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Images in allergy and immunology: Neutrophils in asthma

Published:April 06, 2007DOI:https://doi.org/10.1016/j.jaci.2007.02.006
      Neutrophils are present in much larger numbers than any other inflammatory cell in the circulation and in tissue stores, particularly the lung. This storage of neutrophils is possible because of margination in the lung capillaries, which allows a large reservoir of cells to be rapidly available when required.
      Neutrophils are one of the first inflammatory cells to be recruited into the airways after either allergen exposure or injury. They can be readily found in the sputum of subjects with asthma but are reported in low numbers in bronchoalveolar lavage (BAL) and bronchial biopsies. However, increased numbers are found in the airways during the late-phase reaction after an allergen challenge (Fig 1), in some patients who died within hours after an asthma exacerbation in nocturnal asthma, in some patients with long-standing asthma, and in patients with corticosteroid-dependent asthma.
      Figure thumbnail gr1
      Fig 1Immunostaining for neutrophil elastase (pink) in bronchial biopsy of subject with asthma after allergen challenge shows increased numbers of neutrophils in the submucosa. Magnification ×200.
      The role of neutrophils in asthma has been controversial for some time and remains unclear, whereas their involvement in chronic obstructive pulmonary disease has been well established. Some authors support a role for these cells in asthma, and others do not. Neutrophils may play a role particularly in the more severe spectrum of disease (Fig 2). Wenzel et al
      • Wenzel S.E.
      • Szefler S.J.
      • Leung D.Y.M.
      • Sloan S.I.
      • Rex M.D.
      • Martin R.J.
      Bronchoscopic evaluation of severe asthma: persistent inflammation associated with high dose glucocorticoids.
      showed a consistent increase in neutrophils in BAL fluid and in both bronchial and transbronchial biopsies of patients with chronic steroid-dependent severe asthma who remained symptomatic compared with milder disease. This suggests that airway neutrophilia may reflect asthma severity. Little et al
      • Little S.A.
      • MacLeod K.J.
      • Chalmers G.W.
      • Love J.G.
      • McSharry C.
      • Thomson N.C.
      Association of forced expiratory volume with disease duration and sputum neutrophils in chronic asthma.
      and others have found that measures of chronic asthma severity, such as FEV1, correlate with the degree of neutrophilia in sputum and bronchial biopsy specimens.
      • Louis R.
      • Lau L.C.K.
      • Bron A.O.
      • Roldaan A.C.
      • Radermecker M.
      • Djukanovic R.
      The relationship between airway inflammation and asthma severity.
      • Woodruff P.G.
      • Khashayar R.
      • Lazarus S.C.
      • Janson S.
      • Avila P.
      • Boushey H.A.
      • et al.
      Relationship between airway inflammation, hyperresponsiveness and obstruction in asthma.
      Figure thumbnail gr2
      Fig 2Immunostaining for neutrophil elastase (pink) in bronchial biopsy of patient with severe asthma shows increased numbers of neutrophils. Magnification ×200.
      Neutrophils are recruited to the site of lung inflammation by a number of chemokines, particularly CXCL8 (IL-8; Fig 3), which is mostly produced by the epithelium of the lung.
      • Baggiolini M.
      • Walz A.
      • Kunkel S.L.
      Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils.
      IL-8 is also the most potent activator of neutrophils and triggers the secretion of granular enzymes such as myeloperoxidase, β-glucuronidase, elastase, and gelatinase following their activation. In addition, IL-8 induces the production of leukotriene B4 and oxygen radicals by neutrophils and stimulates neutrophils for phagocytosis.
      Figure thumbnail gr3
      Fig 3Increased expression of IL-8 in the epithelium and submucosa in bronchial biopsy of patient with asthma. Positive staining is brown. Magnification ×200.
      Because neutrophils are relatively steroid-resistant
      • Hauber H.P.
      • Gotfried M.
      • Newman K.
      • Danda R.
      • Servi R.J.
      • Christodoulopoulos P.
      • et al.
      Effect of HFA-flunisolide on peripheral lung inflammation in asthma.
      (Fig 4), it was hypothesized that this increased neutrophilic inflammation might explain the poor response to corticosteroids seen in severe refractory disease. It could be argued, however, that the increased presence of neutrophils in severe asthma may be the result of treatment with high-dose corticosteroids rather than a manifestation of the disease itself. Fukakusa et al
      • Fukakusa M.
