Asthma is accompanied by the accumulation of potentially damaging eosinophils within inflamed airways. How eosinophils may be removed from the airways is not clear. The phagocytic removal of eosinophils in vitro requires that they undergo apoptosis, a form of cell death. We postulated that eosinophil apoptosis may occur in vivo, promoting the removal of airway eosinophils and the resolution of inflammation in asthma. We examined eosinophil apoptosis in sputum samples obtained from 11 subjects during an asthma exacerbation and 2 wk after corticosteroid treatment of the exacerbation. Airway function improved following corticosteroid treatment, and eosinophilic inflammation subsided, with significant decreases occurring in the number of airway eosinophils and the percentage of activated eosinophils. The proportion of apoptotic airway eosinophils increased significantly following corticosteroid treatment, and eosinophil products were apparent within macrophages. Our findings indicate that eosinophil apoptosis is clinically relevant in asthma. Apoptosis may represent a mechanism that promotes the resolution of eosinophilic inflammation in asthma.

We aimed to describe induced sputum cell counts in healthy nonasthmatic children, and to compare these to children with controlled and uncontrolled asthma. Following clinical assessment and spirometry, ultrasonically nebulized hypertonic saline was used to induce sputum from children with asthma (n=50) and without asthma (n=72). Sputum was dispersed and cell counts performed to yield total and differential cell counts. Specific stains were used for eosinophil and mast cell counts. All of the children with asthma were receiving inhaled and/or oral corticosteroids. Current asthma control was assessed in terms of symptoms and lung function. Children were classified as controlled on inhaled corticosteroids (no current symptoms, normal lung function n=15), current symptomatic asthma (n=16) and asthma exacerbation (n=11). It was found that eosinophils comprised a median 0.3% (interquartile range (IQR): 0, 1.05) of cells in sputum from healthy children. Sputum eosinophils (4.3% (IQR: 15, 14.1) p=0.0005) and epithelial cells (14% (IQR: 6, 19.4) p=0.0005) were significantly higher in children with asthma than in nonasthmatic children. Children whose asthma was controlled, as well as those with symptoms, had more sputum eosinophils and epithelial cells than the nonasthmatics. Mast cells were found in the sputum of only four of the 42 children with asthma. This study demonstrates that eosinophilic airway inflammation and epithelial damage can occur in children with asthma. Airway inflammation persists even in those children who are receiving inhaled corticosteroids, have normal lung function and good symptomatic control of their disease.

A As ss se es ss sm me en nt t o of f a ai ir rw wa ay y i in nf fl la am mm ma at ti io on n i in n c ch hi il ld dr re en n w wi it th h a ac cu ut te e a as st th hm ma a u us si in ng g i in nd du uc ce ed d s sp pu ut tu um m ABSTRACT: The aims of this study were: to assess the safety of a sputum induction method using inhaled normal saline in children with acute asthma; and to investigate changes in sputum cell counts between acute exacerbations of asthma and its resolution. Ultrasonically nebulized normal saline was used to induce sputum from children (n=8) presenting with acute asthma within 1 h of arrival and again at least 14 days later, after resolution of the exacerbation. Children received pretreatment with bronchodilator, and peak expiratory flow (PEF) was monitored throughout the procedure. Samples were analysed for total cell count, differential cell counts, and for eosinophils and neutrophils using specific immunochemical stains.Sputum induction was performed without adverse effect in each child with acute asthma. The mean fall in PEF from baseline during sputum induction was 5.3% during the acute attack and 3.4% at resolution. A shorter nebulization time was required to induce sputum in acute asthma than at follow-up (7.8 vs 13.9 min; p=0.04). During acute asthma, there was an intense cellular infiltrate (mean total cell count 34×10 6 cells·mL -1 ), which resolved after recovery (1.9×10 6 cells·mL -1 ) (p=0.04). The infiltrate was heterogenous, comprising eosinophils (6.7×10 6 cells·mL -1 ), neutrophils (5.4×10 6 cells·mL -1 ) and mast cells (0.47×10 6 cells·mL -1 ). Resolution of the exacerbation was accompanied by a significant fall in eosinophils and neutrophils (p≤0.04).Normal saline induction of sputum can be used to assess airway inflammation in acute asthma. Children with acute asthma have intense airway inflammation that is heterogeneous and involves neutrophils, eosinophils and mast cells.

