Sputum Proteomics Identifies Elevated PIGR levels ... - ACS Publications

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Sputum Proteomics Identifies Elevated PIGR levels in Smokers and Mild-to-Moderate COPD Steffen Ohlmeier,† Witold Mazur,‡ Anna Linja-aho,‡ Noora Louhelainen,‡ Mikko R€onty,§ Tuula Toljamo,z Ulrich Bergmann,† and Vuokko L. Kinnula*,‡ †

Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, Oulu, Finland Department of Medicine, Pulmonary Division, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland § Department of Pathology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland z Department of Pulmonary Medicine, Lapland Central Hospital, Rovaniemi, Finland ‡

bS Supporting Information ABSTRACT: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms leading to COPD and its progression are still poorly understood. In this study, induced sputum was analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis. The comparison of nonsmokers, smokers, and smokers with moderate COPD revealed 15 changed proteins with the majority, including polymeric immunoglobulin receptor (PIGR), being elevated in smokers and subjects with COPD. PIGR, which is involved in specific immune defense and inflammation, was further studied in sputum, lung tissue, and plasma by Western blot, immunohistochemistry/image analysis, and/or ELISA. Sputum PIGR was characterized as glycosylated secretory component (SC). Lung PIGR was significantly elevated in the bronchial and alveolar epithelium of smokers and further increased in the alveolar area in mild to moderate COPD. Plasma PIGR was elevated in smokers and smokers with COPD compared to nonsmokers with significant correlation to obstruction. In conclusion, new proteins in smoking-related chronic inflammation and COPD could be identified, with SC/PIGR being one of the most prominent not only in the lung but also in circulating blood. KEYWORDS: chronic obstructive pulmonary disease, polymeric immunoglobulin receptor, proteomics, secretory component, smoking, sputum, 2D DIGE

’ INTRODUCTION Chronic obstructive pulmonary disease (COPD) is a complex disease related mainly to smoking and an increased risk of several comorbidities.1 The pathogenesis of COPD has been strongly connected to protease/antiprotease imbalance and oxidative stress,2 5 typical findings including nearly irreversible airway obstruction with numerous abnormalities in the airways and patchy lung tissue damage (emphysema).6 Inflammation, which can be seen not only in the lung but also in the circulation, persists over months or even years after cessation of smoking.7,8 Diagnosis is based on spirometry, but this does not reflect disease activity. More specific markers not only associated with disease onset but also its pathogenetic mechanisms are required to identify and characterize COPD already in its earlier stages. Proteomics as nonbiased screening technique has been applied in several studies to investigate the effects of smoking and/ or COPD in the lung.9,10 Proteomic screening for COPD markers has been targeted at different sample types such as lung tissue, bronchoalveolar lavage fluid (BALF), and induced sputum.11 16 r 2011 American Chemical Society

However, as those few studies are based on different patient characteristics, sample types, and proteomic approaches, comparison of these findings is difficult. So far, none of these results have been transferred into clinical practice. In recent lung tissue proteomic studies from our laboratory, the major findings included elevation of surfactant protein-A (SP-A) and decrease of specific variants of the receptor for advanced glycation endproducts (RAGE) in COPD lung and decrease of lung hemoglobin subunits and complexes, especially in pulmonary fibrosis.11,17,18 Most potential findings, i.e., SP-A and RAGE, have been validated in several studies on smokers and COPD.19,20 The present proteomic study was initiated by using induced sputum because it reflects mainly airway pathology, it has several advantages compared to heterogeneous lung tissue or invasive bronchofiberoscopy, and international guidelines are available for its collection and processing.21,22 For the proteomic Received: June 3, 2011 Published: November 04, 2011 599

