Am J Physiol Lung Cell Mol Physiol 309: L17–L26, 2015. First published May 15, 2015; doi:10.1152/ajplung.00082.2015.

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Elevated sputum BPIFB1 levels in smokers with chronic obstructive pulmonary disease: a longitudinal study J. Gao,1 S. Ohlmeier,2 P. Nieminen,3 T. Toljamo,4 S. Tiitinen,5 T. Kanerva,1 L. Bingle,6 B. Araujo,6 M. Rönty,7 M. Höyhtyä,5 C. D. Bingle,6 W. Mazur,1 and V. Pulkkinen1 1

HUCH Heart and Lung Center, Department of Pulmonary Medicine, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; 2Proteomics Core Facility, Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland; 3Medical Informatics and Statistics Group, University of Oulu, Oulu, Finland; 4 Department of Pulmonary Medicine, Lapland Central Hospital, Rovaniemi, Finland; 5Medix Biochemica, Kauniainen, Finland; 6Academic Unit of Respiratory Medicine, Department of Infection and Immunity, University of Sheffield, Sheffield, UK; and 7HUSLAB, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland Submitted 13 March 2015; accepted in final form 12 May 2015

Gao J, Ohlmeier S, Nieminen P, Toljamo T, Tiitinen S, Kanerva T, Bingle L, Araujo B, Rönty M, Höyhtyä M, Bingle CD, Mazur W, Pulkkinen V. Elevated sputum BPIFB1 levels in smokers with chronic obstructive pulmonary disease: a longitudinal study. Am J Physiol Lung Cell Mol Physiol 309: L17–L26, 2015. First published May 15, 2015; doi:10.1152/ajplung.00082.2015.—A previous study involving a proteomic screen of induced sputum from smokers and patients with chronic obstructive pulmonary disease (COPD) demonstrated elevated levels of bactericidal/permeability-increasing foldcontaining protein B1 (BPIFB1). The aim of the present study was to further evaluate the association of sputum BPIFB1 levels with smoking and longitudinal changes in lung function in smokers with COPD. Sputum BPIFB1 was characterized by two-dimensional gel electrophoresis and mass spectrometry. The expression of BPIFB1 in COPD was investigated by immunoblotting and immunohistochemistry using sputum and lung tissue samples. BPIFB1 levels were also assessed in induced sputum from nonsmokers (n ⫽ 31), smokers (n ⫽ 169), and patients with COPD (n ⫽ 52) via an ELISA-based method. The longitudinal changes in lung function during the 4-year follow-up period were compared with the baseline sputum BPIFB1 levels. In lung tissue samples, BPIFB1 was localized to regions of goblet cell metaplasia. Secreted and glycosylated BPIFB1 was significantly elevated in the sputum of patients with COPD compared with that of smokers and nonsmokers. Sputum BPIFB1 levels correlated with pack-years and lung function as measured by forced expiratory volume in 1 s (FEV1) % predicted and FEV1/FVC (forced vital capacity) at baseline and after the 4-year follow-up in all participants. The changes in lung function over 4 years were significantly associated with BPIFB1 levels in current smokers with COPD. In conclusion, higher sputum concentrations of BPIFB1 were associated with changes of lung function over time, especially in current smokers with COPD. BPIFB1 may be involved in the pathogenesis of smokingrelated lung diseases. COPD; biomarkers; smoking

(COPD) is one of the leading causes of morbidity and mortality around the world. CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Address for reprint requests and other correspondence: V. Pulkkinen, Biomedicum Helsinki, P.O. Box 63, 00014 Univ. of Helsinki, Helsinki, Finland (e-mail: [email protected]). http://www.ajplung.org

The main risk factor is smoking, which causes persistent inflammation detectable not only in the lung but also in the circulation months or even years after smoking cessation (19, 30). However, it is still not possible to identify individuals who are likely to develop irreversible airflow obstruction (1). Thus more accurate markers and a deeper understanding of the underlying biological mechanisms are required to improve risk prediction and clinical assessment of COPD. Proteomics is a nonbiased screening technique that has been used to investigate various sample types including bronchoalveolar lavage fluid (BALF) (21, 28, 36), induced sputum (10, 14, 23, 24), and lung tissue (17, 26) to identify novel markers for COPD. In recent proteomic studies from our laboratory the major findings included an elevation of surfactant protein-A (SP-A) and a decrease of specific variants of the receptor for advanced glycation end products (RAGE) in lung tissue (25, 26) and polymeric immunoglobulin receptor in sputum samples from patients with COPD (24). Of these, SP-A and soluble RAGE have been validated in longitudinal approaches in larger cohorts (16, 20). Our previous proteomics study also identified elevated levels of bactericidal/permeability-increasing protein fold containing protein B1 (BPIFB1, formerly known as LPLUNC1) in induced sputum from smokers and patients with COPD compared with nonsmokers (24). To date, eleven BPIF/PLUNC genes have been identified in a single locus on chromosome 20, with eight of these generating functional proteins. Proteins that contain a single structural domain have the designation BPIFA and those containing two structural domains have the designation BPIFB. Of these, only BPIFB1 (LPLUNC1) and BPIFA1 (SPLUNC1) are known to be robustly expressed in the lung (3, 5). However, they have distinct expression patterns. BPIFB1 is localized in a population of goblet cells in the nasal passage and airway epithelium and within the serous cells of the airway submucosal glands, whereas BPIFA1 is localized to a population of nonciliated epithelial cells and in the mucous cells of the submucosal glands (4, 6, 7). Both proteins are secreted into the airway lumen and are among the most abundant proteins in respiratory tract secretions (4). BPIFB1 has previously been

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ELEVATED SPUTUM BPIFB1 LEVELS IN SMOKERS WITH COPD

identified in the proteome of nasal mucous (11) and induced sputum (10, 22, 24), and elevated levels of the protein were detected in a proteomic study of BALF from asthmatic patients after segmental allergen challenge (39). BPIFB1 expression is increased in advanced cystic fibrosis (8) and it is also robustly expressed in the bronchiolized epithelium lining the honeycomb cysts in usual interstitial pneumonia (2). BPIFA1 is the best characterized member of the PLUNC family and previous studies have identified multiple protective roles against respiratory infections and inflammation (9). Data on the function of BPIFB1 are much more limited but it has been hypothesized to also function in the innate defense of the airways. The expression and function of BPIFB1 in smoking-related diseases, such as COPD, is an understudied subject and thus very little is known. We hypothesized that BPIFB1 could participate in the development of COPD and thus our aim was to characterize the expression of BPIFB1 and BPIFA1 in COPD using sputum and lung tissue samples. Because of the lack of commercially available ELISA kits for BPIFB1, another aim was to develop a method to reliably measure BPIFB1 levels in sputum and then validate this marker in a larger cohort of smokers with and without COPD as well as to evaluate the association of BPIFB1 with smoking and changes of lung function during a 4-year follow-up. MATERIALS AND METHODS

