Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways

Abstract

Background: Asthma is a heterogeneous inflammatory airway disorder that involves eosinophilic and noneosinophilic phenotypes. Unlike in healthy lungs, eosinophils are often present in atopic asthmatic airways, although a subpopulation of asthmatic subjects predominantly experience neutrophilic inflammation. Recently, it has been demonstrated that eosinophils and neutrophils generate bactericidal extracellular traps consisting of DNA and cytotoxic granule proteins.

Objective: We sought to explore whether living eosinophils and neutrophils infiltrating human atopic asthmatic airways actively form extracellular DNA traps in vivo.

Methods: Quantitative analysis of eosinophils releasing DNA was performed in endobronchial biopsy specimens from 20 human subjects with mild atopic asthma at baseline and after local allergen challenge and 10 healthy subjects. DNA was stained with propidium iodine and major basic protein with specific antibody. Differential cell counts and cytokines/chemokines were assessed in bronchoalveolar lavage fluid.

Results: Asthmatic airways were infiltrated with a significantly higher number of eosinophils than healthy airways (39.3 ± 4.6 vs 0.4 ± 0.9, P < .0001). All asthmatic subjects but only 1 control subject expressed eosinophils releasing DNA that colocalized with major basic protein (33.65 ± 20.33 vs 0.3 ± 0.9 per high-power field, P < .0001). Four asthmatic subjects mostly expressed neutrophilic inflammation and neutrophil DNA traps. Allergen challenge had no significant quantitative effect on eosinophil or neutrophil DNA traps. Airway eosinophils or DNA traps did not correlate with either bronchoalveolar lavage levels of IL-5, IFN-γ, or eotaxin or the provoking doses of methacholine or allergen in asthmatic subjects.

Conclusions: Extracellular DNA traps are generated by eosinophils and neutrophils in human atopic asthmatic airways in vivo. The mechanism and role of this new finding will necessitate further investigation.

Conflict of interest statement

Disclosure of potential conflict of interest: The authors have declared that they have no potential conflict of interest or commercial associations as per journal policy.

Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Figures

Fig 1

Eosinophil and EETs counts per 10 hpf in airway biopsies from asthmatic and control subjects. Values are single numbers as well as mean levels ± SEM.

Fig 2

Representative endobronchial biopsies from atopic asthmatics showing EETs and NETs. A, EETs generated by eosinophils contain DNA and MBP. Biopsies were from three different patients. In the left panels, infiltrating eosinophils expressing MBP are circled by dashed lines. In the middle panels, examples of extracellular DNA are indicated by arrows. In the right panels, co-localization of extracellular DNA and MBP is circled by dashed lines. B, NETs generated by neutrophils contain DNA and elastase. Representative results from one patient are shown. In the left panel, infiltrating neutrophils expressing elastase are circled by dashed lines. In the middle panel, examples of extracellular DNA are indicated by arrows. In the right panel, co-localization of extracellular DNA and elastase is circled by dashed lines. Bars, 10 microM. Control antibodies showed no staining (data not shown).

Fig 3

Extracellular DNA is released from viable eosinophils. A, Positive TUNEL analysis of esophageal biopsy obtained from a patient with eosinophilic esophagitis (EoE, left panel), and asthmatic airway biopsy showing a negative TUNEL assay (right panel). Eosinophils and EETs were identified using anti-ECP antibody (arrows). Lower panels represent magnifications of the indicated fields within the upper panels. B, Fluorescent in situ analysis of asthmatic biopsies. Positive signals within the extracellular DNA (arrows) were seen with an Atp6 but not with a Gapdh probe. The data in each panel are representative of at least three independent experiments. Bars, 10 µM.

Fig 4

No effect of segmental allergen challenge on numbers of EETs and NETs in airway biopsies of atopic asthmatics. A, Eosinophils and EETs before and 24 h after allergen challenge. B, Neutrophils and NETs before and 24 h after allergen challenge in asthmatics with neutrophil infiltration. Values are single numbers as well as mean levels ± SEM per 10 hpf. Changes are not significant.

Fig 5

Effect of segmental allergen challenge on cytokine/chemokine concentrations in BAL fluid of atopic asthmatics. The indicated cytokines/chemokines were measured before and 24 h after allergen challenge. Values are single numbers as well as mean levels ± SEM. P values are indicated.