dx.doi.org/10.1021/pr2006395 | J. Proteome Res. 2012, 11, 599–608

Journal of Proteome Research

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screening, the “saturation difference gel electrophoresis (DIGE)” technology was used. This method is based on cysteine-specific protein labeling with specific Cy dyes, which significantly improves the sensitivity. A further advantage of this method is the use of an internal standard enhancing intergel matching and quantification. In addition, it targets cysteine/thiol-containing proteins, which are generally linked to lung defense, redox balance, and the pathogenesis of several lung diseases.23,24 Changed proteins were identified by peptide mass fingerprinting (PMF) using matrix assisted laser desorption ionization-time-offlight mass spectrometry (MALDI-TOF MS) analysis. Three different study groups (nonsmokers, smokers, and smokers with moderate COPD) were investigated to identify smoking-dependent and -independent changes. Selected proteins were further studied by several techniques. Since recent observations have pointed to a regulatory and protective role for polymeric immunoglobulin receptor (PIGR) in specific immune defense as well as in inflammation,25 PIGR was selected for further investigations. PIGR, present in different variants, is a known scavenger of host and pathogenic factors including microbial and environmental antigens.25,26 In this study, PIGR was analyzed in sputum and lung tissues by several techniques such as Western blotting and immunohistochemistry/image analysis, and its plasma levels were analyzed by ELISA to evaluate whether these alterations display any correlation with obstruction in spirometry and whether they are locally restricted to the lung or are systemic.

For protein identification, excised spots were digested as described.17 Peptide masses were measured with a VOYAGERDE STR (Applied Biosystems, Foster City, CA). Proteins were identified according to their spot-specific peptide mass fingerprint with the bioinformatic tool Aldente (http://au.expasy.org/ tools/aldente/) and UniProtKB/Swiss-Prot as the corresponding peptide/protein database. For detailed information, see the Supporting Information “Materials and Methods” and “MS”. The expected spot position in the 2D-gel according to the known protein sequence was calculated with the Compute pI/ Mw tool (http://ca.expasy.org/tools/pi_tool.html). Western Blot Analysis

Western blot analyses of the sputum supernatants and lung tissue homogenates were performed as described earlier.18 For the Western blot of the sputum supernatants, equal volumes of the supernatants were loaded. Because of problems with the conventional loading markers in COPD,14,30,31 Ponceau S staining was used to standardize the loading of the lung homogenate.17 For detailed information, see the Supporting Information “Materials and Methods”. 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. Next, they were treated with reaction buffer, NP40, and PNGase F and incubated at 37 °C for 1 h according to the instructions of the manufacturer (New England Biolabs 240 County Road, Ipswich, MA). The Western blots of the treated and untreated samples were run as described.

’ MATERIALS AND METHODS Collection of Induced Sputum and Lung Tissue

ELISA

Sputum and plasma specimens were received from volunteers from the area of Lapland Central Hospital; the cohort has been described in detail previously.27 Sputum was processed using the sputum plug method as described;22,28 only sputum samples containing less than 70% of squamous epithelial cells were accepted. Lung tissue specimens were retrieved from the patients of Helsinki University Central Hospital.18 COPD was defined according to GOLD criteria,29 and only mild to moderate (stage I II) cases were included. The study protocol was approved by the ethical committees of Lapland Central Hospital and Helsinki University Central Hospital. For detailed information, see the Supporting Information “Materials and Methods”, including Tables S1 S4.

PIGR in plasma was analyzed by ELISA (E91074Hu, Uscn Life Science Inc., Burlington, NC) according to the manufacturer's instructions. Immunohistochemistry and Image Analysis

Immunohistochemistry was performed as described earlier.11 Digital images of lung tissue sections with normal-looking lung histology by excluding the damaged areas, assessed by an experienced pathologist, were taken using 200 magnification and saved as Photoshop JPG files. The areas of positively vs negatively stained tissue were measured using Image Pro software (Media cybernetics, U. K.) as described.18 For detailed information, see the Supporting Information “Materials and Methods”.

Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) and Mass Spectrometry (MS)

For the proteomic study, purified sputum (5 μg) from nonsmokers (n = 7), “healthy smokers” (n = 7), and smokers with COPD (stage II, n = 7) (Supporting Information “Materials and Methods”: Table S1) was labeled with the “CyDye DIGE Fluor labeling kit (“saturation DIGE”; GE Healthcare, Piscataway, NJ) according to the manufacturer's protocol. Proteins were separated by isoelectric focusing in immobilized pH gradient strips (pH 4 7, 24 cm; GE Healthcare) with the Multiphor II system (GE Healthcare) followed by SDS-PAGE in polyacrylamide gels (12.5%) with the Ettan DALT II system (GE Healthcare). Fluorescence signals were detected with a Typhoon 9400 (GE Healthcare), and 2D gels were analyzed with Delta2D 4.0 (Decodon, Greifswald, Germany). For detailed information, see the Supporting Information “Materials and Methods” and “DIGE”.