Characteristics of participants in the sputum sample studies. The participants were part of a longitudinal study, of a cohort of smokers

and nonsmokers, being conducted in northern Finland. The details of the project and the inclusion and exclusion criteria have been previously published (16, 20, 34, 35). In brief, the exclusion criteria consisted of chronic pulmonary or other diseases requiring regular medication, allergies, and risk factors for other pulmonary diseases, e.g., known exposures, bronchiectasis, malignancies, and also previous lung tuberculosis. The presence of any lung infections during the last 2 mo before entering the study was also an exclusion criterion (35). According to a detailed self-reported questionnaire, all participants considered themselves healthy. The smokers had a cigarette smoking history of ⱖ10 years (Table 1). The flow-volume spirometry was conducted both before and after bronchodilation (BD) with 0.4 mg salbutamol (pre- and post-BD spirometry, respectively). COPD was classified according to the international GOLD 2007 recommendations based on forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC), namely FEV1/FVC ⬍ 0.70. The participants fulfilling the COPD criteria were subdivided into severities by using the BD-FEV1 level expressed as a percentage of the predicted value (37, 38). All COPD diagnoses in the study cohort were confirmed during the study period and none of the participants had any previously prescribed medications for COPD or other diseases. The nonsmoking study participants (nonsmokers) were enrolled if they were ⬎40 years of age, healthy, not taking any medications, and had normal lung function according to the GOLD criteria for obstruction described above. The first visit included individualized smoking counselling by Motivational Interviewing. Two years later, smoking status was assessed and baseline BD spirometry, plasma and sputum samples were collected during 2007–2008 (16, 20, 35). Sputum was processed by using the sputum plug method as described (27, 31). Only represen-

Table 1. Baseline clinical characteristics and longitudinal changes in lung function after the 4-yr follow-up period in participants of the sputum sample study All Baseline

Subjects, n 252 Sex, male/female 143/109 Age, yr 53.9 (9.3) BMI, kg/m2 27.2 (3.9) Smoking status Ex-smoker, n (%) 50 (19.8) Current smoker, n (%) 171 (67.9) Pack-years 31.2 (14.3) Symptoms, n (%) No symptoms 101 (40.1) Only cough 13 (5.2) Only sputum 51 (20.2) Cough and sputum 87 (34.5) Postbronchodilator FVC, liters 4.0 (0.9) FVC % predicted 96.9 (12.4) 3.1 (0.8) FEV1, liters FEV1 % predicted 92.9 (16.9) FEV1/FVC % 78.0 (10.1) Changed lung function ⌬FVC, liters ⌬FVC % predicted ⌬FEV1, liters ⌬FEV1 % predicted ⌬FEV1/FVC % Differential sputum cell counts % Neutrophils 33.2 (20.1) Macrophages 24.4 (20.7)

Nonsmokers

Smokers without COPD

Smokers with COPD

P Value

Follow-up

Baseline

Follow-up

Baseline

Follow-up

Baseline

Follow-up

Baseline/Follow-up

246 138/108 57.4 (9.2) 27.7 (4.1)

31 10/21 55.8 (9.3) 26.8 (4.0)

31 10/21 58.4 (8.5) 27.4 (4.3)

169 88/81 52.1 (8.9) 27.2 (3.9)

145 77/68 54.9 (8.5) 27.7 (4.1)

52 45/7 58.7 (9.0) 27.1 (3.9)

70 51/19 62.2 (9.0) 27.7 (4.1)

⬍0.001/⬍0.001* ⬍0.001/⬍0.001† 0.866/0.900†

53 (21.5) 162 (66.0) 30.4 (18.9)

0 0 0

0 0 0

39 (23.1) 130 (76.9) 29.0 (13.8)

33 (22.8) 112 (77.2) 32.2 (15.4)

11 (21.2) 41 (78.8) 38.6 (13.5)

20 (28.6) 50 (71.4) 40.1 (15.7)

0.772/0.399* ⬍0.001/⬍0.001‡

121 (49.2) 57 (23.2) 21 (8.5) 47 (19.1)

21 (67.7) 1 (3.2) 5 (16.1) 4 (12.9)

27 (87.1) 3 (9.7) 1 (3.2) 0

63 (37.2) 9 (5.3) 40 (23.7) 57 (33.7)

68 (46.9) 31 (21.4) 17 (11.7) 29 (20.0)

17 (32.1) 3 (5.8) 6 (11.5) 26 (50.0)

26 (37.1) 23 (32.9) 3 (4.3) 18 (25.7)

0.006/⬍0.001*

3.8 (0.9) 96.3 (12.7) 2.8 (0.8) 87.7 (17.1) 73.6 (10.6)

3.8 (0.8) 104.1 (13.2) 3.2 (0.6) 107.5 (14.5) 84.5 (5.3)

3.6 (0.8) 102.3 (13.5) 2.9 (0.7) 103.4 (13.1) 81.9 (4.4)

4.0 (1.0) 96.0 (11.8) 3.2 (0.8) 96.3 (12.4) 81.6 (5.4)

3.9 (0.9) 95.9 (11.3) 3.0 (0.7) 92.5 (11.6) 76.3 (7.1)

4.2 (0.9) 96.0 (12.7) 2.6 (0.7) 73.7 (15.2) 62.2 (8.3)

3.8 (0.9) 95.0 (14.2) 2.3 (0.7) 70.8 (15.2) 58.1 (9.3)

0.183/0.312† 0.003/⬍0.018† ⬍0.001/⬍0.001† ⬍0.001/⬍0.001† ⬍0.001/⬍0.001†

⫺0.2 (0.3) ⫺0.7 (7.3) ⫺0.3 (0.2) ⫺6.2 (7.5) ⫺4.9 (5.2) n.a. n.a.

⫺0.2 (0.3) ⫺1.6 (5.8) ⫺0.2 (0.2) ⫺4.2 (6.3) ⫺2.5 (4.9) 22.3 (13.2) 23.0 (19.3)

n.a. n.a.

⫺0.2 (0.3) ⫺0.7 (6.7) ⫺0.3 (0.2) ⫺5.2 (6.9) ⫺4.0 (4.0) 32.3 (19.9) 23.8 (20.8)

n.a. n.a.