      • Bergeron C.
      • Tulic M.
      • Fiset P.O.
      • Al Dewachi O.
      • Laviolette M.
      • et al.
      Oral corticosteroids decrease eosinophil and CC chemokine expression but increase neutrophil, IL-8, and IFN-γ–inducible protein 10 expression in asthmatic airway mucosa.
      showed that treatment of patients with moderate-to-severe asthma with oral corticosteroids led to increased expression of IL-8 mRNA–positive cells in the airway epithelium but decreased IL-8 levels in the submucosal cells of bronchial biopsies (Fig 5).
      Figure thumbnail gr4
      Fig 4Steroid resistance of neutrophils. Bronchial biopsy (A) and graph (B) show increased numbers of elastase-positive neutrophils after treatment with inhaled corticosteroids. Magnification ×200.
      Figure thumbnail gr5
      Fig 5Effect of corticosteroids on neutrophil-associated chemokines. Graph shows the effect of oral corticosteroids on the number of IL-8 mRNA–positive cells in the bronchial subepithelium of patients with moderate-to-severe asthma. Results are presented as means ± SEMs. P < .05 versus expression before steroid treatment.
      Neutrophils produce a number of cytokines, particularly IL-9 (Fig 6), TNF-α, and TGF-α. IL-9 is a regulatory TH2 cytokine that has pleiotropic activity on inflammatory and structural cells associated with asthma, such as mast cells, T cells, eosinophils, and epithelial cells. This cytokine influences eosinophil survival, chemokines, and mucus production. IL-9 has been shown to induce the production and release of IL-8 by human polymorphonuclear cells (PMNs) from subjects with asthma in a dose-dependent manner.
      Figure thumbnail gr6
      Fig 6Increased immunostaining for IL-9 in BAL neutrophils of subject with asthma. Positive staining is indicated with red. Magnification ×400.
      In addition to producing a number of functionally diverse substances, PMNs also express receptors for a number of mediators including IL-8, IL-9 (Fig 7),
      • Soussi-Gounni A.
      • Koussih L.
      • Nutku E.
      • Lamkhioued B.
      • Nicolaides N.C.
      • Levitt R.C.
      • et al.
      Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release.
      and the high-affinity IgE receptor (Fig 8).
      • Soussi-Gounni A.
      • Lamkhioued B.
      • Koussih L.
      • Ra C.
      • Renzi P.M.
      • Hamid Q.
      Human neutrophils express the high-affinity receptor for immunoglobulin E (Fc epsilon RI): role in asthma.
      These receptors have been implicated in different inflammatory reactions, including allergic asthma. IL-9 receptor (IL-9R) is expressed in freshly isolated human peripheral blood PMNs, as well as in BAL-derived PMNs from patients with asthma (Fig 7).
      • Soussi-Gounni A.
      • Koussih L.
      • Nutku E.
      • Lamkhioued B.
      • Nicolaides N.C.
      • Levitt R.C.
      • et al.
      Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release.
      The expression of the IL-9Rα protein on human PMNs was widely heterogeneous, but a significant difference was found between the percentages of IL-9Rα+ PMNs in subjects with asthma compared with normal control subjects, suggesting that the expression of IL-9Rα is under regulatory control.
      Figure thumbnail gr7
      Fig 7Detection of IL-9R immunoreactivity on human peripheral blood and BAL PMNs by immunocytochemistry. Purified PMN cytopreparation from a donor with asthma (A) and a normal control subject (C) are shown. Arrows point to specific staining. Isotype-matched controls (B and D) stained negatively. IL-9Rα–chain immunostaining on BAL cells (E) shows neutrophil morphology (arrows). There is no staining with normal rabbit serum (F).
      Figure thumbnail gr8
      Fig 8Detection of FcɛRIα-chain mRNA in human PMNs by in situ hybridization. A, Positive signal was detected using antisense FcɛRIα-chain riboprobe in PMNs from a patient with asthma. Magnification ×200. The FcɛRIα-chain positive cells (dark field, arrows inB) show neutrophil morphology with phase contrast microscopy (C). Magnification ×400 in B-D. The data represent 9 experiments.
      A number of cellular adhesion molecules are involved in the adhesion of neutrophils to the site of tissue inflammation. Neutrophils must adhere to the endothelium and subsequently migrate through the vessels before entering the tissue. Neutrophil rolling and arrest on endothelium is mediated through successive interactions of selectins and β2-integrins.