Statistical Analysis

SPSS for Windows (Chicago, IL) version 18 software was used for statistical analysis of the Western blot, ELISA, and image analysis data. Data was calculated as mean ( SD or mean ( SEM from at least three concurrent samples, with the differences considered statistically significant if p e 0.05. For detailed information, see the Supporting Information “Materials and Methods”.

’ RESULTS Proteomic Changes in Induced Sputum of Smokers and Smokers with COPD

Since the cysteine-specific “saturation DIGE” is highly sensitive, requiring only 5 μg of protein for the 2DE analysis, this proteomic approach was chosen to allow also the analysis of 600

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Figure 1. Representative 2D gel of sputum from a characteristic COPD patient (stage II). Sputum (5 μg) was labeled by “saturation DIGE” (Cy5). Changed protein spots are indicated with numbers.

sputum samples with low protein levels. In the first stage, the labeling with the cysteine-specific “saturation DIGE” and the separation were optimized for the sputum proteome (for more details, see the Supporting Information “Materials and Methods”). Optimal labeling, indicated by a direct matching of Cy3- and Cy5-labeled protein patterns, was achieved with 0.5 nmol dye/5 μg of protein (Supporting Information “DIGE”). The comparison of different pH gradients for the separation (data not shown) revealed the highest spot numbers at pH 4 7. These conditions were therefore selected for the subsequent analysis. A 2D-gel representative for sputum (5 μg) from a patient with COPD (stage II) is shown in Figure 1. To screen for smoking- and/or COPD-dependent changes, sputum samples of nonsmokers (n = 7), smokers (n = 7), and smokers with moderate COPD (n = 7) (Supporting Information “Materials and Methods”: Table S1) were compared, and representative samples for these three study groups were verified by principal component analysis. Significant changes (p e 0.05; cutoff ratio: (1.5-fold) including mainly COPD-dependent alterations were detected for 38 spots (Figures 1 and 2). These spots were further investigated by mass spectrometry, and 15 different proteins in 34 spots were identified (Table 1 and Supporting Information “MS”). The majority of altered proteins were elevated in smokers with COPD (Figure 2). Although no changes present only in smokers without COPD were detected when compared to nonsmokers, zinc-α-2-glycoprotein, long palate lung and nasal epithelium carcinoma-associated protein, and transthyretin were significantly elevated in both smokers and subjects with COPD (Table 1). In agreement with the protective properties of α-1antitrypsin against COPD,32,33 its level was elevated in smokers with COPD (Table 1). Polymeric immunoglobulin receptor (PIGR), which was elevated in COPD, is a potential regulator of specific immune defense and inflammation,25 but its role in COPD is still poorly understood.34,35 Therefore, this receptor protein was investigated in more detail.

Glycosylated Secretory Component (SC) of PIGR is Elevated in Induced Sputum of Subjects with COPD

PIGR exists in various variants with different functions: fulllength PIGR mediates transcytosis of IgA, whereas a cleaved PIGR form (secretory component, SC) in complex with polymeric IgA or as free form represents a scavenger for environmental and microbial antigens.25,26 Verification of the changed PIGR variant is essential to elucidate its role in COPD pathogenesis. Removal of the signal sequence from the full-length PIGR (amino acids 1 764) results in a smaller PIGR molecule (amino acids 19 764) and further removal of the transmembrane segment in the formation of SC (amino acids 19 603), which represents the ligand-binding part of PIGR. Interestingly, mass spectrometry analyses of the PIGR spots detected only peptides matching amino acids 19 597 (Figure 3a), suggesting that sputum PIGR is present in its SC form. In agreement with the observations from 2DE, sputum PIGR was detected by Western blot at 86 kDa with the level elevated in COPD in comparison to nonsmokers (1.8-fold, p = 0.01) (Figure 3b). According to the calculated theoretical position, this could indicate the presence of full (83 kDa) or mature (81 kDa) PIGR, whereas the expected size of the SC is 64 kDa. However, PIGR might exist also as a glycosylated form, which alters its migration in the gel. To verify this possibility, N-linked glycosylation of sputum PIGR was investigated by PNGase F treatment. The deglycosylation resulted in a shift from 86 to 60 kDa (Figure 3c). Together with the sequence coverage detected by mass spectrometry, this is evidence for the presence of glycosylated SC. PIGR/SC is Localized in Specific Lung Cells and Elevated in Subjects with COPD Compared to Smokers and Nonsmokers