42.5 (20.6) 26.8 (21.3)

⫺0.2 (0.4) ⫺0.4 (8.8) ⫺0.4 (0.3) ⫺9.1 (8.3) ⫺7.6 (6.3)

0.996† 0.747† 0.003† ⬍0.001† ⬍0.001†

n.a. n.a.

⬍0.001† 0.617†

Values are means ⫾ SD unless stated otherwise, and P values were compared across all subgroups at baseline and follow-up, respectively. COPD, chronic obstructive pulmonary disease; BMI, body mass index; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s; ⌬, change. *␹2 test, †ANOVA F-test, ‡independent sample t-test; n.a., not available. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00082.2015 • www.ajplung.org

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ELEVATED SPUTUM BPIFB1 LEVELS IN SMOKERS WITH COPD

tative sputum samples containing less than 70% squamous epithelial cells were accepted. Follow-up BD spirometries were conducted 4 years later, from 2011 to 2012; 295 participants had baseline samples, baseline spirometry, and follow-up spirometry. Of these, representative sputum samples were obtained from 252 participants for ELISA measurements. In addition, 14 samples from each study group (nonsmokers, smokers, and COPD patients) were randomly selected for immunoblotting analyses. Postbronchodilation values were used for the assessment of longitudinal change of lung function. The study was approved by the Ethics Committee of Lapland Central Hospital (4th June 2003 and 31st October 2006), and all participants provided written, informed consent. Characteristics of participants in the lung tissue sample studies. Lung tissue specimens for immunoblotting and for immunohistochemistry were retrieved from the patients of Helsinki University Central Hospital (15, 24). Tissue specimens were obtained from lung surgery from hamartomas or from the surgery for local tumors (controls), or from lung transplantations (COPD Stage IV and idiopathic pulmonary fibrosis, IPF). The smokers with and without COPD had a cigarette smoking history of ⱖ10 years whereas the four IPF patients had no pack-years (Table 2). The study was approved by ethical committee of Helsinki University Central Hospital, and all participants provided written, informed consent. Identification of BPIFB1 by proteomics. Identification of BPIFB1 by two-dimensional (2D) difference gel electrophoresis (DIGE) and mass spectrometry (MS) using proteins (5 ␮g) extracted from purified sputum from nonsmokers (n ⫽ 7), smokers without COPD (n ⫽ 7), and smokers with COPD (Stage II, n ⫽ 7) has previously been described (24). Western blot analysis. Western blot analyses of lung tissue homogenates from nonsmokers (n ⫽ 14), smokers without COPD (n ⫽ 14), and smokers with COPD (n ⫽ 14) were performed as described (15). For Western blotting, equal volumes of the sputum supernatants (n ⫽ 14/group) were loaded. Ponceau S staining was used to standardize the loading of the sputum and lung homogenates (25) to avoid problems with the conventional loading markers in COPD (10, 13, 29). The samples were run as two sets of samples on the gels: nonsmokers (n ⫽ 7) vs. smokers (n ⫽ 7) and nonsmokers (n ⫽ 7) vs. COPD (n ⫽ 7). The mean value of relative expression in nonsmokers was set to a value of 1, and the results were combined after two different rounds of Western blots. The polyclonal BPIFB1-1 and BPIFB1-2 antibodies have been characterized previously (4) and BPIFA1 (MAB1897) monoclonal antibodies were purchased from R&D Systems. Deglycosylation by PNGase F. Sputum supernatants from two smokers were studied to detect possible glycosylation. The samples were incubated with glycoprotein denaturing buffer at 100°C for 10

min before being treated with reaction buffer, Nonidet P-40, and PNGase F and incubated at 37°C for 1 h according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA). Western blots of the treated and untreated samples were carried out as described. BPIFB1 ELISA. BPIFB1-1 and BPIFB1-2 antibodies against C⫹AQATIRMDTSASGPTRLVLS (corresponding to residues 137– 156) and C⫹KDALVLTPASLWKPSSPVSQ (corresponding to residues 465– 484), respectively, were produced in rabbits by Genscript (Piscataway, NJ). These represent the same epitopes used previously to generate species-specific BPIFB1 antibodies (4). The antibodies were purified by affinity chromatography and the rabbit antiBPIFB1-2 antibody was biotinylated with Lightning Link kit (7040010, Innova Biosciences) according to the manufacturer’s instructions. The anti-BPIFB1-1 antibody, 1 ␮g/ml, was captured on microtiter plates (MaxiSorp NUNC immuno module, Thermo Scientific, 468667) overnight at ⫹4°C. Nonspecific binding sites were blocked with 1% BSA-PBS for 2 h at room temperature. Standards (recombinant human BPIFB1, 13275-H08H, Sino Biological) and samples were diluted 1:20 in assay buffer or furthermore 1:200 if concentration exceeded the detection range and incubated as duplicates for 1 h with gentle shaking. Bound BPIFB1 was detected with biotinylated rabbit anti-BPIFB1-2 and Eu-labeled streptavidin (1244-360, Perkin Elmer, Finland). Finally, the Delfia enhancement solution (118-100, Perkin Elmer) was added and the fluorescence of Europium was measured with an EnVision Xcite fluorometer (Perkin Elmer). Immunohistochemistry. Serial sections (4 ␮m) were cut from formalin-fixed and paraffin-embedded tissue and stained as previously described (4, 5, 7). The following antibodies were used in this study: rabbit polyclonal antibodies against BPIFB1 (final dilution of 1:600) (4) and rabbit polyclonal antibodies against MUC5B (SC-20119, Santa Cruz Biotechnology, final dilution 1:500). A standard antigenretrieval procedure using trisodium citrate in a microwave for 8 min was used for both antibodies. Sections were incubated with 100% normal goat serum at room temperature for 30 min and then at 4°C overnight with the primary antibodies diluted as indicated above with 100% normal serum. Rabbit IgG (DAKO) was used as a negative control on replicate slides. A Vectastain Elite ABC kit (Vector Laboratories) containing an anti-rabbit biotin-labeled secondary antibody was used according to the manufacturer’s instructions. Peroxidase enzymatic development was performed by use of a Vector NovaRed substrate kit, resulting in red staining in positive cells. Sections were counterstained with hematoxylin, dehydrated to xylene, and mounted in DPX. Alcian blue staining of acidic mucins was performed by a standard histological method.

