asthma and chronic obstructive pulmonary disease ( copd ) are chronic inflammatory airway diseases , characterised by airflow limitation obstruction that is reversible in asthma yet , under current therapeutics , progressive and not completely reversible in copd . asthma and copd are recognized for their heterogeneous nature , particularly in regard to the type of inflammation present . four major phenotypes ( eosinophilic , neutrophilic , paucigranulocytic , and mixed granulocytic ) have been described and characterised by the proportions of eosinophils and neutrophils [ 2 , 3 ] . these inflammatory phenotypes are associated with differences in disease severity and response to corticosteroids [ 4 , 5 ] , yet their underlying biology remains poorly understood [ 6 , 7 ] . the migration and activity of eosinophils and neutrophils are influenced by a range of host factors , including a class of antimicrobial peptides also known as alarmins . these antimicrobial peptides are small ( < 100 amino acids ) proteins , which play an important role in influencing and modulating the immune response through the receptor - mediated chemotaxis and activation of a range of innate and adaptive immune cells . the - and -defensins constitute the two major classes of human defensins , classified based on the differential organisation of six cysteine motifs . -defensin-1 is constitutively expressed by the epithelial cells of the respiratory tract and is both broadly antimicrobial and able to influence the immune response . -defensin-1 polymorphisms have been associated with both asthma [ 11 , 12 ] and copd [ 13 , 14 ] . increased gene expression of -defensin-1 has been observed in bronchial epithelium and bal fluid cells in copd and was negatively associated with lung function and airflow limitation . -defensin-1 protein , however , has yet to be examined in either asthma or copd . this study characterises the protein levels of -defensin-1 in induced sputum samples in both asthma and copd compared with healthy controls . we investigate the relationship of -defensin-1 to inflammatory phenotypes and disease severity . to understand the potential source of aberrant -defensin-1 expression , we also investigate its production in primary bronchial epithelial cells exposed to cigarette smoke extract in culture . we hypothesised that protein expression would be greater in asthma and copd , associated with disease severity and inflammatory phenotype , and altered in epithelial cells after cigarette smoke exposure . adults with copd and asthma were recruited from the john hunter hospital ambulatory care clinic , nsw , australia . participants with copd ( n = 43 and n = 10 for the primary bronchial epithelial cell ( pbec ) study ) had a diagnosis of copd and postbronchodilator fev1/fvc < 70% . participants with asthma ( n = 94 ) were diagnosed according to american thoracic society guidelines based upon current ( past 12 months ) episodic respiratory symptoms , doctor 's diagnosis , and demonstrated evidence of airway hyperresponsiveness to hypertonic saline . healthy controls ( n = 28 and n = 4 for the pbec study ) were nonsmokers and had fev1 > 80% predicted and were recruited by advertisement . exclusion criteria included a respiratory tract infection , exacerbation of respiratory disease or change in maintenance therapy in the past month , and current smoking ( except for the pbec study where current smoking in the copd group was not exclusion criteria ) . all participants gave written informed consent and the hunter new england area health service and the university of newcastle research ethics committees approved this study . spirometry ( koko pd instrumentation , louisville , colorado , usa ) and sputum induction with hypertonic saline ( 4.5% ) were performed in participants with fev1 > 1.3  l and with 0.9% saline in participants whose fev1 was below this level . the protocol specified a fixed sputum induction time of 15.5 minutes . for inflammatory cell counts , selected sputum was dispersed using dithiothreitol ( dtt ) , suspension was filtered , and a total cell count and cell viability count were performed . cytospins were prepared and stained ( may - grunwald giemsa ) and a differential cell count was obtained from 400 nonsquamous cells . the concentration of -defensin-1 was determined by elisa ( 100 - 240-bd1 , alpha diagnostic international , san antonio , texas , usa ) as per manufacturer 's instructions . the measurement of -defensin-1 protein in sputum supernatant was validated by determining the following : the inhibitory effect of dtt , matrix effects of sample dilution , and the recovery of spiked protein . matrix effects were minimised when samples were diluted 1/10 . on average , 104% of spiked -defensin-1 protein ( n = 6 ) was recovered . the granulocyte cut - offs to determine inflammatory phenotype used were 3% for sputum eosinophils and 61% for sputum neutrophils . severe asthma