subjects were recruited from hospital staff and general respiratory and  difficult asthma  clinics at glenfield hospital , leicester ; local primary health care ; and by local advertising . asthma was defined according to the current global initiative for asthma ( gina ) guidelines . subjects with asthma had typical symptoms and the presence of one or more of the following objective criteria : significant bronchodilator reversibility of fev1 > 200 ml , a provocation concentration of methacholine causing a 20% fall in fev1 ( pc20 ) of < 8 mg / ml , or a peak flow amplitude percent mean over 2 weeks of > 20% . asthma severity was classified using the gina treatment steps ( mild to moderate 1 - 3 , severe 4 - 5 ) . copd was diagnosed and severity categorized by using global initiative for chronic obstructive lung disease criteria . subjects were recruited as three independent cross - sectional groups , to assess il-17 a , f , and il-17r expression in proximal airways in asthma ( group 1 ) and copd ( group 2 ) , and sputum il-17 concentration in asthma and copd ( group 3 ) . healthy control subjects had normal spirometry and some smokers with > 10 pack - year history were included to enable comparisons between healthy smokers and subjects with copd . subjects with asthma and healthy control subjects in group 1 and 2 also underwent methacholine inhalation test using the tidal breathing method and allergen skin prick tests for dermatophagoides pteronyssinus , dog , cat , and grass pollen . sputum induction was performed in all subjects in groups 1 and 2 . in group 1 , subjects underwent bronchoscopy conducted according to the british thoracic society guidelines , and biopsies were taken from the right middle and lower lobe carinae . in group 2 , all bronchial mucosal specimens were fixed in acetone and embedded in glycomethacrylate as described previously . sputum was selected , dispersed using the mucolytic dithiothreitol ( dtt ) , and processed to generate a sputum differential cell count , and cell - free supernatants were stored at -80c for later analysis as described previously.sputum il-17 was measured by enzyme - linked immunosorbent assay ( elisa ) ( r&d systems europe ltd ; abingdon , england ) . the il-17 assay was validated in line with european respiratory society recommendations to assess the effect of dtt and the recovery of exogenous spiking with recombinant cytokine . il-17 recovery was not affected by dtt and mean recovery of exogenous spiked il-17 was 93% ( n = 16 ) . il-17 was also measured in a further independent population of subjects with copd and asthma using the mesoscale device platform . two - micrometer sections were cut and stained using polyclonal antibodies against il-17a and il-17f ( r&d systems europe ltd ) , monoclonal antibodies against eosinophils ( major basic protein ) , neutrophils ( neutrophil elastase ) , and mast cells ( tryptase ) , with appropriate isotype controls mouse igg1 ( dako uk ltd ; cambridge , england ) and goat immunoglobulins ( r&d systems europe ltd ) . the number of positive nucleated cells was enumerated per mm of bronchial submucosa by a blinded observer as described previously . in a subgroup of six subjects with asthma , il-17a and il-17f statistical analysis was performed using prism version 4 ( graphpad ; la jolla , ca ) . parametric data were expressed as mean ( sem ) , data that had a normal log distribution were log transformed and described as geometric mean ( 95% ci ) , and nonparametric data were described as median ( interquartile range [ iqr ] ) . one - way analysis of variance ( kruskal - wallis for nonparametric data ) was used for across - group comparisons with tukey and dunn post hoc tests for between - group comparisons , respectively . correlations were assessed by spearman rank ( rs ) and pearson ( r ) correlation coefficients . subjects were recruited from hospital staff and general respiratory and  difficult asthma  clinics at glenfield hospital , leicester ; local primary health care ; and by local advertising . asthma was defined according to the current global initiative for asthma ( gina ) guidelines . subjects with asthma had typical symptoms and the presence of one or more of the following objective criteria : significant bronchodilator reversibility of fev1 > 200 ml , a provocation concentration of methacholine causing a 20% fall in fev1 ( pc20 ) of < 8 mg / ml , or a peak flow amplitude percent mean over 2 weeks of > 20% . asthma severity was classified using the gina treatment steps ( mild to moderate 1 - 3 , severe 4 - 5 ) . copd was diagnosed and severity categorized by using global initiative for chronic obstructive lung disease criteria . subjects were recruited as three independent cross - sectional groups , to assess il-17 a , f , and il-17r expression in proximal airways in asthma ( group 1 ) and copd ( group 2 ) , and sputum il-17 concentration in asthma and copd ( group 3 ) . healthy control subjects had normal spirometry and some smokers with > 10 pack - year history were included to enable comparisons between healthy smokers and subjects with copd . healthy control subjects in group 1 and 2 also underwent methacholine inhalation test using the tidal breathing method and allergen skin prick tests for dermatophagoides pteronyssinus , dog , cat , and grass pollen . sputum induction was performed in all subjects in groups 1 and 2 . in group 1 , subjects underwent bronchoscopy conducted according to the british thoracic society guidelines , and biopsies were taken from the right middle and lower lobe carinae . in group 2 , all bronchial mucosal specimens were fixed in acetone and embedded in glycomethacrylate as described previously . sputum was selected , dispersed using the mucolytic dithiothreitol ( dtt ) , and processed to generate a sputum differential cell count , and cell - free supernatants were stored at -80c for later analysis as described previously.sputum il-17 was measured by enzyme - linked immunosorbent assay ( elisa ) ( r&d systems europe ltd ; abingdon , england ) . the il-17 assay was validated in line with european respiratory society recommendations to assess the effect of dtt and the recovery of exogenous spiking with recombinant cytokine . il-17 recovery was not affected by dtt and mean recovery of exogenous spiked il-17 was 93% ( n = 16 ) . il-17 was also measured in a further independent population of subjects with copd and asthma using the mesoscale device platform . two - micrometer sections were cut and stained using polyclonal antibodies against il-17a and il-17f ( r&d systems europe ltd ) , monoclonal antibodies against eosinophils ( major basic protein ) , neutrophils ( neutrophil elastase ) , and mast cells ( tryptase ) , with appropriate isotype controls mouse igg1 ( dako uk ltd ; cambridge , england ) and goat immunoglobulins ( r&d systems europe ltd ) . the number of positive nucleated cells was enumerated per mm of bronchial submucosa by a blinded observer as described previously . in a subgroup of six subjects with asthma , il-17a and il-17f statistical analysis was performed using prism version 4 ( graphpad ; la jolla , ca ) . parametric data were expressed as mean ( sem ) , data that had a normal log distribution were log transformed and described as geometric mean ( 95% ci ) , and nonparametric data were described as median ( interquartile range [ iqr ] ) . one - way analysis of variance ( kruskal - wallis for nonparametric data ) was used for across - group comparisons with tukey and dunn post hoc tests for between - group comparisons , respectively . correlations were assessed by spearman rank ( rs ) and pearson ( r ) correlation coefficients . clinical characteristics of group 1 are as shown in table 1 . examples of il-17a and il-17f cells in the bronchial submucosa in asthma are as shown in figures 1a1c . the median ( iqr ) il-17a cells / mm submucosa was increased in mild to moderate asthma ( 2.1 [ 2.4 ] ) compared with healthy control subjects ( 0.4 [ 2.8 ] ) but not compared with asthma ( 0.9 [ 4.2 ] ) ( p = .04 , kruskal - wallis ; p < .05 mild to moderate asthma vs control subjects ) ( fig 1d ) . the number of il-17f cells / mm submucosa was increased in severe asthma ( 2.7 [ 3.6 ] ) and mild to moderate asthma ( 1.6 [ 1.0 ] ) compared with healthy control subjects ( 0.7 [ 1.4 ] ) ( p = .001 kruskal - wallis ; p < .01 severe asthma vs control subjects , and p < .05 mild to moderate vs control subjects ) ( fig 1e ) . the median ( iqr ) proportion 50% ( 27% ) and 24% ( 19% ) of the il-17a and 44% ( 40% ) and 0 ( 25% ) of il-17f cells colocalized to neutrophils and eosinophils , respectively . clinical and sputum characteristics of biopsy group asthma bd = bronchodilator ; gina = global initiative for asthma ; iqr = interquartile range ; pc20 = provocation concentration causing a 20% fall in fev1 ; se = standard error . p < .05 compared with control subjects . il-17a and il-17f expression in the submucosa in asthma . d , the number of il-17a and e , il-17f cells in the bronchial submucosa of healthy control subjects , subjects with mild to moderate asthma ( global initiative for asthma [ gina ] 1 - 3 ) , and subjects with severe asthma ( gina 4 - 5 ) . p values for across - group comparison by kruskal - wallis test and dunn post hoc test for between - group comparisons are as shown . . examples of il-17a and il-17f cells in the bronchial submucosa in copd are as shown in figures 2a2c . the median ( iqr ) il-17a cells / mm submucosa was increased in copd ( 0.5 [ 3.7 ] ) compared with nonsmoking control subjects ( 0 ) , but not compared with smoking control subjects , ( p = .046 , kruskal - wallis ; p < .05 copd vs nonsmoking control subjects ) ( fig 2d ) . there were no differences in the number of il-17f cells / mm submucosa in lung resection tissue from subjects with copd and control subjects with and without a significant smoking history ( fig 2e , table 2 ) . clinical characteristics and il-17a and il-17f expression in copd see table 1 legend for expansion of the abbreviation . p < .05 compared with nonsmoking control subjects . il-17a and il-17f expression in the submucosa in copd . representative photomicrographs of proximal airway resections from copd tissue , illustrating isotype control . a , goat igg . d , the number of il-17a and e , il-17f cells in the submucosa of healthy control subjects ( never smokers ) , healthy control subjects ( smokers ) , and subjects with copd . p values for across - group comparison by kruskal - wallis test and dunn post hoc test for between - group comparisons are as shown . the number of il-17a cells was correlated with fev1% predicted ( rs = 0.38 ; p = .04 ) , and the sputum neutrophil count ( rs = 0.43 , p = .03 ) in asthma but was not associated with the number of neutrophils or eosinophils in the bronchial mucosa ( table 3 ) . the number of il-17f was positively correlated with the number of eosinophils in tissue ( rs = 0.50 ; p = .005 ) but not significantly correlated with lung function or neutrophil number in tissue or sputum ( table 3 ) . correlations between il-17a / f , granulocytic inflammation , and lung function in asthma rs = spearman rank correlation coefficient . sputum il-17 was below the limit of detection in all samples from subjects with asthma ( n = 22 ) , subjects with copd ( n = 22 ) , and control subjects ( n = 12 ) measured by elisa . using the mesoscale device platform the sputum il-17 concentration in copd was increased compared with asthma ( 2 [ 0 - 7 ] pg / ml vs 0 [ 0 - 2 ] pg / ml , p < .0001 ) and was correlated with post - bronchodilator fev1% predicted ( r = 0.5 , p = .008 ) and fev1/fvc ( r = 0.4 , p = .04 ) ( figs 3a3c).the clinical characteristics of these subjects with asthma and copd are as shown ( table 4 ) . b , correlations between sputum il-17 concentration in subjects with copd and postbronchodilator fev1% predicted . clinical characteristics and il-17a and il-17f sputum concentration in asthma and copd see table 1 legend for expansion of the abbreviation . clinical characteristics of group 1 are as shown in table 1 . examples of il-17a and il-17f cells in the bronchial submucosa in asthma are as shown in figures 1a1c . the median ( iqr ) il-17a cells / mm submucosa was increased in mild to moderate asthma ( 2.1 [ 2.4 ] ) compared with healthy control subjects ( 0.4 [ 2.8 ] ) but not compared with asthma ( 0.9 [ 4.2 ] ) ( p = .04 , kruskal - wallis ; p < .05 mild to moderate asthma vs control subjects ) ( fig 1d ) . the number of il-17f cells / mm submucosa was increased in severe asthma ( 2.7 [ 3.6 ] ) and mild to moderate asthma ( 1.6 [ 1.0 ] ) compared with healthy control subjects ( 0.7 [ 1.4 ] ) ( p = .001 kruskal - wallis ; p < .01 severe asthma vs control subjects , and p < .05 mild to moderate vs control subjects ) ( fig 1e ) . the median ( iqr ) proportion 50% ( 27% ) and 24% ( 19% ) of the il-17a and 44% ( 40% ) and 0 ( 25% ) of il-17f cells colocalized to neutrophils and eosinophils , respectively . clinical and sputum characteristics of biopsy group asthma bd = bronchodilator ; gina = global initiative for asthma ; iqr = interquartile