Table 2. Clinical characteristics of the participants examined in the lung tissue studies Sample Set 1 Nonsmokers

Subjects, n Sex, male/female Age, yr BMI, kg/m2 Smoking status Ex-smoker, n (%) Current smoker, n (%) Pack-years Postbronchodilator FVC, liters FEV1, liters FEV1/FVC %

14 7/7 64.3 (12.5) 26.2 (6.6) 3 (21%) 0 2.9 (0.8) 3.0 (0.7) 81.9 (10.9)

Smokers without COPD

Sample Set 2

Smokers with COPD

14 9/5 60.7 (8.2) 26.5 (7.1)

14 7/7 61.4 (5.4) 27.4 (10.0)

1 (7%) 13 (93%) 30.0 (15.4)

11 (79%) 39.7 (9.3)

3.2 (0.9) 3.3 (0.9) 80.4 (6.0)

1.5 (0.6) 1.8 (1.5) 44.4 (15.6)

P value

0.537* 0.557† 0.979†

Nonsmokers

IPF

4 0/4 69.8 (7.3) n.a.

4 4/0 55.8 (13.4) n.a.

5 3/2 59.6 (4.5) n.a.

P value

0.782* 0.114†

3 (60%) 1 (20%) 27.0 (11.5)

0.128‡,§ ⬍0.001† 0.004† ⬍0.001†

Smokers with COPD

2.7 (0.2) 2.4 (0.5) 83.0 (13.5)

2.3 (1.2) 1.6 (0.3) 76.7 (21.4)

2.6 (1.0) 1.0 (0.5) 36.3 (11.1)

0.818† 0.006† 0.009†

Values are means ⫾ SD unless stated otherwise, and P values were compared across subgroups in sample set 1 and set 2, respectively. *␹2 test, †ANOVA F-test, ‡independent sample t-test, §comparison between smokers and COPD. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00082.2015 • www.ajplung.org

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ELEVATED SPUTUM BPIFB1 LEVELS IN SMOKERS WITH COPD

Statistical analysis. The distribution of basic subject characteristics is summarized by mean and standard deviation (SD) values or with percentages. Intra-assay coefficient of variation (CV) of the ELISA assay was calculated by averaging CV % values across CV % values for all samples within one assay, and interassay CV % were calculated by averaging the CV % values of the controls between the different plates. Because of the skew of the distribution of BPIFB1 the median values with interquartiles are reported for BPIFB1 measurements. Comparison between participant characteristics and sputum BPIFB1 among the study groups was evaluated by the Kruskal-Wallis test followed by a Mann-Whitney U-test, when appropriate. Spearman’s rank correlation was used to evaluate the associations of sputum BPIFB1 concentrations with other variables. IBM SPSS Statistics version 21 (SPSS) and GraphPad Prism version 6.00 (GraphPad Software) were used for the statistical analyses.

The baseline demographic and clinical characteristics of participants examined in the lung tissue studies are shown in Table 2. The mean age and BMI were similar in comparisons between nonsmokers, smokers without COPD, and smokers with COPD. By definition post-BD FEV1/FVC (%) was reduced in COPD (⬍70%) and it was markedly lower than in nonsmokers and smokers. The four IPF patients had normal post-BD FEV1/FVC, i.e., mean value ⬎70% (Table 2). Secreted and glycosylated BPIFB1 isoform 1 is elevated in sputum of smokers with and without COPD. We have previously observed elevated BPIFB1 levels in the sputum of smokers with and without COPD (24). The theoretical molecular weight of BPIFB1 according to the protein sequence is 52 kDa. In the 2D gel sputum BPIFB1 was identified in two spots (Fig. 1A) with similar expression profiles, indicating that both spots are regulated by the same mechanisms. The localization of both spots at 54 kDa (Fig. 1A) suggests the presence of full-length BPIFB1 isoform 1. Further MS analyses with specific peptides revealed that isoform 1 was indeed present in both spots (Fig. 1B). Reproducible sequence coverages were obtained for amino acids 22 to 465 whereas none of the detected peptides covered the NH2-terminal signal peptide (amino acids 1–21). This suggests the presence of secreted BPIFB1 in sputum. Another mechanism that could affect the molecular weight of BPIFB1 is N-linked glycosylation, which was further investigated by PNGase F treatment and immunoblotting. The shift of the 55-kDa band to a lower molecular weight after treatment

RESULTS

Participant characteristics. The baseline demographic and clinical characteristics of participants of the sputum sample study in each of the study groups (nonsmokers, smokers without COPD, and smokers with COPD) are shown in Table 1. The smokers without COPD were significantly younger than the nonsmokers and the smokers with COPD. There were no significant differences in body mass index (BMI) among the three groups. By definition the smokers with COPD had a post-BD FEV1/FVC ⬍70% and their FEV1 was lower than in the other two groups with normal BD spirometries. However, post-BD FEV1/FVC was lower in smokers without COPD than in nonsmokers. pH 4

7

A

kDa 100 80 70 60 50

B 90 80

40

BPIFB1

NS S C 1

NS S C 2

20

70

% Intensity

30

3.6E+4

100

60

*

50 40 30 20

1

2

10

10

0 767.0

1506.6

2246.2

2985.8

3725.4

0 4465.0

Mass (m/z)

C

PNGase F 70 kDa 55 kDa 35 kDa

+

SP 1 21 Gly

Gly

Gly

484

* 22

Matched peptides

465

Fig. 1. Secreted bactericidal/permeability-increasing fold-containing protein B1 (BPIFB1) is upregulated in sputum of smokers without and with chronic obstructive pulmonary disease (COPD). A: representative 2D gel of sputum [5 ␮g protein labeled by “saturation DIGE” (difference gel electrophoresis)] characteristic for a COPD patient (Stage II). The enlarged gel part shows the position of both changed BPIFB1 spots. The expression profiles of both spots indicate the detected protein levels in nonsmokers (NS), smokers (S), and COPD patients (C). B: representative mass spectrum (MS) of the changed BPIFB1. Protein-specific peptides are marked with gray arrows that match to both isoforms and black arrows that match only to isoform 1. The schematic presentation of BPIFB1 shows the signal peptide (SP), glycosylation sites (Gly) and the sequence coverage of the spot-specific peptides. The NH2-terminal peptide of mature BPIFB1 is marked by an asterisk. As an example mass spectrum and matched peptides are presented for spot 1. MS scores, sequence coverage, and matched peptides, respectively, 225.46, 60%, 23 peptides for spot 1 and 153.87, 49%, 18 peptides for spot 2. C: representative blot of detection of BPIFB1 in a sputum sample from a smoker by Western blot with (⫹) and without (⫺) PNGase F treatment for deglycosylation. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00082.2015 • www.ajplung.org