PIGR was also investigated in lung tissue by Western blot and immunohistochemistry. Similar to sputum, PIGR could be detected by Western blot in tissue homogenates exclusively in one band at 86 kDa, indicative of the presence of glycosylated SC (Figure 3d). However, in contrast to sputum PIGR, 601



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Figure 2. Proteins with elevated or decreased levels in smokers and/or smokers with COPD (stage II) detected by 2D-DIGE. Enlarged gel parts corresponding to spot numbers indicated in Figure 1 and characteristic for a nonsmoker (NS) as well as expression profiles indicating protein levels in nonsmokers (NS), smokers (S), and subjects with COPD stage II (COPD) are presented. Subject characteristics are shown in Table S1 (Supporting Information “Materials and Methods”).

no significant differences between the groups were detected by Western blot of the lung tissue homogenates (data not shown). This might be explained by the extensive heterogeneity present in lung homogenate, which does not reflect changes in individual pulmonary cells. Therefore, PIGR was further investigated by immunohistochemistry of tissue specimens of nonsmokers, smokers, and subjects with COPD (stage I II). PIGR was widely expressed in the epithelium and in alveolar macrophages (Figures 4a and 5a). PIGR immunoreactivity was further quantified by image analysis. In the alveolar-interstitial area, elevated PIGR levels were detected in smokers (relative positivity mean value: 10.7 ( 2.6%) and especially in COPD (35.8 ( 3.2%) when compared to nonsmokers (1.1 ( 0.3%, all p < 0.0001) (Figure 4b). A significant difference was also detected between “healthy smokers” and subjects with COPD (p < 0.0001). In normal-looking bronchial epithelium, PIGR levels were higher in smokers (12.2 ( 1.8%; p = 0.007) and subjects with COPD (11.7 ( 1.9%; p = 0.007) compared to nonsmokers (4.4 ( 1.2%) (Figure 5a,b). Elevated PIGR Levels in Plasma of Smokers and Smokers with COPD are Associated with Airway Obstruction

To verify the role of PIGR as a potential local or systemic inflammatory regulator, plasma from nonsmokers (n = 36),

smokers (n = 52), and smokers with COPD (stage I II, n = 42) was investigated by ELISA. PIGR levels were significantly higher in plasma of smokers (mean ( SD: 5451 ( 2228 ng/mL, p < 0.0001) and smokers with COPD (6601 ( 3306 ng/mL, p < 0.0001) compared to nonsmokers (3818 ( 2046 ng/mL) (Figure 6a). Although there were no associations between the plasma PIGR levels of the smokers with or without COPD and the baseline demographics including age, BMI and smoking (pack years), plasma levels of PIGR did correlate significantly with air flow obstruction (correlation coefficient = 0.240, p = 0.006) (Figure 6b).

