Asthma and lower airway disease
Analyses of asthma severity phenotypes and inflammatory proteins in subjects stratified by sputum granulocytes

https://doi.org/10.1016/j.jaci.2010.02.008Get rights and content

Background

Patients with severe asthma have increased granulocytes in their sputum compared with patients with mild to moderate asthma.

Objective

We hypothesized that inflammatory granulocytes in sputum may identify specific asthma severity phenotypes and are associated with different patterns of inflammatory proteins in sputum supernatants.

Methods

This hypothesis was tested in 242 patients with asthma enrolled in the Severe Asthma Research Program who provided sputum samples for cell count, differential cell determinations, cell lysates for Western blot, and supernatant analyses by inflammatory protein microarrays and ELISAs. ANOVA and multiple linear regression models tested mediator associations.

Results

Stratified by sputum granulocytes, <2% or ≥2% eosinophils and <40% or ≥40% neutrophils, subjects with both increased eosinophils and neutrophils had the lowest lung function and increased symptoms and health care use. Subjects with elevated eosinophils with or without increased neutrophils had significantly increased fraction exhaled nitric oxide (FeNO) and serum eosinophils and greater frequency of daily β-agonist use. Microarray data stratified by granulocytes revealed 25 to 28 inflammatory proteins increased >2-fold in sputa with ≥40% neutrophils. Microarray analyses stratified by severity of asthma identified 6 to 9 proteins increased >2-fold in sputa in subjects with severe asthma compared with nonsevere asthma. ELISA data stratified by sputum granulocytes showed significant increases in brain-derived neurotrophic factor, IL-1β, and macrophage inflammatory protein 3α/CCL20 for those with ≥40% neutrophils; these mediators demonstrated positive associations with neutrophil counts.

Conclusion

Combined increased sputum eosinophils and neutrophils identified patients with asthma with the lowest lung function, worse asthma control, and increased symptoms and health care requirements. Inflammatory protein analyses of sputum supernatants found novel mediators increased in patients with asthma, predominantly associated with increased sputum neutrophils.

Section snippets

Methods

Characterization of severe and not severe asthma was performed according to the SARP protocol.15 Nonsmoking subjects (<5 pack-years) met American Thoracic Society criteria for diagnosis of asthma and provided informed consent approved by the institutional review board. Comprehensive evaluation included spirometry, bronchodilator reversibility and bronchial responsiveness, assessment of atopy, collection of blood, exhaled nitric oxide, sputum induction, and an administered questionnaire that

Subject stratification on the basis of sputum inflammation percent eosinophils and percent neutrophils

There were no significant differences in sputum cell percentages for severe compared with nonsevere subjects (see this article's Table E2 in the Online Repository at www.jacionline.org), and therefore, these were combined for stratification by granulocytes: <2% eosinophils + <40% neutrophils, <2% eosinophils + ≥40% neutrophils, ≥2% eosinophils + <40% neutrophils, and ≥2% eosinophils + ≥40% neutrophils (Table I and see Table E7 in this article's Online Repository at www.jacionline.org). The

Discussion

Sputum represents the best available noninvasive assessment of bronchial inflammation in asthma and reflects underlying pathology caused by infiltrating cells and soluble mediators. Instead of comparing sputum cellular and biochemical components between healthy subjects and subjects with asthma, our objective was to assess whether comprehensive analysis of induced sputum over a spectrum of asthma severity improves our understanding of the factors that characterize different asthma phenotypes.

References (53)

  • K. Asai et al.

    Imbalance between vascular endothelial growth factor and endostatin levels in induced sputum from asthmatic subjects

    J Allergy Clin Immunol

    (2002)
  • S.-W. Park et al.

    Interleukin-13 and interleukin-5 in induced sputum of eosinophilic bronchitis: comparison with asthma

    Chest

    (2005)
  • C. Hahn et al.

    Airway epithelial cells produce neurotrophins and promote the survival of eosinophils during allergic airway inflammation

    J Allergy Clin Immunol

    (2006)
  • A.T. Hastie et al.

    IL-1b release from cultured bronchial epithelial cells and bronchoalveolar lavage cells from allergic and normal humans following segmental challenge with ragweed

    Cytokine

    (1996)
  • M. Pichavant et al.

    Asthmatic bronchial epithelium activated by the proteolytic allergen Der p 1 increases selective dendritic cell recruitment

    J Allergy Clin Immunol

    (2005)
  • W.A. Nockher et al.

    Neurotrophins and asthma: novel insight into neuroimmune interaction

    J Allergy Clin Immunol

    (2006)
  • A.J. Fox et al.

    Release of nerve growth factor by human pulmonary epithelial cells: role in airway inflammatory diseases

    Eur J Pharmacol

    (2001)
  • O. Schulte-Herbruggen et al.

    Tumor necrosis factor-a and interleukin-6 regulate secretion of brain-derived neurotrophic factor in human monocytes

    J Neuroimmunol

    (2005)
  • H.S. Carlsen et al.

    Monocyte-like and mature macrophages produce CXCL13 (B cell-attracting chemokine 1) in inflammatory lesions with lymphoid neogenesis

    Blood

    (2004)
  • M.C. Subauste et al.

    Effects of tumor necrosis factor-alpha, epidermal growth factor and transforming growth factor-alpha on interleukin-8 production by, and human rhinovirus replication in, bronchial epithelial cells

    Internat Immunopharcacol

    (2001)
  • R. Polosa et al.

    Expression of c-erbB receptors and ligands in the bronchial epithelium of asthmatic subjects

    J Allergy Clin Immunol

    (2002)
  • R. Louis et al.

    The relationship between airways inflammation and asthma severity

    Am J Respir Crit Care Med

    (2000)
  • The ENFUMOSA cross-sectional European multicentre study of the clinical phenotype of chronic severe asthma

    Eur Respir J

    (2003)
  • R.H. Green et al.

    Analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids

    Thorax

    (2002)
  • A. Jatakanon et al.

    Neutrophilic inflammation in severe persistent asthma

    Am J Respir Crit Care Med

    (1999)
  • A. ten Brinke et al.

    Refractory" eosinophilic airway inflammation in severe asthma: effect of parenteral corticosteroids

    Am J Respir Crit Care Med

    (2004)
  • Cited by (380)

    • GLUT1 mediates the release of HMGB1 from airway epithelial cells in mixed granulocytic asthma

      2024, Biochimica et Biophysica Acta - Molecular Basis of Disease
    • Sputum microbiota and inflammatory subtypes in asthma, COPD, and its overlap

      2024, Journal of Allergy and Clinical Immunology: Global
    View all citing articles on Scopus

    Supported by the NHLBI Severe Asthma Research Program Awards (HL69167) and the Wake Forest University General Clinical Research Center (MO1 RR07122).

    Disclosure of potential conflict of interest: D. A. Meyers and S. P. Peters have received research support from the National Institutes of Health/National Heart, Lung, and Blood Institute. The rest of the authors have declared that they have no conflict of interest.

    View full text