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by the heterogeneity of the lung tissue due to the presence of different cell types. In sputum, BPIFB1 was detected as a doublet band at around 55 kDa by immunoblotting (Fig. 2, C and D). This indicates the presence of both glycosylated and nonglycosylated secreted BPIFB1. Image analysis of both bands indicated a 1.5-fold increase in BPIFB1 levels in COPD compared with nonsmokers (P ⬍ 0.05) and smokers (P ⫽ 0.006) (Fig. 2D). In lung tissue homogenates, BPIFB1 was detected by Western blot exclusively in one band at 54 kDa (Fig. 2, C and E). Comparison of the study groups revealed 1.5-fold higher BPIFB1 levels in smokers with COPD (P ⫽ 0.006) and 1.3-fold higher levels in smokers without COPD (P ⫽ 0.167) compared with those of nonsmokers (Fig. 2E). We also compared another set of COPD samples with lung tissue lysates from IPF patients and from nonsmokers. Again, there was a trend toward higher levels of lung BPIFB1 in COPD compared with nonsmokers (P ⫽ 0.016) and IPF (P ⫽ 0.064) (Fig. 2F). These experiments were repeated with polyclonal antibodies against BPIFA1 and polyclonal antibodies detecting another epitope in BPIFB1 (4). The results were similar to those described above (data not shown). Sputum BPIFB1 levels are smoking dependent and elevated in COPD. Sputum BPIFB1 was further investigated in a larger cohort (n ⫽ 252) to validate the results and to verify whether the elevation observed in COPD is smoking related. Since no commercial ELISA kit was available, antibodies were raised

revealed the presence of glycosylated BPIFB1 in sputum (Fig. 1C). The observed separation of both BPIFB1 spots at a similar molecular weight but with different charges indicates the presence of differentially phosphorylated BPIFB1. Indeed, several putative phosphorylation sites have been identified in BPIFB1 (http://www.phosphosite.org); however, the similar expression profiles point to a change of protein levels in general rather than changes in phosphorylation or glycosylation. Altogether this indicates that the upregulated BPIFB1 in smokers and subjects with COPD is the secreted BPIFB1 isoform 1 and that this upregulation is likely the result of either higher expression or processing. Expression of BPIFBA1 and BPIFB1 in induced sputum and lung tissue. BPIFA1 and BPIFB1 expression was further investigated in sputum and lung tissue by immunoblotting using commercial monoclonal antibodies against BPIFA1 and polyclonal antibodies against BPIFB1 (4). BPIFA1 was detected as a single band at 26 kDa in nonsmokers, smokers, and patients with COPD, which, since the theoretical molecular weights of the intact and processed protein are 26.7 and 24.7 kDa, respectively, indicates the presence of secreted protein. In a subset of sputum samples randomly selected for analysis there was a trend toward higher expression of BPIFBA1 in smokers and patients with COPD although the difference was not statistically significant compared with nonsmokers (Fig. 2A). In lung tissue, there were no differences between nonsmokers, smokers, and patients with COPD (Fig. 2B). This might be explained

A

B

C Non-smoker

Sputum

2.5

26

BPIFA1

2.0

55

BPIFB1

Lung BPIFA1

Relative units

Relative units

4 3 2 1

1.5

Lung

1.0 0.5

26

BPIFA1

55

BPIFB1

0.0

0

NS

D

S

COPD

NS

E

Sputum BPIFB1

**

2 1 0

F

2.0 1.5 1.0 0.5

S

COPD

*

2

1

0

0.0

NS

Lung BPIFB1 3

Relative units

3

COPD

*

2.5

Relative units

4

S

Lung BPIFB1

* Relative units

COPD

kDa

Sputum BPIFA1

NS

S

COPD

NS

IPF

COPD

Fig. 2. Detection of bactericidal/permeability-increasing fold-containing protein A1 (BPIFA1) and BPIFB1 in sputum and lung tissue samples. Immunoblotting of sputum (A) and lung tissue samples (B) from nonsmokers (NS, n ⫽ 14), smokers (S, n ⫽ 14), and patients with COPD (COPD, n ⫽ 14) with monoclonal antibodies against BPIFA1. C: representative blots of BPIFA1 and BPIFB1 in sputum and lung tissue samples from nonsmokers (n ⫽ 7) and COPD patients (n ⫽ 7). Immunoblotting of sputum (D) and lung tissue samples (E) from nonsmokers (n ⫽ 14), smokers (n ⫽ 14), and patients with COPD (n ⫽ 14) with polyclonal antibodies against BPIFB1. F: Western blot of lung tissue homogenates from nonsmokers (n ⫽ 4) and patients with idiopathic pulmonary fibrosis (IPF, n ⫽ 4) and COPD (n ⫽ 5) with antibodies against BPIFB1. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00082.2015 • www.ajplung.org

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ELEVATED SPUTUM BPIFB1 LEVELS IN SMOKERS WITH COPD

A

B

4000

4000

***

*** BPIFB1 (µg/mL)

Fig. 3. Sputum levels of BPIFB1 measured by a newly developed method using an ELISAbased assay. A: BPIFB1 levels in sputum samples from nonsmokers (n ⫽ 31), smokers (n ⫽ 169), and smokers with COPD (n ⫽ 52) at baseline. B: effects of smoking on BPIFB1 levels in the smoker and COPD groups stratified according to their smoking status into current smokers (n ⫽ 130 and n ⫽ 41, respectively) and ex-smokers (n ⫽ 39 and n ⫽ 11, respectively). Boxes represent the 25th to 75th percentiles and the solid lines within the boxes show the median values; whiskers are 10th and 90th percentiles; and points represent outliers. **P ⬍ 0.01 and ***P ⬍ 0.001.

Current smokers Ex-smokers

**

3000

3000

2000

2000

1000

1000

0

0 non-smokers

smokers

against two distinct sequences in BPIFB1 (4) and both were used in establishing a functional ELISA to detect levels of BPIFB1 in sputum. The limit of detection for the assay was 3,661 counts per second, corresponding to the lowest standard used (312 ng/ml). The detection range of the assay was 0.5–20 ␮g/ml. The intra-assay CV % was ⱕ13% and the interassay CV % was ⱕ18%. The baseline sputum levels of BPIFB1 (median, interquartile range) were significantly higher in smokers with COPD (332.9, 128.6 – 673.3 ␮g/ml) than in smokers without COPD (167.4, 24.2– 488.7 ␮g/ml, P ⫽ 0.007) and in nonsmokers (28.9, 3.8 –118.4 ␮g/ml, P ⬍ 0.001) (Fig. 3A). In addition, the sputum levels of BPIFB1 were significantly higher in smokers without COPD than in nonsmokers (P ⬍ 0.001). To further investigate the effects of smoking on BPIFB1 levels, the smoker and COPD groups were divided into current smokers and ex-smokers according to their smoking status at baseline. The levels of BPIFB1 were significantly higher in COPD patients who continued smoking compared with the levels of current smokers without COPD (P ⫽ 0.004), whereas there were no significant differences in BPIFB1 levels between ex-smokers with and without COPD (Fig. 3B).