      The neutrophil plays a key role in acute severe or fatal asthma. It is the dominant leukocyte in airway secretions from patients with acute severe asthma requiring mechanical ventilation. IL-8 is an important mediator of this neutrophilia, and chemokine levels correlate positively with the number of neutrophils, elevated free neutrophil elastase, and severity of exacerbation. Elevated IL-8 and free neutrophil elastase activity have also been demonstrated in sputum of subjects with acute asthma not requiring ventilation. Levels of both total and active sputum elastase have been shown to be significantly correlated with the number of sputum neutrophils.
      Neutrophils may be important mediators of the prominent mucus hypersecretion seen in acute severe and chronic asthma because neutrophil elastase (Fig 1, Fig 2) is an important secretagogue for goblet cells and submucosal gland cells. Neutrophil products may also be important mediators of epithelial cell activation, eosinophil activation, and heightened vascular permeability in acute severe asthma.
      Because neutrophils have the ability to participate in tissue damage, they may have a potential role to play in the airway remodeling process that occurs in severe asthma. Neutrophils are an important cellular source of TGF-β (Fig 9), and neutrophils from individuals with asthma are able to release higher levels of this profibrotic cytokine than those from subjects without asthma.
      • Chu H.W.
      • Trudeau J.B.
      • Balzar S.
      • Wenzel S.E.
      Peripheral blood and airway tissue expression of transforming growth factor beta by neutrophils in asthmatic subjects and normal control subjects.
      It is probable therefore that the neutrophil contributes directly to abnormal repair mechanisms and to the development of airway remodeling in asthma.
      Figure thumbnail gr9
      Fig 9Positive immunostaining for TGF-β1,2,3 in a neutrophil (A) in an asthmatic endobronchial biopsy; TGF-β1 immunostaining in peripheral blood neutrophils of a subject with asthma (B) and negative control without TGF-β1 antibody incubation (C). Magnification ×400.
      Matrix metalloproteinases (MMPs) selectively degrade extracellular matrix components and have been implicated in angiogenesis and smooth muscle hyperplasia. MMPs are also involved in the trafficking of inflammatory and structural cells. MMP-9 is the major MMP expressed in BAL fluid, sputum, and bronchial mucosal biopsies in asthma.
      • Hoshino M.
      • Nakamura Y.
      • Sim J.
      • Shimojo J.
      • Isoqai S.
      Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation.
      • Wenzel S.E.
      • Balzar S.
      • Cundall M.
      • Chu H.W.
      Subepithelial basement membrane immunoreactivity for matrix metalloproteinase-9: association with asthma severity, neutrophilic inflammation and wound repair.
      • Cundall M.
      • Yongchang S.
      • Miranda C.
      • Trudeau J.B.
      • Barnes S.
      • Wenzel S.E.
      Neutrophil-derived matrix metalloproteinase-9 is increased in severe asthma and poorly inhibited by glucocorticoids.
      BAL neutrophils are the main source of MMP-9 in severe asthma.
      • Cundall M.
      • Yongchang S.
      • Miranda C.
      • Trudeau J.B.
      • Barnes S.
      • Wenzel S.E.
      Neutrophil-derived matrix metalloproteinase-9 is increased in severe asthma and poorly inhibited by glucocorticoids.
      Untreated subjects with stable asthma demonstrate a higher tissue inhibitor of metalloproteinase-1/MMP-9 molar ratio in their BAL fluid and sputum compared with treated subjects with asthma and control subjects. This ratio has been shown to be negatively correlated with FEV1 values.
      • Vignola A.M.
      • Riccobono L.
      • Mirabella A.
      • Profita M.
      • Chanez P.
      • Bellia V.
      • et al.
      Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis.
      The fact that increased neutrophil numbers are present in the airways during the late-phase reaction after allergen challenge, in fatal or nocturnal asthma, and in subjects with severe disease suggests that these cells play a role in the development of well defined clinical subphenotypes. However, their precise role in the pathogenesis of asthma remains to be clarified. Further studies examining the mechanisms by which neutrophils may lead to airway remodeling are required as well as studies investigating the potential beneficial effects of antineutrophil therapies in severe asthma. In addition, a better understanding of the mechanisms that modulate the recruitment and activation of neutrophils in the airways of subjects with asthma may provide novel targets for the development of future therapeutic strategies.

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