range ; pc20 = provocation concentration causing a 20% fall in fev1 ; se = standard error . p < .05 compared with control subjects . il-17a and il-17f expression in the submucosa in asthma . d , the number of il-17a and e , il-17f cells in the bronchial submucosa of healthy control subjects , subjects with mild to moderate asthma ( global initiative for asthma [ gina ] 1 - 3 ) , and subjects with severe asthma ( gina 4 - 5 ) . p values for across - group comparison by kruskal - wallis test and dunn post hoc test for between - group comparisons are as shown . . examples of il-17a and il-17f cells in the bronchial submucosa in copd are as shown in figures 2a2c . the median ( iqr ) il-17a cells / mm submucosa was increased in copd ( 0.5 [ 3.7 ] ) compared with nonsmoking control subjects ( 0 ) , but not compared with smoking control subjects , ( p = .046 , kruskal - wallis ; p < .05 copd vs nonsmoking control subjects ) ( fig 2d ) . there were no differences in the number of il-17f cells / mm submucosa in lung resection tissue from subjects with copd and control subjects with and without a significant smoking history ( fig 2e , table 2 ) . clinical characteristics and il-17a and il-17f expression in copd see table 1 legend for expansion of the abbreviation . p < .05 compared with nonsmoking control subjects . il-17a and il-17f expression in the submucosa in copd . representative photomicrographs of proximal airway resections from copd tissue , illustrating isotype control . a , goat igg . d , the number of il-17a and e , il-17f cells in the submucosa of healthy control subjects ( never smokers ) , healthy control subjects ( smokers ) , and subjects with copd . p values for across - group comparison by kruskal - wallis test and dunn post hoc test for between - group comparisons are as shown . the number of il-17a cells was correlated with fev1% predicted ( rs = 0.38 ; p = .04 ) , and the sputum neutrophil count ( rs = 0.43 , p = .03 ) in asthma but was not associated with the number of neutrophils or eosinophils in the bronchial mucosa ( table 3 ) . the number of il-17f was positively correlated with the number of eosinophils in tissue ( rs = 0.50 ; p = .005 ) but not significantly correlated with lung function or neutrophil number in tissue or sputum ( table 3 ) . correlations between il-17a / f , granulocytic inflammation , and lung function in asthma rs = spearman rank correlation coefficient . sputum il-17 was below the limit of detection in all samples from subjects with asthma ( n = 22 ) , subjects with copd ( n = 22 ) , and control subjects ( n = 12 ) measured by elisa . using the mesoscale device platform the sputum il-17 concentration in copd was increased compared with asthma ( 2 [ 0 - 7 ] pg / ml vs 0 [ 0 - 2 ] pg / ml , p < .0001 ) and was correlated with post - bronchodilator fev1% predicted ( r = 0.5 , p = .008 ) and fev1/fvc ( r = 0.4 , p = .04 ) ( figs 3a3c).the clinical characteristics of these subjects with asthma and copd are as shown ( table 4 ) . b , correlations between sputum il-17 concentration in subjects with copd and postbronchodilator fev1% predicted . clinical characteristics and il-17a and il-17f sputum concentration in asthma and copd see table 1 legend for expansion of the abbreviation . we report here that il-17a expression in the bronchial submucosa was increased in mild to moderate asthma , and il-17f was increased in mild to moderate and severe asthma . in contrast , in ex - smoking subjects with mild to moderate copd the number of il-17a cells in the bronchial submucosa , but not il-17f , was increased compared with nonsmoking control subjects . in asthma there was a weak relationship between the number of il-17a cells and the fev1% predicted , and the sputum neutrophil count . there was no association between neutrophilic inflammation and il-17a or f expression in the bronchial submucosa . however , there was a good correlation between the number of il-17f cells and eosinophils in the bronchial submucosa . in copd there was a highly significant , albeit small , increase in sputum il-17 compared with asthma , and this was correlated with airflow obstruction and lung function impairment , but not airway inflammation . our study therefore supports our hypothesis that il-17 may play a role in asthma and copd . there is an increasing body of evidence supporting a role for th17 cells in asthma . animal