**

COPD

smokers

COPD

Correlations between BPIFB1 and characteristics at baseline and after a 4-year follow-up. Sputum BPIFB1 levels at baseline correlated with BMI, pack-years, FEV1 % predicted, and FEV1/FVC % in all participants (Table 3). In a subgroup analysis, there were significant correlations between sputum BPIFB1 concentrations and BMI in nonsmokers and smokers. Interestingly, BPIFB1 levels correlated with pack-years only in smokers with COPD whereas there was no correlation in smokers without COPD. There was no significant correlation between the presence of chronic cough and/or sputum at the baseline visit and the sputum levels of BPIFB1 in any of the subjects or among any of the study groups. The levels of BPIFB1 were similar in male and female subjects, and there were no significant sex differences in sputum BPIFB1 levels within the study groups (data not shown). We also analyzed the correlation of baseline BPIFB1 levels with demographics after the 4-year follow-up period. Details of six participants were lacking in the follow-up; one had moved away from the region and five had died because of cancer (n ⫽ 1), coronary artery disease (n ⫽ 3), or other reasons (n ⫽ 1). Similar to baseline, sputum BPIFB1 levels correlated with packyears, FEV1 % predicted, and FEV1/FVC after the 4-year fol-

Table 3. Correlation of sputum BPIFB1 with demographics at baseline and with demographics after the 4-yr follow-up All

Baseline (n ⫽ 252) Age BMI Pack-years FVC, liters FVC % predicted FEV1, liters FEV1 % predicted FEV1/FVC % Neutrophils % Follow-up (n ⫽ 246) Pack-years FVC, liters FVC % predicted FEV1, liters FEV1 % predicted FEV1/FVC %

Nonsmokers

r

P value

0.050 0.211 0.154 0.059 ⫺0.098 ⫺0.025 ⫺0.174 ⫺0.158 0.093

0.431 0.001 0.022 0.352 0.122 0.693 0.006 0.012 0.138

0.265 0.042 ⫺0.106 ⫺0.033 ⫺0.169 ⫺0.128

⬍0.0001 0.513 0.096 0.612 0.008 0.046

r

Smokers without COPD

Smokers with COPD

P value

r

P value

r

P value

0.345 0.391

0.057 0.030

⫺0.246 ⫺0.014 ⫺0.175 ⫺0.029 0.062 0.426

0.182 0.943 0.347 0.883 0.739 0.017

⫺0.063 0.228 0.012 0.079 ⫺0.015 0.089 ⫺0.014 0.026 ⫺0.083

0.412 0.003 0.878 0.310 0.846 0.248 0.852 0.739 0.281

0.199 0.125 0.446 ⫺0.09 ⫺0.189 ⫺0.085 ⫺0.155 0.061 0.219

0.157 0.377 0.001 0.524 0.179 0.548 0.273 0.669 0.119

⫺0.219 ⫺0.053 ⫺0.192 ⫺0.046 ⫺0.002

0.236 0.776 0.301 0.807 0.991

0.007 0.091 0.005 0.126 0.052 0.097

0.934 0.274 0.952 0.130 0.532 0.246

0.410 ⫺0.074 ⫺0.250 ⫺0.142 ⫺0.294 ⫺0.199

⬍0.0001 0.542 0.037 0.240 0.013 0.099

BPIFB1, bactericidal/permability-increasing fold-containing protein B1. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00082.2015 • www.ajplung.org

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ELEVATED SPUTUM BPIFB1 LEVELS IN SMOKERS WITH COPD

B 20 10 0 -10 -20 - Smokers with COPD r=-0.480 p-value=0.001

-30 -40 0

1000

2000

3000

4000

Change of FEV1/FVC during 4 years (%)

Change of FEV1 % during 4 years (%)

A

20 10 0 -10 -20 - Smokers with COPD r=-0.367 p-value=0.012

-30

Fig. 4. A: relationship between sputum BPIFB1 levels at baseline and the change of forced expiratory volume in 1 s (FEV1) % during a 4-year follow-up in smokers with COPD. B: relationship between sputum BPIFB1 levels at baseline and the change of FEV1-to forced vital capacity (FVC) ratio during a 4-year follow-up in smokers with COPD.

-40 0

1000

Baseline BPIFB1 (µg/mL)

2000

3000

4000

Baseline BPIFB1 (µg/mL)

low-up in all participants (Table 3). In subgroup analysis, BPIFB1 concentration was significantly associated with FVC % predicted and FEV1 % predicted in smokers with COPD. Correlation between elevated BPIFB1 and changes of lung function over a 4-year follow-up period. No correlation was found between sputum BPIFB1 concentration and longitudinal changes (⌬) of FEV1 in all participants, nonsmokers, smokers without COPD, and smokers with COPD during the 4-year study period (data not shown). Importantly, a subgroup analysis showed that a higher BPIFB1 concentration was significantly associated with a longitudinal change in FEV1 (P ⫽ 0.003) and FEV1 % predicted (P ⫽ 0.001; Fig. 4A) and a longitudinal decline of FEV1/FVC (⌬FEV1/FVC %) in smokers with COPD (P ⫽ 0.012; Fig. 4B). To further explore this association, the levels of BPIFB1 in smokers with and without COPD were stratified according to their smoking status into current smokers and ex-smokers and correlated with longitudinal changes in lung function over the 4-year study period. Most importantly, BPIFB1 levels correlated with the longitudinal changes in lung function (⌬FEV1, ⌬FEV1 % predicted, and ⌬FEV1/FVC %) only in current smokers with COPD. These correlations were not observed in other study groups (Table 4). However, an inverse correlation between BPIFB1 levels and changes in lung function (⌬FVC and ⌬FVC % predicted) was observed in ex-smokers with COPD. BPIFB1 staining is elevated in remodeled airway epithelium in severe COPD. We used immunohistochemistry to complement the quantitative data and to provide some information on the potential source of the production of BPIFB1. Consistent

with our previous reports (4), sections taken from patients without COPD and who had no smoking history showed limited staining for BPIFB1 throughout the sections (Fig. 5A). Higher magnification images clearly demonstrate that the epithelium of the airways is negative for staining due to a relative lack of goblet cells in these sections (Fig. 5B). A serial section stained with the goblet cell marker MUC5B was also negative (Fig. 5C). In contrast, in patients with severe COPD, strong staining of BPIFB1 was seen in the airways (Fig. 5, D–F), where it appeared to be associated with cells that were also strongly positive for the goblet cell marker MUC5B (Fig. 5, G–I). Importantly, staining was never seen in peripheral lung tissue or within the subepithelial and fibrotic regions. DISCUSSION

