Background Asthma is a heterogeneous inflammatory airway disorder which involves eosinophilic and non-eosinophilic phenotypes. Unlike in normal lungs, eosinophils are often present in atopic asthmatic airways although a subpopulation of asthmatic subjects predominantly develops neutrophilic inflammation. Recently, it has been demonstrated that eosinophils and neutrophils generate bactericidal extracellular traps consisting of DNA and cytotoxic granule proteins. Objective To explore if 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 biopsies from 20 mild human atopic asthmatics at baseline and after local allergen challenge, and 10 normal subjects. DNA was stained with propidium iodine and major basic protein (MBP) with specific antibody. Differential cell counts and cytokines/chemokines were assessed in bronchoalveolar fluids. Results Asthmatic airways were infiltrated with a significantly higher number of eosinophils than normal airways (39.3±4.6 vs. 0.4±0.9, p<0.0001). All asthmatics but only one control subject expressed eosinophils releasing DNA that colocalized with MBP (33.65±20.33 vs. 0.3±0.9 per hpf, p<0.0001). Four asthmatics 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 BAL levels of IL-5, IFN-γ, or eotaxin, or the provoking doses of methacholine or allergen in asthmatics. 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. Clinical implications Eosinophil extracellular traps (EETs) (1) are frequently seen in atopic asthma, (2) consist of DNA and granule proteins, and (3) might be a new useful biomarker reflecting eosinophil activation.

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The mechanism of steroid action in asthma is unknown. Because steroids have effects in vitro on eicosanoid synthesis, we determined the effect of oral prednisone for 6 to 9 days on eicosanoid levels in bronchoalveolar lavage (BAL) fluid of 14 atopic asthmatic volunteers at baseline and after allergen instillation. We also determined the effect of prednisone on the ex vivo release of eicosanoids from macrophage-rich BAL-fluid cells. Prednisone reduced symptoms and inhaler use but had no significant effect on BAL-fluid eicosanoid levels. At baseline, prostaglandin D2 (PGD2) levels were 101 +/- 37 pg/ml in BAL fluid (mean +/- SEM), versus 66 +/- 18 pg/ml after prednisone; likewise, 5-hydroxy eicosatetraenoic acid (5-HETE) levels were 59 +/- 15 versus 78 +/- 21; leukotriene E4 (LTE4) levels were 35 +/- 13 versus 51 +/- 21, and 15-hydroxy eicosatetraenoic acid (15-HETE) levels were 29 +/- 8 versus 19 +/- 7. Allergen-stimulated levels of PGD2 were 1274 +/- 565 versus 1468 +/- 679 after prednisone; likewise, allergen-stimulated 5-HETE levels were 95 +/- 21 versus 82 +/- 21; those of LTE4 were 54 +/- 20 versus 91 +/- 51; and those of 15-HETE were 63 +/- 19 versus 60 +/- 25. Prednisone reduced the synthesis of eicosanoids in macrophage-rich BAL-fluid cells in vitro. LTB4 levels fell significantly from 35 +/- 6.4 ng/10(6) BAL-fluid cells to 17 +/- 5.4 after prednisone; likewise, levels of thromboxane B2 (TXB2) fell from 35.7 +/- 7.5 to 20.7 +/- 6.6. Part of the action of steroids may involve alteration in macrophage eicosanoid synthesis.

Further definition of the role of leukotrienes (LT) and prostaglandins (PG) in asthma would be helped by a noninvasive method for assessing airway production. The supernatant from sputum induced with hypertonic saline and dispersed using dithiotrietol has been successfully used to measure other molecular markers of airway inflammation and might be a useful method. We have measured induced sputum supernatant LTC(4)/D(4)/E(4) concentrations using enzyme immunoassay and PGE(2), PGD(2), TXB(2), and PGF(2alpha) using gas chromatography-negative ion chemical ionization-mass spectroscopy in 10 normal subjects and in 26 subjects with asthma of variable severity. Sputum cysteinyl-leukotrienes concentrations were significantly greater in subjects with asthma (median, 9.5 ng/ml) than in normal control subjects (6.4 ng/ml; p < 0.02) and greater in subjects with persistent asthma requiring inhaled corticosteroids (median, 11.4 ng/ml) or studied within 48 h of an acute severe exacerbation of asthma (13 ng/ml) than in subjects with episodic asthma treated with inhaled beta(2)-agonists only (7.2 ng/ml). There were no significant differences in the concentrations of other eicosanoids between groups, although there was a negative correlation between the percentage sputum eosinophil count and sputum PGE(2) concentration (r = -0.48; p < 0.01) in subjects with asthma. We conclude that induced sputum contains high concentrations of eicosanoids and that sputum LTC(4)/D(4)/E(4) concentrations are significantly greater in subjects with asthma than in normal subjects. The inverse relationship between eosinophilic airway inflammation and sputum PGE(2) concentration would be consistent, with the latter having an anti-inflammatory role.