subjects had uncontrolled asthma ( measured by juniper asthma control questionnaire ( acq ) , score  1 ) and/or poor lung function ( fev1% predicted 80 and fev1/fvc%  70 ) despite prescription of high - dose inhaled corticosteroids ( ics ) ( > 1000  g beclomethasone equivalents per day ) in combination with long acting -agonists . if participants had poor lung function or poor symptom control but did not meet the treatment requirements for severe asthma , they were classified as uncontrolled asthma . controlled asthma was defined as normal lung function ( fev1% predicted > 80 and/or fev1/fvc% > 70 ) and controlled symptoms ( acq score < 1 ) . copd severity was defined according to the global obstructive lung disease ( gold ) initiative stage . cigarette smoke extract ( cse ) was prepared by bubbling smoke from one filterless kentucky research cigarette , 3r4f containing 9.5  mg tar and 0.8  mg nicotine , through 10  ml of cell culture medium bronchial epithelial basal medium ( bebm , lonza ) at a speed of 5 minutes per cigarette and used in the following cell culture experiments immediately . human pbecs were obtained by endobronchial brushing during fibre - optic bronchoscopy and cultured as described previously . cells were seeded onto placental collagen ( sigma ) coated 24-well plates ( nunclon ) and used at passage 2 once they reached 80% confluency . after exposure to 1% cse the pbecs were maintained in bebm ( lonza ) containing 1x insulin , transferrin , and sodium selenite liquid media supplement ( sigma ) . we have previously determined that this concentration of cse via dose response curves causes minimal toxicity to the cells whilst still inducing an immune response . clinical and cell count data were analysed using stata / ic 11.1 ( stata corporation , college station , texas , usa ) and graphpad prism 5.0 ( graphpad software , inc . , california , usa ) and reported as mean ( sd ) for normally distributed data and median ( q1 and q3 ) for nonparametric data . for normally distributed data , t - test or anova test with a bonferroni post hoc test to adjust for multiple comparisons was applied . for nonparametric data , a mann - whitney test or kruskal - wallis test with dunn 's post hoc test to adjust for multiple comparisons was applied . receiver operating characteristic ( roc ) curves were generated and the area under the curve ( auc ) was calculated to assess the relationship between the presence of copd and -defensin-1 levels . participants with copd were moderate to severe ( with 2 mild and 2 very severe ) , were significantly older , had a greater prevalence and smoking history , and a higher daily dose of ics . as expected , fev1% predicted and the fev1/fvc were significantly lower in asthma subjects than in healthy controls and lower in copd subjects than in both asthma subjects and healthy controls . changes in inflammatory cells were present in asthma and copd , including increases in total cell count , neutrophils , and eosinophils . -defensin-1 protein in sputum was significantly higher in copd subjects ( median ( q1 and q3 ) : 63.0 ( 43.6 and 81.9 )  ng / ml ) than in asthma subjects ( 26.3 ( 18.3 and 40.8 )  ng / ml ) and healthy controls ( 18.2 ( 14.2 and 27.8 )  ng / ml ; figure 1 ) . there was no significant difference in -defensin-1 protein between inflammatory phenotypes of asthma or copd ( figure 2 ) . -defensin-1 protein was significantly higher in those with severe asthma ( figure 3 ) but not different between copd gold stages ( data not shown ) . airway -defensin-1 protein level was correlated with smoking history ( pack years smoked ) in copd subjects ( spearman r = 0.52 and p = 0.003 , figure 4 ) but not in asthma subjects or healthy controls . airway -defensin-1 protein level was weakly correlated with ics dose in asthma ( spearman r = 0.31 and p = 0.006 ) but not in copd , most likely reflecting the relationship to asthma severity . airway -defensin-1 protein was predictive of copd from healthy controls with an accuracy ( auc ) of 88.3% ( 95% ci : 79.397.4% ; figure 5(a ) ) . the best cut - off point for -defensin-1 level to predict copd from healthy controls was > 29.3  ng / ml , with a sensitivity of 87.5% , specificity of 83.3% , and a positive likelihood ratio of 5.3 . airway -defensin-1 protein was predictive of copd from asthma with an accuracy ( auc ) of 80.8% ( 95% ci : 72.589.1% ; figure 5(b ) ) . the best cut - off point for -defensin-1 level to predict copd from asthma was > 42.1  ng / ml , with a sensitivity of 80.0% , specificity of 78.3% , and a positive likelihood ratio of 3.7 . airway -defensin-1 protein was predictive of copd from severe asthma with an accuracy ( auc ) of 68.7% ( 95% ci : 55.282.2% , figure 5(c ) ) . the best cut - off point for -defensin-1 level to predict copd from severe asthma was > 47.3  ng / ml , with a sensitivity of 70.0% , specificity of 72.4% , and a positive likelihood ratio of 2.5 . the level of -defensin-1 released from untreated media control pbecs was similar between healthy controls and participants with copd . however , upon stimulation with cigarette smoke extract ( cse ) -defensin-1 production was reduced by a mean of 2.8-fold in healthy control pbecs , whereas copd pbecs continued to produce the same level of -defensin-1 ( mean 2.8-fold versus 0.1-fold , p = 0.003 ) . figure 6 shows the levels of -defensin-1 in culture , further illustrating this point , with the copd group releasing significantly more -defensin-1 than the healthy control pbecs after 1% cse stimulation . to the authors ' knowledge , this was the first study to examine -defensin-1 protein in sputum in either asthma or copd . we report an elevated level of -defensin-1 protein in copd and severe asthma , with no relationship to inflammatory phenotype . airway levels of -defensin-1 could significantly discriminate the presence of copd from both asthma subjects and healthy controls . production of -defensin-1 was reduced in healthy pbecs but maintained in copd pbecs after cse exposure , which may account for the persistent and heightened -defensin-1 levels . factors driving persistent inflammation in asthma and copd , contributing to worsening of symptoms and lung function , are of interest to increase knowledge of underlying mechanisms and identify potential novel treatment targets . a class of multifunctional antimicrobial proteins known as alarmins , of which the defensins and the cathelicidins constitute two of the major families , may be of importance . human -defensin-1 is a small cationic peptide that is expressed constitutively by the epithelial cells of the respiratory tract . research into the functions of -defensin-1 has primarily centred around its antimicrobial properties ; however other functions have been reported . -defensin-1 has immunomodulatory effects , promoting the activation and maturation of monocyte derived dendritic cells through upregulation of cell surface expression of costimulatory molecules and maturation markers , as well as the promotion of proinflammatory cytokine production . by engaging a number of cell surface receptors including ccr6 , -defensin-1 promotes the chemotaxis of immature dendritic cells and t cells . through these immunomodulatory functions , -defensin-1 may influence the pathogenesis of copd , by promoting t cell and dendritic cell mediated inflammation . polymorphisms in the gene encoding -defensin-1 ( defb1 ) have been shown to affect the concentrations of -defensin-1 protein detected in saliva , indicating that these gene polymorphisms do influence expression and therefore could modify innate immune responses , and may influence disease susceptibility . defb1 gene polymorphisms have been associated with both asthma [ 11 , 12 ] and copd susceptibility [ 13 , 14 ] . additionally , increase in defb1 gene expression in bronchial epithelial and bal fluid cells of patients with copd is negatively correlated with fev1% predicted and fev1/fvc . the distinct increase in -defensin-1 protein seen in the current study supports a model in which -defensin-1 is dysregulated in copd and severe asthma . long term corticosteroid treatment can lead to changes in the balance between innate and adaptive immune responses , including cell migration and the chemokine network in macrophages . further study is required to determine the effects of long term ics treatment on -defensin-1 levels . the development of new diagnostic tools and treatments for copd has not kept pace with understanding of the disease . as an airway sample , sputum provides appealing means to study potential biomarkers as it contains a multitude of inflammatory mediators involved in copd disease processes . this study also shows that -defensin-1 protein levels in the sputum supernatant may be a useful biomarker for the detection of copd . -defensin-1 was able to distinguish copd from both asthma subjects and healthy controls with high levels of accuracy . for many years , spirometry and clinical symptoms have been the predominant tools to manage copd ; however this approach is not optimal . sputum eosinophils and exhaled nitric oxide can predict eosinophilic inflammation in airways disease   but do not differentiate asthma and copd . blood biomarkers including c - reactive protein , il-6 , and fibrinogen can detect a systemic inflammatory component of copd , important in predicting patients at risk of exacerbations , but these markers are not specific to copd and have been determined to be elevated in the neutrophilic asthma phenotype . a plasma protein signature of 2-macroglobulin , haptoglobin , and hemopexin was shown to discriminate between asthma and copd with 84% accuracy . this combination of 3 markers was only slightly better than sputum -defensin-1 alone in the current study at 81% . follow - up studies are warranted to investigate -defensin-1 for its capability for predicting copd diagnosis