’ DISCUSSION The present study identified 15 elevated/decreased proteins in sputum of nonsmokers, smokers, and/or smokers with COPD. The major interest was forwarded to PIGR because it plays a crucial role in mucosal immunity. For these studies, larger numbers of cases and several methodologies were included. In general, our results are partly in agreement with a recent sputum 2DE study, which compared the sputum obtained from smokers and COPD patients.16 Our study, however, applied a different 2DE strategy (2D-DIGE) involving the highly sensitive detection of cysteine-containing proteins and an internal standard for 602



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Table 1. Elevated or Decreased Proteins in Sputum of Nonsmokers (NS), Smokers (S) and Smokers with COPD Stage II Disease proteina name

description

ratiob UniProt-KB

#

NS vs S

NS vs COPD

t-testc S vs COPD

NS vs S

NS vs COPD

S vs COPD

Elevated in COPD ACTG

γ-actin

P63261

18

(1.75)

3.27

(1.87)

92.52

99.17

87.29

AZGP1

zinc-α-2-glycoprotein

P25311

19

2.74

2.86

(1.04)

97.98

96.02

7.85

20

3.86

3.78

( 1.02)

99.40

98.92

4.65

21

2.64

3.64

(1.38)

95.33

96.94

48.32

22

(2.16)

2.61

(1.21)

91.48

96.60

31.88

23

(1.85)

1.96

(1.06)

92.77

96.10

13.21

24

(1.74)

2.03

(1.16)

92.49

95.83

32.23

25 5

(1.42) ( 1.61)

1.79 (1.51)

(1.27) 2.78

91.57 66.26

98.56 69.29

62.66 97.15 96.29

C3

complement C3 β

P01024

GC

vitamin D-binding protein

P02774

IGKC

LPLUNC1

Ig k chain C region

long palate, lung and nasal epithelium

P01834

9

(1.19)

2.65

2.23

41.03

99.56

10

( 1.17)

2.42

2.83

39.68

99.13

98.53

26

(1.05)

1.65

1.57

18.11

99.71

98.62 97.34

27

( 1.01)

1.47

1.50

2.66

98.09

28

( 1.08)

1.42

1.54

29.98

97.59

99.13

95.38

98.08

33.67

Q8TDL5

16

2.33

2.02

( 1.16)

carcinoma-associated protein 17

(2.37)

2.54

(1.07)

89.88

98.55

15.63

MSMB

β-microseminoprotein (PSP94)

P08118

32

(2.13)

2.33

(1.09)

82.03

98.56

17.30

PIGR

polymeric immunoglobulin receptor

P01833

1

(2.33)

3.03

(1.30)

88.53

95.67

41.23

2

(2.43)

2.94

(1.21)

86.06

97.14

30.91

3

(2.04)

2.49

(1.23)

80.33

95.78

33.78

(1.69)

2.98

87.71

94.67

97.68

SERPINA1

α-1-antitrypsin

P01009

6

( 1.76)

7

( 1.51)

2.75

4.15

92.57

95.78

95.34

TF TTR

serotransferrin transthyretin

P02787 P02766

4 30

( 1.34) 4.71

(2.37) 6.18

3.54 (1.31)

58.05 97.72

92.44 99.39

95.19 44.60

31

(1.16)

2.26

(1.95)

29.34

99.69

94.61

8

(1.54)

3.12

(2.03)

64.04

99.94

93.39

36

( 1.06)

1.53

1.62

16.29

95.37

97.23

37

( 1.09)

1.47

1.59

28.76

97.11

98.70

3.04

(1.04)

95.16

98.26

7.89

( 1.10) ( 1.29)

1.69 2.27

( 1.53) ( 1.75)

26.39 55.05

96.28 99.17

63.63 66.01

13

(1.05)

1.88

( 1.98)

11.92

95.39

72.74

14

( 1.26)

2.68

( 2.12)

47.52

99.62

71.59

15

( 1.45)

2.90

( 2.00)

75.35

99.91

76.31

38

2.91

Decreased in COPD AMY1

α-amylase 1

P04745

11 12

CST1

cystatin-SN

P01037

35

(1.14)

3.42

( 3.38)

16.48

95.37

74.28

CST4

cystatin S

P01036

33

( 1.15)

4.25

( 3.20)

22.13

95.15

90.08

34

( 1.44)

4.62

( 2.79)

49.68

95.15

87.53

29

( 1.57)

3.29

73.73

96.05

97.34

PIP

prolactin-inducible protein

P12273

2.09

a UniProtKB accession numbers and spot numbers (#) are shown. Details about the protein identification are presented in the Supporting Information “MS”. b Ratio represents the significant (bold) or not significant (parentheses) change in the protein level of the second study group. c t-test indicates statistical significance (95 100% = p e 0.05).

improved statistical analysis. In addition, nonsmokers were included to evaluate smoking-dependent and -independent effects separately. One of the major findings was that the PIGR elevation detected in smokers and mild to moderate COPD could be confirmed not only quantitatively in specific lung cells but also in plasma, pointing to a role of PIGR also in the systemic inflammation associated with smoking and COPD.