models of asthma suggest that th17 cytokines promote neutrophilic inflammation , which in concert with th2 cells is important in the development of airway hyperresponsiveness . previous reports have demonstrated increased il-17a and il-17f bronchial submucosa expression in moderate to severe asthma , which were attenuated by systemic corticosteroids . the data for sputum il-17 is conflicting with one study demonstrating no differences in the sputum il-17 concentration between asthma and healthy volunteers , whereas in another study il-17 was increased in moderate to severe asthma . in the latter study sputum il-17 importantly , consistent with our findings previous studies have found that the sputum il-17 concentration in sputum is low and in some studies samples have needed to be concentrated to detect il-17 . we found that il-17a and il - f were increased in mild to moderate disease , whereas il-17f but not il-17a was increased in severe asthma . our subjects with severe asthma were treated with high - dose inhaled and/or oral corticosteroids , and therefore this may have attenuated the il-17a expression in this group . il-17a and il-17f expression was not associated with smoking status but importantly this study was not powered to fully explore the effects of smoking in asthma . we report for the first time , to our knowledge , the relationship between il-17a and f expression and neutrophilic inflammation in tissue and sputum . we found that there was no association between il-17a and il-17f expression neutrophilic inflammation in tissue . intriguingly , there was a weak inverse correlation between submucosal il-17a expression and the sputum neutrophil count . sputum il-17 was also markedly attenuated by inhaled corticosteroids , but the sputum neutrophil count was unchanged , questioning the role of il-17 in persistent neutrophilic airway inflammation . however , others have demonstrated an association between il-17 and il-8 mrna in sputum cells and the number of sputum neutrophils , suggesting that the site of il-17 expression may be important . consistent with our finding in a recent comparison between subjects with severe asthma with and without persistent airflow obstruction , il-17 was among the dominant mediators associated with the absence of persistent airflow obstruction . together these findings do not support a role for il-17 in the development of fixed airflow obstruction in asthma . to date hence , it is predictable that il-17 may play an important role in this disease . there is a single report of il-17a and f expression in bronchoscopic biopsies obtained across the spectrum of copd disease severity compared with smoking and nonsmoking control subjects . il-17a was increased equally across the severity of copd compared with the nonsmoking control subjects , but was not different from the smoking control subjects . study the number of neutrophils in tissue was increased in the copd groups with a marked increase in severe copd . our findings of increased il-17a expression in mild to moderate copd compared with nonsmoking control subjects , but not smoking control subjects , is therefore entirely consistent with these earlier reports . however , we were unable to detect il-17 by elisa , and with a more sensitive mesoscale device platform a substantial proportion still remained below the limit of detection . the difference between copd and asthma was highly significant , but small , questioning whether this difference is biologically important . interestingly , the sputum il-17 concentration in copd was related to the degree of airflow limitation and obstruction . therefore , the potential role of il-17 , particularly in severe disease , and its synergy with the development of airway inflammation in response to cigarette smoke exposure or alternatively through interactions with other cytokines , such as tumor necrosis factor or il-1 , needs to be further explored . neutrophils were the predominant source of il-17 as evidenced by colocalization using immunohistochemistry . however , there was no correlation between the number of il-17 cells and neutrophils in the bronchial submucosa . in contrast to the established role of il-17a and il-17f in the initiation of neutrophilic inflammation , neither has been implicated in the development of eosinophilic inflammation . indeed , il-17f transgenic mice develop an