Proteomics of Human Lung Tissue Identifies Surfactant Protein A as

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Proteomics of Human Lung Tissue Identifies Surfactant Protein A as a Marker of Chronic Obstructive Pulmonary Disease Steffen Ohlmeier,† Minna Vuolanto,‡ Tuula Toljamo,| Katri Vuopala,⊥ Kaisa Salmenkivi,§ Marjukka Mylla¨rniemi,‡ and Vuokko L. Kinnula*,‡ Department of Biochemistry, Proteomics Core Facility, Biocenter Oulu, University of Oulu, Oulu, Finland, Department of Medicine, Pulmonary Division, Department of Pathology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland, and Departments of Medicine, Pulmonary Division and Pathology, Lapland Central Hospital, Finland Received June 10, 2008

Chronic Obstructive Pulmonary Disease (COPD), a lung disease related to smoking, is one of the leading causes of chronic morbidity and mortality around the world. One goal in COPD research is the identification of biomarkers for early diagnosis of the disease. Here, we sought COPD-specific changes in the proteome from human lung tissue. This revealed increased levels of surfactant protein A (SP-A) in COPD but not in the normal or fibrotic lung. The results were confirmed by immunohistochemistry, morphometry and Western blotting. Furthermore, elevated SP-A protein levels were detected from the induced sputum supernatants of COPD patients. The levels of other surfactant proteins (SP-B, SPC, SP-D) were not altered. Our results suggest that SP-A is linked to the pathogenesis of COPD and could be considered as a potential COPD biomarker. Keywords: Surfactant Protein A • Chronic Obstructive Pulmonary Disease • Biomarker • Two-dimensional gel electrophoresis • Human lung • Usual Interstitial Pneumonia • Idiopathic Pulmonary Fibrosis

Introduction COPD is one of the leading causes of chronic morbidity and mortality around the world.1 The disease is almost always related to smoking; thus, COPD is generally preventable. The prognosis of COPD is improved after smoking cessation, though in some individuals inflammation persists.2 Anti-inflammatory strategies have only marginal beneficial effects on COPD, especially when the responses are compared to the impact of these drugs in asthma.3 Therefore, one major focus in COPD research is the characterization of mechanisms involved in COPD pathogenesis to find new approaches for early diagnosis. However, despite extensive efforts, no specific or sensitive marker has been discovered so far. In this study, we used a proteomic approach to identify potential COPD biomarkers. This revealed SP-A as a promising candidate. SP-A is the major pulmonary surfactant-associated protein with multiple functions, for example, in innate host defense and the regulation of inflammatory processes in the lung.4,5 Human SP-A is encoded by two genes, SP-A1 and SPA2, that exhibit genetic and splicing variability.6 Interestingly, * Corresponding author. Vuokko Kinnula, MD, PhD, Professor of Pulmonary Medicine, Department of Medicine, P.O. Box 22 (Haartmaninkatu 4), FI-00014 University of Helsinki, Finland. Phone: 358 9 4717 2255, Fax: 358 9 4717 4049, Email:[email protected]. † Department of Biochemistry, University of Oulu. ‡ Department of Medicine, University of Helsinki and Helsinki University Central Hospital. | Department of Medicine, Lapland Central Hospital. ⊥ Department of Pathology, Lapland Central Hospital. § Department of Pathology, University of Helsinki and Helsinki University Central Hospital. 10.1021/pr800423x CCC: $40.75