At present, only a few proteomic studies have been conducted on sputum, and these have utilized different pathological conditions and techniques including gel-free MS-based analyses (LC MS/MS,14 SELDI-MS15) as well as gel-based 2DE16 using postelectrophoretic staining instead of the 2D-DIGE technology used in the present study. This might explain why only four identified proteins, i.e., zinc-α-2-glycoprotein, β-microseminoprotein 603



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Figure 3. Detection of glycosylated SC in sputum and lung tissue. (a) Schematic presentation of PIGR. Signal peptide (SP), transmembrane sequence (TMS), glycosylation sites (Gly), Ig-like V-type domains (V1 5), as well as sequence coverage of the spot-specific peptides are indicated. (b) Western blot of sputum from nonsmokers (NS, n = 7) and smokers with COPD stage II (COPD, n = 7). (c) Detection of PIGR in sputum of two smokers by Western blot with (+) and without ( ) PNGase F treatment for deglycosylation of SC. (d) Western blot of lung tissue homogenate from a nonsmoker reveals presence of SC. Subject characteristics are shown in Table S2 (Supporting Information “Materials and Methods”).

Figure 4. Immunohistochemistry localizes PIGR in alveoli. (a) Lung tissues representative for nonsmokers (NS), smokers (S), and subjects with COPD stage I II (COPD) are shown. Specific localization of PIGR (brown color) is indicated by arrows (magnification: 200). Negative controls showed no detectable staining (not shown). (b) Quantification of PIGR immunoreactivity by the image analysis. Data are presented as mean ( SEM. Subject characteristics are shown in Table S3 (Supporting Information “Materials and Methods”).

(isoform PSP94), cystatin S, and transthyretin, have been detected earlier by proteomic studies investigating COPD.14,16 In our study, zinc-α-2-glycoprotein was elevated, whereas in the earlier 2DE study, no change in this protein in smokers/COPD was detected.16 The elevation of PSP94 in COPD was found in both studies, but in our study, this was detectable only in subjects with COPD compared to nonsmokers. Transthyretin was elevated in COPD in our study, and the same change was reported in the study of Nicholas and co-workers.16 The apparent differences between these two studies may be partly explained by the variation of the sputum protein levels in smokers as well as by differences in the study groups and the proteomic approach applied. COPD pathology can be studied in different local or systemic sample types, each having their own advantages and disadvantages.

A detailed overview about the pros and cons of the different sample types for proteomic studies on parenchymal lung diseases including COPD has been recently described by Kinnula and co-workers.9 Bronchial biopsies and BALF represent invasive sampling techniques of a patchy and heterogeneous disease, which in turn may result in additional variations apart from COPD pathology. In contrast, induced sputum, which reflects mainly large airway pathology, is nearly noninvasive and relatively easy to obtain. International guidelines are available for sputum collection and processing,21,22 and the sampling has been optimized for 2DE.36 Therefore, induced sputum was chosen for the initial proteomic screening. Additional sample types of the present study included both lung tissue and plasma to identify not only local changes of PIGR in the lung but also 604



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Figure 5. Immunohistochemistry localizes PIGR in the bronchial epithelium. (a) Lung tissues representative for nonsmokers (NS), smokers (S), and subjects with COPD stage I II (COPD) are shown. Specific localization of PIGR (brown color) is indicated by arrows (magnification: 200). Negative controls showed no detectable staining (not shown). (b) Quantification of PIGR immunoreactivity by image analysis. Data are presented as mean ( SEM. Subject characteristics are shown in Table S3 (Supporting Information “Materials and Methods”).

