic ulcer and Helicobacter pylori.
      ). There is serologic evidence for infection with Chlamydia pneumoniae in asthma, and evidence linking infection with more severe disease (
      • ten Brinke A.
      • van Dissel J.T.
      • Sterk P.J.
      • et al.
      Persistent airflow limitation in adult-onset nonatopic asthma is associated with serological evidence of Chlamydia pneumoniae infection.
      ,
      • Black P.N.
      • Scicchitano R.
      • Jenkins C.R.
      • et al.
      Serological evidence of infection with Chlamydia pneumoniae is related to the severity of asthma.
      ). Furthermore, there is polymerase chain reaction–derived evidence of airway infection with Mycoplasma pneumoniae in asthmatic patients (
      • Kraft M.
      • Cassell G.H.
      • Henson J.E.
      • et al.
      Detection of Mycoplasma pneumoniae in the airways of adults with chronic asthma.
      ). However, it is too early to confirm that chronic airway infection plays a major role in chronic asthma, and antibiotic treatment fails to show sustained improvement in pulmonary function, even in asthmatic patients with serologic evidence for C. pneumoniae infection (
      • Black P.N.
      • Blasi F.
      • Jenkins C.R.
      • et al.
      Trial of roxithromycin in subjects with asthma and serological evidence of infection with Chlamydia pneumoniae.
      ).
      Another possible mechanism of airway neutrophilia in chronic severe asthma is that airway epithelial cells are chronically activated and hypersecrete neutrophil chemoattractants such as interleukin (IL) 8. Indeed, primary culture of lower airway epithelial cells from healthy and asthmatic subjects show that asthmatic epithelial cells constitutively hypersecrete IL-8 and show a heightened sensitivity to environmental pollutants, such as diesel exhaust particles (
      • Bayram H.
      • Devalia J.L.
      • Khair O.A.
      • et al.
      Comparison of ciliary activity and inflammatory mediator release from bronchial epithelial cells of nonatopic nonasthmatic subjects and atopic asthmatic patients and the effect of diesel exhaust particles in vitro.
      ). Such observations have led to speculation that lower airway epithelial cells in asthma have an unknown but intrinsic functional abnormality that is central to the pathogenesis of asthma (
      • Evans M.J.
      • Van Winkle L.S.
      • Fanucchi M.V.
      • Plopper C.G.
      Cellular and molecular characteristics of basal cells in airway epithelium.
      ,
      • Holgate S.T.
      • Lackie P.M.
      • Howarth P.H.
      • et al.
      Invited lecture activation of the epithelial mesenchymal trophic unit in the pathogenesis of asthma.
      ). In fact, a fundamental question is whether airway epithelial cells are abnormally sensitive to the actions of Th2 cytokines, particularly because a Th2 T-cell phenotype occurs in atopic patients, but only a minority of atopic patients develop asthma. It has been hypothesized that a combination of susceptibilities is required for the development of asthma. Immune deviation of CD4+ T cells to a Th2 phenotype is one susceptibility, but an additional susceptibility involving epithelial cells may also be required. Epithelial cells may also become aberrantly programmed in infancy because of viral infection (
      • Holtzman M.J.
      • Morton J.D.
      • Shornick L.P.
      • et al.
      Immunity, inflammation, and remodeling in the airway epithelial barrier epithelial-viral-allergic paradigm.
      ). Resolution of the viral infection may not restore a normal epithelial function, rather, epithelial cells continue to be activated in ways that mimic the acute response to the original viral infection (
      • Holtzman M.J.
      • Morton J.D.
      • Shornick L.P.
      • et al.
      Immunity, inflammation, and remodeling in the airway epithelial barrier epithelial-viral-allergic paradigm.
      ). Support for this hypothesis comes from studies of experimental viral infections in rodents, where acute infection causes lifelong abnormalities in airway pathology and function (
      • Castleman W.L.
      • Sorkness R.L.
      • Lemanske R.F.
      • McAllister P.K.
      Viral bronchiolitis during early life induces increased numbers of bronchiolar mast cells and airway hyperresponsiveness.
      ). The persistence of epithelial cell dysfunction for prolonged periods following resolution of the initial injury may result from permanent changes in the interactions of innate (epithelial cell) and adaptive (T cells) immune mechanisms in the airways. Although the specific mechanisms of any such permanent changes are unknown, it has been shown recently that T memory cells promote the production of neutrophil chemoattractants by epithelial cells. Specifically, they produce IL-17, an interleukin that is linked to airway neutrophilia and to IL-8 production by airway epithelial cells in both in vitro and in vivo studies of airway inflammation (
      • Linden A.
      Role of interleukin-17 and the neutrophil in asthma.
      ).
      In conclusion, the evidence from observational studies suggests that chronic severe asthma is associated with neutrophilic inflammation. Chronic severe asthma and incompletely reversible airway obstruction in asthmatic patients remain clinical problems with unmet therapeutic needs. Hence, further studies are required to determine the mechanisms through which neutrophils may lead to airway remodeling and to determine if antineutrophil therapies might be beneficial in severe asthma. Human and murine studies of asthma that focused on acute T-cell and eosinophil responses to allergen have advanced our understanding of allergic mechanisms in asthma, but as they are not characterized by neutrophilic inflammation, they may ultimately be limited as models of chronic severe asthma.

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