 2008 American Chemical Society

SP-A1 and SP-A2 proteins have been proposed to mediate different functions (reviewed by Oberley7). Since SP-A1 and SPA2 are very similar, and differ, depending on the allele, by up to 10 amino acids, most studies have investigated total SP-A protein instead of SP-A1 and SP-A2 separately. Several but not all studies have revealed elevated SP-A levels in serum samples of smokers, patients with COPD and idiopathic pulmonary fibrosis (IPF),8-14 while the levels of SP-A in bronchoalveolar lavage (BAL) fluid of COPD and IPF appear to be decreased.15-19 These findings are controversial, and do not explain why specimens from different individuals display such variable levels of SP-A. The level of SP-A has not been evaluated in the lung tissues or induced sputum samples of COPD patients. In the first phase of this study, healthy and diseased tissue was compared by 2-DE (two-dimensional gel electrophoresis) analyses. Several spots were increased in all COPD specimens and all of them were identified as SP-A by mass spectrometry (MS). In the second phase, levels of SP-A were detected from the normal lung and from patients with COPD of variable severity or R-1-antitrypsin (AAT) deficiency, which represents a genetically inherited susceptibility for COPD with emphysema predominance. As induced sputum is widely used and validated method for the assessment of obstructive lung diseases, the levels of SP-A were further investigated from the induced sputum of nonsmokers, smokers and COPD patients. Lung specimens of patients with idiopathic pulmonary fibrosis (IPF) with lung histopathology of usual interstitial pneumonia (UIP) were used as disease control samples for SP-A expression. The detailed MS-analyses were performed to reveal which distinct form of SP-A protein is involved in COPD. Journal of Proteome Research 2008, 7, 5125–5132 5125 Published on Web 10/30/2008

research articles Materials and Methods For detailed information see Supporting Information. Lung Tissues and Sputum Samples. Tissue specimens were retrieved from the patients of Helsinki University Central Hospital and sputum specimens from volunteers or patients of the Divisions of Pulmonary Medicine, Helsinki University Central Hospital and Lapland Central Hospital. COPD was defined according to GOLD criteria.20 For more detailed information, see the text and Supplemental tables S1-S5 (Supporting Information). Two-Dimensional Gel Electrophoresis (2-DE) and Protein Identification. Frozen tissue samples from control, Stage IV (very severe disease) COPD and IPF lungs were powdered and further purified by acetone precipitation. The protein extract was resuspended in urea buffer (7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 0.15% [w/v] DTT, 0.5% [v/v] carrier ampholytes 3-10, Complete Mini protease inhibitor cocktail [Roche]), incubated for 10 min in an ultrasonic bath, and centrifuged. Protein aliquots (100 µg) were stored at -20 °C. The protein separation for each sample (control lung and Stage IV COPD), done in triplicate, was performed as described earlier.21 In brief, the protein solution was adjusted with urea buffer to a final volume of 350 µL and in-gel rehydration was performed overnight. Isoelectric focusing (IEF) was carried out in IPG strips (pH 4-7, 18 cm, GE Healthcare) with the Multiphor II system (GE Healthcare) under paraffin oil for 55 kVh. SDSPAGE was performed overnight in polyacrylamide gels (12.5% T, 2.6% C) with the Ettan DALT II system (GE Healthcare) at 1-2 W per gel and 12 °C. The gels were silver stained22 and analyzed with the 2-D PAGE image analysis software Melanie 3.0 (GeneBio), and reproducible changes in spot intensity (at least 2-fold) were marked in the gel. The exact position (isoelectric point, molecular weight) of the SP-A spots was determined from our reference 2-D gel of human lung (pH 4-7) with identified marker proteins. For protein identification, excised spots were digested as described.21 Peptide masses were measured with a VOYAGERDE STR (Applied Biosystems), and proteins identified by full database search (Aldente database version 11/02/2008 (http:// ca.expasy.org/tools/aldente/) according to the following parameters (20 ppm; 1 missed cut; [M + H]; +CAM; +MSO). SP-A was identified in all selected nine spots upon matching of at least 3 major peptides and a sequence coverage of at least 20%. Since the SP-A peptides in the mass spectrum matched to SPA1 and SP-A2, the spot-specific minor peptides were also analyzed to reveal which SP-A protein might be present. These peptides were compared with the theoretical peptides specific for SP-A1 and SP-A2 calculated according to PeptideMass (http://au.expasy.org/tools/peptide-mass.html). Western Blot Analysis. Tissue samples were homogenized in lysis buffer (QIAzol, Qiagen Sciences, MD). Both tissue samples and sputum supernatants were adjusted to 2-4 µg/ µL protein concentration. Protein concentrations were analyzed by Dc Protein Assay kit -method (Bio-Rad, Hercules, CA). The samples were dissolved to 5% 2-mercaptoethanol in sample buffer (Bio-Rad, Hercules, CA). Twenty to 40 µg of protein was loaded per lane to polyacrylamide gels. Electrophoretic protein separation was done in a 12% polyacrylamide gel at 100 V for 1 h and the protein bands were transferred to nitrocellulose membranes (VWR International, Inc., Dassel, Germany) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA). Membranes were blocked overnight in 10% 5126