Figure 6. PIGR in plasma and its correlation with air flow obstruction. (a) Comparison of plasma PIGR concentration in nonsmokers (NS, n = 36), smokers (S, n = 52), and smokers with COPD (stage I II, n = 42). Data are expressed as medians with interquartile range (box) and overall range (whiskers). (b) Relationship between plasma (n = 130) PIGR levels and obstruction indicated by the postbronchodilator FEV1 (forced expiratory volume in one second)/FVC % (predicted forced vital capacity) (correlation coefficient = 0.240, p = 0.006). Subject characteristics are shown in Table S4 (Supporting Information “Materials and Methods”).

its potential significance associated with systemic manifestations of COPD. The proteomic screening of sputum was performed with the cysteine-specific “saturation DIGE” because its high sensitivity allows the analysis of sputum with low protein levels, and it is not biased toward sputum with high protein content. Although this approach is restricted to cysteine-containing proteins, they represent more than 90% of the human proteome.37 In this technique, cysteine-free proteins and proteins with all cysteines modified in an irreversible reaction (e.g., sulfonic acid) are undetectable. The number of the spots that could be detected here in the gels with 5 μg of protein is comparable with a recent sputum 2DE study that used 450 μg of protein/gel and visualized total protein by SYPRO Ruby.16 Their study detected and

analyzed 363 spots that were present in at least 80% of the gels in both investigated study groups (smokers and smokers with COPD); in our study, 684 spots could be detected and analyzed in all study groups (nonsmokers and smokers without or with COPD). A common problem in the separation of sputum proteins is the disturbance of the electrophoresis by the abundance of high-molecular-weight and highly charged mucins. In our study, a slight vertical disturbance in the protein separation could be observed in several gels between pH 5.5 and 6.5, in agreement with the expected pI of several mucins.36 However, since the protein separation was performed in narrow and long pH gradients (pH 4 7, 24 cm), these problems remained minor. Elevated PIGR levels (2.5 3.0-fold) in sputum were found initially by 2D-DIGE of subjects with COPD when compared to 605


Journal of Proteome Research nonsmokers, and these findings were confirmed (1.8-fold) by Western blotting. The comparison between smokers and nonsmokers by 2D-DIGE showed a similar trend (2.0 2.4-fold), but the difference was not significant. By immunohistochemistry/ image analysis, lung PIGR elevation was seen both in subjects with COPD and smokers without COPD. Importantly, PIGR was highly elevated in the normal-looking alveolar region in COPD (32.5-fold) and in smokers (9.7-fold) when compared to nonsmokers. Corresponding changes in the bronchial epithelium were remarkably lower, i.e., in COPD 2.7-fold and in smokers 2.8-fold, compared to nonsmokers. On the basis of these observations, the alteration in PIGR appears to be related to smoking and COPD, which is not surprising because PIGR has several protective functions at mucosal surfaces.25,26 Given that the changes in sputum PIGR levels were very similar to those observed in the bronchial epithelium and clearly smaller than in the alveolar region, sputum results underestimate the real changes of PIGR in the lung periphery. Not surprisingly, PIGR was also elevated in the plasma samples of smokers and smokers with COPD with a significant correlation to obstruction. Further prospective follow-up studies will be required to verify the role of PIGR as a potential marker for COPD-related systemic inflammation. Several PIGR variants with different functions have been described: full-length, membrane-bound PIGR mediates the transcytosis of polymeric immunoglobulin A (pIgA) to mucosal surfaces; its processed secretory component (SC) is released either in complex with pIgA as “secretory IgA”, which has a major role in nonspecific immune response, or as free SC, which has additional functions such as nonspecific scavenging of various pathogenic factors.26 PIGR was identified by 2D-DIGE and Western blot as glycosylated SC, which can either be present in complexed or free form. Since the level of IgA detected in sputum by 2D-DIGE was not significantly changed in smokers or subjects with COPD (data not shown), this points to the presence of free SC rather than the complex with IgA. This is further supported by the identification of glycosylated SC because binding of IgA to a specific SC domain is not dependent on glycosylation.38 In contrast, glycosylation is required for free SC to bind to host and pathogenic factors.26 One of the inflammatory cytokines associated with COPD is interleukin 8 (IL-8).39,40 Interestingly, SC has been shown to inactivate IL-8, though the binding can only take place with glycosylated SC.41,42 Therefore, elevation of SC in mild to moderate COPD might represent a mechanism to reduce local inflammation. In agreement with our findings, elevated SC levels have been reported in the sputum samples from subjects with other inflammatory lung diseases including asthma and cystic fibrosis,42 further supporting an involvement of SC in airway defense and inflammation. In contrast, a recent study showed PIGR decrease in structurally abnormal epithelium and airway remodeling areas in COPD; those abnormalities are mainly associated with severe to very severe end stage COPD (stage III IV).35 That particular study did not include current smokers, and total PIGR rather than processed, soluble SC was investigated. Decreased PIGR has also been reported by another study but, again, in very severe COPD.34 Altogether, the results of our laboratory and the others suggest that PIGR is initially elevated by smoking, inflammation, and early COPD but declines toward the end stage disease in the abnormal epithelium. These differences might also be partly related to complex SC/PIGR regulation, which has been shown to be dependent on numerous factors, e.g., neutrophils,