Studies on BPIFB1 are very limited and levels of BPIFB1 in COPD have not been previously analyzed. In this study we carefully characterized sputum levels of BPIFB1 in nonsmokers, smokers without COPD, and smokers with COPD and subsequently examined the association between BPIFB1 and airflow limitation by using baseline demographic data and the longitudinal decline of lung function during a 4-year follow-up period. This study represents the first quantitative analysis of BPIFB1 in human lung disease. Baseline sputum BPIFB1 levels were significantly higher in smokers with COPD than in nonsmokers and smokers without COPD. Moreover, sputum BPIFB1 concentrations were significantly associated with longitudinal declines of lung function in patients with COPD.

Table 4. Correlation of baseline sputum BPIFB1 with the longitudinal changes in lung function over the 4-yr follow-up period in smokers with and without COPD stratified according to their smoking status Smokers without COPD Current smokers (n ⫽ 130)

⌬FVC, liters ⌬FVC % predicted ⌬FEV1, liters ⌬FEV1 % predicted ⌬FEV1/FVC %

Smokers with COPD

Ex-smokers (n ⫽ 39)

Current smokers (n ⫽ 41)

Ex-smokers (n ⫽ 11)

r

P value

r

P value

r

P value

r

P value

0.032 0.026 0.120 0.095 0.075

0.716 0.767 0.173 0.281 0.399

⫺0.194 ⫺0.176 ⫺0.074 ⫺0.076 0.204

0.237 0.284 0.656 0.647 0.214

0.031 0.047 ⫺0.375 ⫺0.451 ⫺0.541

0.861 0.789 0.026 0.007 0.001

⫺0.678 ⫺0.808 ⫺0.538 ⫺0.524 ⫺0.073

0.022 0.003 0.088 0.098 0.831

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00082.2015 • www.ajplung.org

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ELEVATED SPUTUM BPIFB1 LEVELS IN SMOKERS WITH COPD

A BPIFB1

B BPIFB1

500 µm

C MUC5B

100 µm

E BPIFB1

D BPIFB1

500 µm

G MUC5B

100 µm

F BPIFB1

100 µm

H MUC5B

500 µm

100 µm

I MUC5B

100 µm

100 µm

Fig. 5. Localization of BPIFB1 in the airway epithelium. Immunohistochemistry for BPIFB1 (A, B, D, E, F) and MUC5B (C, G, H, I) was performed as described in MATERIALS AND METHODS. Sections show staining of representative samples from lung sections taken from a nonsmoker (A–C) and from a patient with severe COPD (D–I). Scale bars are present on each individual panel.

Finally, we showed that BPIFB1 is localized to regions of goblet cell metaplasia. In addition to our previous proteomic study of COPD patients (24), elevated levels of BPIFB1 have been discovered in proteomic screening for other respiratory diseases, such as in the lung tissue of patients with cystic fibrosis (8), in sputum from patients with severe uncontrolled asthma (18), and in BALF from asthma patients after segmental allergen challenge (39). It is likely that sputum BPIFB1 levels reflect the homeostasis of the respiratory epithelium and may be upregulated in COPD as a result of smoking, chronic infection, and enhanced immune responses. We have previously shown that BPIFB1 is found in a population of airway goblet cells (4). It is therefore not surprising that sputum BPIFB1 levels are elevated in more severe COPD where ciliated epithelial cells are replaced by goblet cells as a physiological response to increased airway irritation. Our data clearly confirm that BPIFB1 staining is markedly increased in the COPD airways compared with normal tissue. The localization of the protein to regions enriched in MUC5B staining suggests that it is associated with goblet cells. These cells are largely absent in normal lung. It is important to note that BPIFB1 staining is restricted to the

airway epithelium, suggesting that the precise spatial expression of the protein is maintained in severe disease. These data are also consistent with our previous reports of elevated staining of BPIFB1 in goblet cells in the airways of patients with cystic fibrosis (8) and IPF (2). Presence of the glycosylated form of BPIFB1 in sputum is in agreement with the suggested function of BPIFB1 in airway defense, since effective clearance of mucus requires a balance of sputum volume and viscoelasticity. With one of the suggested functions of BPIFB1 being associated with antimicrobial activity, it would be interesting to correlate the expression of BPIFB1 with the microbiome of the lung. Although the data on the expression of BPIFB1 in COPD are extremely limited and are largely confined to proteomic studies, BPIFA1 expression has been studied in greater detail. Lung biopsies from four subjects with uncharacterized COPD had higher BPIFA1 mRNA and protein expression levels than those from healthy controls (12). In airway epithelial cells, BPIFA1 was detected only in current smokers but not in nonsmokers (33), and a MS-based study identified BPIFA1 only in sputum of patients with COPD but not in nonsmokers, smokers, or patients with chronic bronchitis (10). A gel-based

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00082.2015 • www.ajplung.org