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Ohlmeier et al. skimmed milk in Tris-buffered saline (TBS) containing 1% Tween. To verify if the new protein findings were dependent on the specificity of the antibody, two different SP-A antibodies were used, both of which showed similar results. The antibodies were mouse monoclonal SP-A antibodies (MAB3270 Chemicon, Temecula, CA or HYB-238 Santa Cruz Biotechnology, Inc. Santa Cruz, CA, respectively). Sheep anti-mouse (IgG) horseradish peroxidase conjugated secondary antibody (Amersham Biosciences, U.K.) was used at 1:50 000 dilution. Immunodetection was performed with the chemiluminescent HPR-substrate immunodetection kit (Millipore, Billerica, MA). Membranes were exposed to X-ray film (Kodak, Rochester, NY). Since the expression of housekeeping proteins (e.g., β-actin but possibly also others) vary in airway and parenchymal lung diseases including COPD,23-26 the protein concentration was verified by measuring it in triplicate and equal loading was ensured by Ponceau S staining of the membranes (Sigma Aldrich, St. Louis, MO). Densitometric analysis for the Western blot bands was done with the ImageJ 1.37V software (National Institutes of Health, Bethesda, MD). Western blotting from the same control and COPD homogenates were also investigated for the expression of other three surfactants, SP-B, SP-C and SP-D (Sc-53137, Sc-13979, Sc-59695, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, respectively). Immunohistochemistry and Morphometry. Digital images for morphometric analysis were taken using 200× magnification and saved as Photoshop JPG files. The area of positively vs negatively stained tissue was measured using Image Pro software (Media cybernetics, U.K.). Statistical Analysis. SPSS for Windows (Chicago, IL) was used for statistical analysis and the significance of the associations between two variables was assessed with Mann-Whitney U test. Data was calculated as mean ( SEM from at least three concurrent samples of several tissue sections of COPD and control; p e 0.05 was considered statistically significant. Ethical Considerations. The study protocol was accepted by the ethical committees of Helsinki University Hospital and Lapland Central Hospital and is in accordance with the ethical standards of the Helsinki declaration of 1975.

Results Screening for COPD Markers from the Human Lung by 2-DE. First, homogenates from control (n ) 4) and COPD lung tissue (stage IV) (n ) 4) were separated by 2-DE in pH 4-7 and pH 3-10 (data not shown) and the protein pattern studied for COPD specific changes. This approach revealed nine spots localized in the same gel area within two “spot trains” which were only detectable in 2-D gels of COPD lungs (Figure 1). MS analyses revealed that all changed spots correspond to SP-A (Table 1). Interestingly, modified SP-A has been detected earlier within several spots in human specimens.18,27-34 This might be explained by the numerous co- or post-translational modifications to which SP-A can be subjected.27,28,31,32,35 On the basis of these earlier observations and the detected positions, the upper “spot train” (spots 1-5: pI 4.35-4.85/ 31-33 kDa) may correspond to the N-glycosylated and the lower “spot train” (spots 6-9: pI 4.42-4.78/ 29-32 kDa) to the nonglycosylated protein.27,28 Since all spots were elevated, this suggests general differences in expression or localization rather than a specific change in one of the post-translational modifications. Western Blot Analysis of SP-A Expression in Different Stages of COPD. Since 2-DE analyses were performed only to very severe COPD (Stage IV), the expression of SP-A was

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Surfactant Protein A in COPD

Figure 1. Two-dimensional gel electrophoresis (2-DE) reveals overexpression of SP-A in Stage IV COPD. A 2-D gel representative for a lung with COPD is shown left. Representative SP-A expression (spots 1-9) in normal diseased lungs (Control, n ) 4; COPD, Stage IV, n ) 4) are shown right. Lung homogenates were separated by 2-DE (pH 4-7, 100 µg) and the protein pattern was visualized by silver staining. SP-A was identified by mass spectrometry (Table 1). Patient characteristics are shown in the Supplemental Table S2 (Supporting Information). Table 1. Identification of SP-A in Spots 1-9 protein

Swiss-Prot

spot

score

SC (%)