Recombinant human deoxyribonuclease (rhDNase) has been shown to reduce sputum viscoelasticity and to improve lung function in patients with cystic fibrosis (CF). The aim of this study was to determine whether airway inflammation would decrease after administration of rhDNase. Twenty patients with CF and chronic suppurative lung disease inhaled 2.5 mg of rhDNase daily for 1 month. Before and after the 1‐month trial, lung function was measured and sputum was obtained, either after spontaneous expectoration or after sputum induction with hypertonic saline. Sputum total cell and differential counts were measured using techniques previously described. The mean age of the patients was 16.8 years (range, 6.7–27.5). After 1 month of rhDNase, mean FEV1 increased from a baseline of 62.3% predicted to 70.8% (P = 0.02, paired t test); and FVC increased from 74.4% to 83.9% predicted (P = 0.007). No significant differences were found in sputum cytology before or after rhDNase (median total cell counts 16.0 × 106/ml vs. 19.3 × 106/ml, P = 0.68). Thirteen patients had a 10% or greater increase in FEV1 after rhDNase (responders). Initial lung function was less in responders than in nonresponders (53.5% vs. 78.6%, P = 0.007). There was no significant change in total cell count and neutrophil count after rhDNase in either responders or nonresponders. We conclude that airway inflammation, as measured by total cell counts in sputum, was a prominent feature in cystic fibrosis, and neutrophils were the dominant inflammatory cells. Although the administration of rhDNase resulted in significant improvements in FEV1, there was no evidence of accompanying changes in airway inflammation. Pediatr Pulmonol. 1998;26:97–100. © 1998 Wiley‐Liss, Inc.

Summary. Sputum analysis is a useful technique for the study of airway inflammation. In asthma, dithiothreitol (DlT) is used to disperse cells from surrounding mucus; however, the applicability of these processing methods to cystic fibrosis (CF) sputum is unknown. In order to compare two methods for processing sputum of patients with CF, sputum was obtained from 11 subjects with CF (8 female, aged 9-21 years). The sample was split into 2 portions and sputum dispersal using DlT was compared with an enzyme mixture (E) of deoxyribonuclease, hyaluronidase, and galactosidase. Outcomes assessed were sample quality, cell viability (percent cells excluding trypan blue), total cell count (TCC), neutrophil count, and elastase immunoreactivity (percent cells positive). Sample quality (enzymes vs. DTT, 8.3 2 0.3 vs 7.6 ? 0.4, mean 2 SEM) and cell viability (enzymes vs. DTT, 75.0% vs. 68.O%, median) were similar for both methods.Sputum total cell count (20.5 x 106/ml vs. 12.0 x 106/ml, median; P = 0.01) and neutrophil count (13.4 x lOYml vs. 5.5 x 106/ml, median; P = 0.02) were significantly higher with E. Elastase immunoreactivity was lost after processing with E (19.OY0 vs. 39.5%, median; P = 0.04). When purified peripheral blood neutrophils were incubated with DTT and E, there was no reduction in neutrophil viability, suggesting that the reduced neutrophil number in CF sputum was not due to a toxic effect of DTT, but rather incomplete dispersal. We conclude that published sputum processing methods for asthma using DTT give false results when applied to CF sputum, which should be processed using an enzyme mixture.

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Sputum analysis is a useful technique for the study of airway inflammation. In asthma, dithiothreitol (DTT) is used to disperse cells from surrounding mucus; however, the applicability of these processing methods to cystic fibrosis (CF) sputum is unknown. In order to compare two methods for processing sputum of patients with CF, sputum was obtained from 11 subjects with CF (8 female, aged 9–21 years). The sample was split into 2 portions and sputum dispersal using DTT was compared with an enzyme mixture (E) of deoxyribonuclease, hyaluronidase, and galactosidase. Outcomes assessed were sample quality, cell viability (percent cells excluding trypan blue), total cell count (TCC), neutrophil count, and elastase immunoreactivity (percent cells positive). Sample quality (enzymes vs. DTT, 8.3 ± 0.3 vs 7.6 ± 0.4, mean ± SEM) and cell viability (enzymes vs. DTT, 75.0% vs. 68.0%, median) were similar for both methods. Sputum total cell count (20.5 × 106/ml vs. 12.0 × 106/ml, median, P = 0.01) and neutrophil count (13.4 × 106/ml vs. 5.5 × 106/ml, median; P = 0.02) were significantly higher with E. Elastase immunoreactivity was lost after processing with E (19.0% vs. 39.5%, median; P = 0.04). When purified peripheral blood neutrophils were incubated with DTT and E, there was no reduction in neutrophil viability, suggesting that the reduced neutrophil number in CF sputum was not due to a toxic effect of DTT, but rather incomplete dispersal. We conclude that published sputum processing methods for asthma using DTT give false results when applied to CF sputum, which should be processed using an enzyme mixture. Pediatr Pulmonol. 1996; 22:402–407. © 1996 Wiley‐Liss, Inc