ELEVATED SPUTUM BPIFB1 LEVELS IN SMOKERS WITH COPD

proteomic study on sputum did, however, detect BPIFA1 in smokers as well as patients with COPD, indicating slightly elevated protein levels in COPD (23). These contradictory results suggest that systematic studies are required to clarify the levels of the protein in COPD samples. Our results suggest a more important function for BPIFB1 in COPD, but further studies in larger cohorts will be needed to clarify the importance of BPIFA1 and BPIFB1 in COPD. Increased mRNA levels of BPIFA1 were recently shown to be associated with decreased lung function measured by prebronchodilator FEV1 % predicted in a group of 739 patients with smoking-related lung disease (primarily lung cancer and COPD) (32). In our present study, sputum BPIFB1 levels were associated with BMI, pack-years, and lung function as measured by FEV1 % predicted. In addition, a weaker correlation between BPIFB1 levels and FEV1/FVC was detected. These associations were not observed in COPD patients at baseline, but the associations between baseline BPIFB1 levels and lung function became significant in COPD patients after a 4-year follow-up. This is probably because of the design of our present study since most of the participants in the COPD group had only mild to moderate airflow limitation at baseline. Importantly, the association of increased sputum BPIFB1 levels with changes in lung function was most apparent in current smokers with COPD, and they also had higher sputum BPIFB1 levels than current smokers without COPD. In addition, sputum BPIFB1 levels showed a strong correlation with packyears only in smokers with COPD, but not in smokers without COPD. These observations further indicate that BPIFB1 is associated not only with smoking but also with smokingrelated pathological changes in the airway wall. Although the observed correlation between BMI and BPIFB1 in nonsmokers and smokers without COPD is intriguing, there were no significant differences in BMI between the study groups. Unlike smoking, BMI appears not to be a confounding factor for elevated BPIFB1 levels in COPD. There were some limitations in our study. First, the number of participants in the nonsmoker and COPD groups was relatively small. Second, we did not perform high-resolution computed tomography or diffusion capacity studies in all participants to assess lung tissue damage and emphysema. Third, the follow-up period of 4 years was relatively short, but this was due to the study design of a longitudinal analysis among apparently healthy middle-aged to elderly individuals. Aside from these limitations our study has significant strengths, including that none of the participants had any other exposures or comorbidities and they were not taking any medications at the time of enrolment (35). Furthermore, analysis of induced sputum is a relatively safe, noninvasive, and repeatable method for the assessment of bronchial inflammation. The results obtained in this study were in agreement with the results from the proteomic screening as shown with immunoblotting of sputum and lung tissue with several antibodies detecting distinct epitopes as well as with an ELISA-based method of a larger amount of sputum samples. In conclusion, we have shown that elevated sputum BPIFB1 levels are associated with the changes of airflow limitation over a 4-year follow-up period, especially in current smokers with COPD. These findings are in agreement with the known function of BPIFB1 in pulmonary immunity. However, further studies are required to confirm our present findings and to

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clarify the relationships between BPIFB1 and its role in the pathophysiology of COPD. ACKNOWLEDGMENTS We thank Vuokko Kinnula for substantial contribution to the manuscript prior to her death on 17th November 2012. Tiina Lapinkari, Eeva-Liisa Stefanius, Marjo Kaukonen, Merita Salmela, Lea Lipasti, and Suvi Suutari are acknowledged for help and excellent technical assistance. GRANTS This work was financially supported by the SalWe Research program for IMO (Tekes, the Finnish Funding Agency for Technology and Innovation grant 648/10), and by a governmental subsidy from the Ministry of Social Affairs and Health, a governmental subsidy for health science research (EVO) in Rovaniemi, the EVO funding of the Helsinki University Central Hospital, Research Funds of the University of Helsinki, Sigrid Jusélius Foundation, Finnish Anti-Tuberculosis Association Foundation, and the Jalmari and Rauha Ahokas Foundation. J. Gao is further supported by the China Scholarship Council (CSC), the Finnish Cultural Foundation, CIMO, the Research Foundation of the Pulmonary Diseases, and Väinö and Laina Kivi Foundation. B. Araujo was supported by an ERS Long Term Research Fellowship. DISCLOSURES Sari Tiitinen and Matti Höyhtyä are employed by Medix Biochemica and have no conflict of interest. AUTHOR CONTRIBUTIONS J.G., S.O., S.T., T.K., L.B., and B.A. performed experiments; J.G., S.O., P.N., S.T., T.K., and V.P. analyzed data; J.G., S.O., P.N., S.T., M.H., C.D.B., and V.P. interpreted results of experiments; J.G., S.O., P.N., C.D.B., and V.P. prepared figures; J.G., S.O., P.N., C.D.B., and V.P. drafted manuscript; J.G., S.O., P.N., T.T., S.T., L.B., M.H., C.D.B., W.M., and V.P. edited and revised manuscript; J.G., S.O., P.N., T.T., S.T., T.K., L.B., B.A., M.R., M.H., C.D.B., W.M., and V.P. approved final version of manuscript; S.O., P.N., T.T., M.H., C.D.B., W.M., and V.P. conception and design of research. REFERENCES 1. Agusti A, Soriano JB. COPD as a systemic disease. COPD 5: 133–138, 2008. 2. Bingle CD, Araujo B, Wallace WA, Hirani N, Bingle L. What is top of the charts? BPIFB1/LPLUNC1 localises to the bronchiolised epithelium in the honeycomb cysts in UIP. Thorax 68: 1167–1168, 2013. 3. Bingle CD, Craven CJ. PLUNC: a novel family of candidate host defence proteins expressed in the upper airways and nasopharynx. Hum Mol Genet 11: 937–943, 2002. 4. Bingle CD, Wilson K, Lunn H, Barnes FA, High AS, Wallace WA, Rassl D, Campos MA, Ribeiro M, Bingle L. Human LPLUNC1 is a secreted product of goblet cells and minor glands of the respiratory and upper aerodigestive tracts. Histochem Cell Biol 133: 505–515, 2010. 5. Bingle L, Barnes FA, Cross SS, Rassl D, Wallace WA, Campos MA, Bingle CD. Differential epithelial expression of the putative innate immune molecule SPLUNC1 in cystic fibrosis. Respir Res 8: 79, 2007. 6. Bingle L, Bingle CD. Distribution of human PLUNC/BPI fold-containing (BPIF) proteins. Biochem Soc Trans 39: 1023–1027, 2011. 7. Bingle L, Cross SS, High AS, Wallace WA, Devine DA, Havard S, Campos MA, Bingle CD. SPLUNC1 (PLUNC) is expressed in glandular tissues of the respiratory tract and in lung tumours with a glandular phenotype. J Pathol 205: 491–497, 2005. 8. Bingle L, Wilson K, Musa M, Araujo B, Rassl D, Wallace WA, LeClair EE, Mauad T, Zhou Z, Mall MA, Bingle CD. Bpifb1 (Lplunc1) is upregulated in cystic fibrosis lung disease. Histochem Cell Biol 138: 749 –758, 2012. 9. Britto CJ, Cohn L. Bactericidal/permeability-increasing protein foldcontaining family member A1 in airway host protection and respiratory disease. Am J Respir Cell Mol Biol 52: 525–534, 2015. 10. Casado B, Ladarola P, Pannell LK, Luisetti M, Corsico A, Ansaldo E, Ferrarotti I, Boschetto P, Baraniuk JN. Protein expression in sputum of smokers and chronic obstructive pulmonary disease patients: a pilot study by CapLC-ESI-Q-TOF. J Proteome Res 6: 4615–4623, 2007.

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