P

SP-A

Q8IWL2/Q8IWL1

1 2 3 4 5 6 7 8 9

1.55 1.55 2.79 2.32 2.32 5.89 5.53 1.12 2.32

21 21 23 21 21 35 32 25 21

4 4 4 4 4 5 5 3 4

a Aldente score, sequence coverage (SC) and matched peptides (P) are listed. A typical mass spectrum indicating the SP-A-specific peptides is shown in the Supporting Information Figure S1.

investigated by Western blot analyses of control lung tissue, and lung of various COPD severities (Stage II-IV). Stage II-III and Stage IV COPD displayed higher SP-A levels than the control lung (Figure 2A,B), revealing that SP-A level is already elevated at an early state of COPD. In addition, lung tissues with AAT deficiency, compared to control lungs, showed highly elevated SP-A levels (Figure 2C). SP-A Localization and Quantitation by Immunohistochemistry. The distribution of SP-A in lung tissue was further investigated to specify its role in COPD. SP-A was detected in alveolar type II cells and macrophages both in the control and in the COPD lungs (Stages II-IV) (Figure 3A) indicating no differences in SP-A localization between the patient groups. SP-A -positive cells were also observed in the large airways near the basement membrane. Double staining with antibodies against the epithelial cell marker TTF-1 (Thyroid Transcription Factor 1) and SP-A showed that alveolar and bronchiolar epithelial cells were both TTF-1 and SP-A positive (Figure 3C). This finding is in agreement with earlier results that have shown that SP-A transcription is dependent on the activation of TTF1.36 Morphometrical quantification of SP-A positive areas revealed a 3.7-fold elevation in Stage II-III, 3.4-fold in Stage IV COPD and 7.5-fold in AAT deficiency in comparison to the control lung (Figure 3B,D). SP-A Levels in the Induced Sputum Specimens of COPD Patients. The role of SP-A as a potential noninvasive biomarker for COPD was investigated from 32 sputum specimens of COPD

patients, smokers and nonsmokers. All smokers were current smokers and the sample collection was started at least 12 h after the last cigarette had been smoked. Western blot analysis revealed that some nonsmokers and healthy smokers showed sporadic SP-A level elevation, but in general, increased SP-A levels were detected in the sputum of the COPD patients. In agreement with our recent study, the cell differential counts confirmed increased numbers of neutrophils and macrophages in induced sputa of COPD patients.37 In the COPD patients, SP-A levels were elevated even when compared to healthy smokers (Figure 4). Detection of the SP-A-Isoforms. Human SP-A consists of two proteins (SP-A1 and SP-A2) which differ, depending on the allele, only by up to 10 amino acids. All major peptides detected in the mass spectra matched to both isoforms (see Supporting Information Figure S1). To verify which specific SP-A protein is affected in the gels, all detected, spot-specific peptides were compared with the theoretical SP-A1- and SP-A2-specific peptides calculated with PeptideMass (http://au.expasy.org/ tools/peptide-mass.html). The spot-specific peptides matched to several peptides characteristic for SP-A2 (e.g., 932.4432, 3129.4516, 3500.6320), while no SP-A1-specific peptides could be detected. This suggests that SP-A2 is affected by COPD. Since the majority, but not all, of the detected SP-A in BAL fluid corresponds to SP-A2,38 the presence of SP-A1 in our specimens though at a lower abundance cannot be excluded. Expression of Other Surfactant Proteins in the Lung Specimens of Patients with COPD. Given that not only SP-A but also SP-D levels have been shown to be changed by oxidative stress,39 the levels of other surfactant proteins, SP-B, SP-C, and SP-D, were evaluated in the control and COPD lung. Western blot analyses revealed that the levels of these surfactant proteins were not affected by COPD indicating SP-A as the only COPD-specific surfactant protein (Figure 5). SP-A Expression in IPF/UIP Lungs. Controversial results on the expression of SP-A in the serum and BAL samples of IPF patients have been reported.10-12,17-19,38 The levels of SP-A in the IPF/UIP lungs were analyzed as a disease control to verify whether the level of SP-A elevation in the lung tissue would be disease-specific to COPD. Western blot of IPF/UIP lung homogenates showed no consistent increase in SP-A levels though Journal of Proteome Research • Vol. 7, No. 12, 2008 5127

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Figure 2. Relative intensities of SP-A in control and COPD lungs determined by Western blot analysis: (A) control (n ) 5) and Stage II-III COPD (n ) 4), (B) control (n ) 5) and Stage IV COPD (n ) 4), and (C) control (n ) 4) and AAT deficiency (n ) 5). For patient characteristics see Supplemental Table S2 (Supporting Information).

variability could be observed. Because of this variability, IPF/ UIP lungs were further investigated by 2-DE, immunohistochemistry and morphometry. Here, 2-DE revealed no elevation in the levels of SP-A in IPF/UIP lungs (Figure 6A). Immunohistochemistry detected SP-A both in the control and IPF/UIP lung, but the SP-A level was not changed (Figure 6B). The Western blot analyses of SP-A levels are shown in figure 6C.