airway neutrophilia after allergen challenge , whereas knockout mice have an impaired neutrophilic response and enhanced th2 cytokine production . therefore , we were surprised by the relationship between the number of il-17f cells and eosinophils in the bronchial submucosal . however , a similar relationship has been described between sputum il-17 mrna and il-5 mrna , possibly suggesting a hitherto undescribed association between eosinophilic airway inflammation and the th17 axis . thus , the potential interactions between eosinophilic inflammation and up - regulation of the th17 axis require further study . whether il-17a and il-17f expression is related to longitudinal clinical outcomes , such as disease progression , lung function decline , and exacerbations , requires further examination . similarly , we are unable to determine whether differences observed between mild and severe asthma reflect disease severity or are a consequence of differences in treatment . current literature suggests that il-17a is sensitive to corticosteroid therapy , in contrast to neutrophilic inflammation , which is corticosteroid resistant . importantly , the number of il-17a or il-17f cells was expressed as cells / mm of submucosa rather than as a proportion of the total number of cells in the submucosa . therefore , whether the changes in cell number between groups is a reflection of increased expression or an increase in the total number of cells needs to be examined in future studies . we are confident that our assays to assess il-17 were robust as recovery was unaffected by the mucolytic dtt and recovery of exogenous spikes of il-17 to sputum samples was good . however , a number of samples were below the limit of detection ; therefore , the sputum data do need to be interpreted cautiously . therefore , findings with a marginal level of significance also need to be interpreted with caution . in conclusion , we found that il-17a expression in bronchial submucosa was increased in mild to moderate asthma and in copd , although this was not independent of smoking . il-17f expression was increased in mild to moderate and severe asthma , but not in copd . our findings therefore do support a potential role for il-17a and il-17f in asthma and possibly copd . efficacy studies of therapeutic strategies targeted at the il-17 axis are eagerly awaited that will further define the functional importance of il-17a and il-17f in airways disease .
background : asthma and copd are characterized by airway dysfunction and inflammation . 
 neutrophilic airway inflammation is a common feature of copd and is recognized in asthma , particularly in severe disease . 
 the t helper ( th ) 17 cytokines il-17a and il-17f have been implicated in the development of neutrophilic airway inflammation , but their expression in asthma and copd is uncertain.methods:we assessed il-17a and il-17f expression in the bronchial submucosa from 30 subjects with asthma , 10 ex - smokers with mild to moderate copd , and 27 nonsmoking and 14 smoking control subjects . sputum il-17 concentration was measured in 165 subjects with asthma and 27 with copd.results:the median ( interquartile range ) il-17a cells / mm2 submucosa was increased in mild to moderate asthma ( 2.1 [ 2.4 ] ) compared with healthy control subjects ( 0.4 [ 2.8 ] ) but not in severe asthma ( p = .04 ) . in copd , 
 il-17a+ cells / mm2 submucosa were increased ( 0.5 [ 3.7 ] ) compared with nonsmoking control subjects ( 0 [ 0 ] ) but not compared with smoking control subjects ( p = .046 ) . 
 il-17f+ cells / mm2 submucosa were increased in severe asthma ( 2.7 [ 3.6 ] ) and mild to moderate asthma ( 1.6 [ 1.0 ] ) compared with healthy controls subjects ( 0.7 [ 1.4 ] ) ( p = .001 ) but was not increased in subjects with copd . il-17a and 
 il-17f were not associated with increased neutrophilic inflammation , but il-17f was correlated with the submucosal eosinophil count ( rs = 0.5 , p = .005 ) . 
 the sputum il-17 concentration in copd was increased compared with asthma ( 2 [ 0 - 7 ] pg / ml vs 0 [ 0 - 2 ] pg / ml , p < .0001 ) and was correlated with post - bronchodilator fev1% predicted ( r = 0.5 , p = .008 ) and fev1/fvc ( r = 0.4 , p = .04).conclusions : our findings support a potential role for the th17 cytokines il-17a and il-17f in asthma and copd , but do not demonstrate a relationship with neutrophilic inflammation .