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redox balance, and TGF-β, which have been associated with COPD.26,34,43

’ CONCLUSIONS The level of PIGR is elevated in sputum and lung tissue of smokers and subjects with early stage COPD, suggesting a smoking-dependent induction of PIGR. The characterization of the changed PIGR as glycosylated secretory component suggests a role of PIGR/SC in the regulation of inflammation during COPD pathogenesis. The elevated PIGR levels in the plasma of smokers and subjects with COPD, with a correlation to airway obstruction, point to an involvement of PIGR in the systemic effects associated with this disease. Furthermore, this proteomic screening identified numerous other proteins, which will be further investigated prospectively to characterize their roles in COPD. ’ ASSOCIATED CONTENT

bS

Supporting Information Three parts (Materials and Methods, DIGE, and MS) containing supplementary tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org

’ AUTHOR INFORMATION Corresponding Author

*University of Helsinki and Helsinki University Hospital, Department of Medicine, Pulmonary Division, BOX 440 (Stenb€ackinkatu 9, Room 327), 00029 Helsinki, Finland. Phone: +3589 47172255, +358405055026. Fax: +3589 47176107. E-mail: Vuokko.Kinnula@helsinki.fi.

’ ACKNOWLEDGMENT Tiina Marjomaa, Sari Nummijoki, Eeva-Liisa Stefanius, Marjo Kaukonen, P€aivi Sortti, and Katri Vuopala are acknowledged for their help and/or excellent assistance. This work was financially supported by the special governmental subsidy for health sciences research (EVO) funding of the Helsinki University Central Hospital, University of Helsinki and Lapland Central Hospital, Finnish Antituberculosis Association Foundation and partly funded by the SalWe Program for IMO (Tekes the Finnish Funding Agency for Technology and Innovation Grant 648/10). ’ REFERENCES (1) Agusti, A.; Soriano, J. B. COPD as a systemic disease. COPD 2008, 5, 133–138. (2) Elkington, P. T; Friedland, J. S. Matrix metalloproteinases in destructive pulmonary pathology. Thorax 2006, 61, 259–266. (3) Kinnula, V. L. Focus on antioxidant enzymes and antioxidant strategies in smoking related airway diseases. Thorax 2005, 60, 693–700. (4) Rahman, I.; Adcock, I. M. Oxidative stress and redox regulation of lung inflammation in COPD. Eur. Respir. J. 2006, 28, 219–242. (5) Rabe, K. F.; Hurd, S.; Anzueto, A.; Barnes, P. J.; Buist, S. A.; Calverley, P.; Fukuchi, Y.; Jenkins, C.; Rodriguez-Roisin, R.; van Weel, C.; Zielinski, J. Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am. J. Respir. Crit. Care Med. 2007, 176, 532–555. (6) Hogg, J. C.; Timens, W. The pathology of chronic obstructive pulmonary disease. Annu. Rev. Pathol. 2009, 4, 435–459. 606



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Sputum Proteomics Identifies Elevated PIGR levels ... - ACS Publications

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