and prognosis and future risk of exacerbations . the epithelium is likely the major source of -defensin-1 that is secreted in the airways . until recently , only four -defensins ( 1 , 2 , 3 , and 4 ) , with their genes clustered on chromosome 8 , were known to be expressed by epithelial cells . however , additional -defensins are predicted to be expressed , though their biological functions are not clear . regulation of the expression of -defensin-1 is usually constitutive ; however it can be induced in vitro by stimulation with lps and ifn- , poly i : c , bacterial components , and tnf- and il-1 . -defensin-2 is induced by bacterial products through activation of toll - like receptors ( tlrs ) or by proinflammatory cytokines including tnf- or il-1 . the expression of -defensins may therefore produce an environment whereby inflammation is enhanced , and there is increased vascular permeability . in this way dysregulation of -defensin-1 in severe asthma and copd is likely to be more harmful than beneficial . although the production of -defensins is crucial to proper immune function , altered expression of these molecules may contribute to disease progression . exposure of the epithelium to cigarette smoke , important in the pathogenesis of copd , has been shown to influence the production of -defensins . our study shows that production of -defensin-1 was decreased upon exposure to cse in healthy pbecs ; however , when copd pbecs were exposed , -defensin-1 production was maintained . this abnormal response of pbecs from copd patients may be contributing to the higher level of -defensin-1 seen in the sputum , which may be triggered by exposure to cigarette smoke . other studies have shown that -defensin-1 gene expression is also decreased after cse exposure in a549 cells , a lung alveolar epithelial cell line . further investigation is required to understand the mechanisms causing the abnormal -defensin-1 production in copd . there needs to be further investigation regarding the stimuli responsible for the elevated -defensin-1 levels in copd and the mechanisms involved . given the results of the pbec culture , we would suspect that the epithelial cells are reprogrammed so that their production of -defensin-1 does not alter as it should when exposed to different stimuli . we also did not investigate the relationship of -defensin-1 levels to genotype in this population . future studies will need to confirm the biomarker potential of -defensin-1 in a larger population of subjects with copd , which will also be necessary for detecting associations with disease severity . further studies should also investigate the levels of -defensin-1 in those patients with asthma copd overlap . this study shows that -defensin-1 protein is increased in the airways in copd and severe asthma but not associated with inflammatory phenotype . in fact , -defensin-1 expression was a strong biomarker for predicting copd from both asthma subjects and healthy controls . the level of -defensin-1 was correlated with smoking history and , in vitro , -defensin-1 was decreased upon smoke exposure in epithelial cells from healthy participants but maintained in epithelial cells from participants with copd , suggesting a differential response or tolerance to smoke exposure . this study has identified dysregulated -defensin-1 production , indicating that this protein may be a therapeutic target for copd and severe asthma .
background . 
 innate immune antimicrobial peptides , including -defensin-1 , promote the chemotaxis and activation of several immune cells . 
 the role of -defensin-1 in asthma and chronic obstructive pulmonary disease ( copd ) remains unclear . 
 methods . 
 induced sputum was collected from healthy controls and individuals with asthma or copd . 
 -defensin-1 protein in sputum supernatant was quantified by elisa . 
 biomarker potential was examined using receiver operating characteristic curves . 
 -defensin-1 release from primary bronchial epithelial cells ( pbecs ) was investigated in culture with and without cigarette smoke extract ( cse ) . results . 
 airway -defensin-1 protein was elevated in copd participants compared to asthma participants and healthy controls . 
 inflammatory phenotype had no effect on -defensin-1 levels in asthma or copd . 
 -defensin-1 protein was significantly higher in severe asthma compared to controlled and uncontrolled asthma . 
 -defensin-1 protein could predict the presence of copd from both healthy controls and asthma patients . 
 exposure of pbecs to cse decreased -defensin-1 production in healthy controls ; however in pbecs from copd participants the level of -defensin-1 remanied unchanged . 
 conclusions . 
 elevated -defensin-1 protein is a feature of copd and severe asthma regardless of inflammatory phenotype . 
 -defensin-1 production is dysregulated in the epithelium of patients with copd and may be an effective biomarker and potential therapeutic target .