Discussion Pulmonary surfactant reduces surface tension at the air-liquid interface. The functions of various surfactant proteins differ both in their localization and biological action, for example, 5128

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Ohlmeier et al. SP-A has been connected to the host defense of the lung. Considering that SP-A is an abundant, constantly secreted protein in the alveolar space and even larger airways,5,7 we were surprised to detect highly elevated levels of SP-A in lung tissues of COPD patients. This finding was verified by several different approaches including 2-DE, Western blot, immunohistochemistry and morphometry. Furthermore, elevated levels of SP-A were also found from induced sputum supernatants of Stage II COPD. In contrast, no such change in the levels of other surfactant proteins (SP-B, SP-C, SP-D) could be observed in tissue samples of COPD patients. On the other hand, SP-A levels were not elevated in the lung tissue of patients suffering from another kind of pulmonary disorder, IPF. Altogether, this indicates a specific role for SP-A in COPD, but the exact mechanisms of SP-A regulation are not entirely clear. There are numerous co- or post-translational modifications to which SP-A can be subjected, for example, signal peptide cleavage, acetylation, proline hydroxylation, N-linked glycosylation, sialylation, glutamic acid carboxylation and sulfation.27,28,31,32,35 However, since all protein spots were altered, the COPD-dependent changes are likely caused by changes in protein expression rather than modification. Interestingly, human SP-A consists of two proteins (SP-A1, SP-A2) which bear clear similarity but are differentially regulated at the transcriptional level and have been proposed to mediate different functions.7 SP-A2 has been shown to bind to carbohydrates with greater affinity and to stimulate higher levels of TNF-R and IL-8 release from macrophages than SP-A1.7,34,40,41 In addition, SPA-2 has been shown to be more abundant than SP-A1 in BAL fluid, and the ratio between SP-A1 and SP-A2 is also age-dependent.38 However, as far as we are aware, no studies have been performed on specific SP-A1 or SP-A2 proteins in COPD. Our observations reveal that COPD evokes elevation of the level of SP-A, especially SP-A2. SP-A levels have been found to be elevated in serum samples of smokers and patients with COPD.8-10 Controversially, some studies have suggested that serum levels of SP-A in smokers can remain unchanged14 and most BAL or bronchial fluid studies report declined SP-A levels in COPD.15,16,42 This finding suggests that lung damage caused by smoking may not be detectable from the serum or the BAL fluid, at least if SP-A is used as a marker. BAL reflects fluids in the alveoli and small airways (areas that are destroyed in COPD/emphysema), whereas sputum includes secretions from more proximal airways, that is, areas where also SP-A has been detected.5 Moreover, induced sputum contains a significantly lower percentage of macrophages but a higher percentage of neutrophils than BAL fluid.43,44 BAL fluid is difficult to collect from patients with COPD; it requires an invasive procedure (bronchofiberoscopy) and the recovery is often poor and not representative. In contrast, analysis of induced sputum is a widely recognized and validated method in investigating lung pathophysiology in obstructive lung diseases, asthma and COPD. The detection of increased SP-A levels in the induced sputum of Stage II COPD indicates sputum as a useful target to screen for COPD already at an early stage of the disease. Serum samples of IPF patients have been shown to contain higher SP-A levels than the sera of healthy controls or the sera of patients with other interstitial lung diseases, although variable results have been noted as well.10,11,13 The observations on SP-A levels in BAL fluids of IPF patients are controversial.17-19,45,46 In a recent proteomic study on lung tissues, no elevation of level of SP-A in the IPF lung could be detected.47

Surfactant Protein A in COPD

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Figure 3. SP-A localization in human lung tissue specimens. The digital images for morphometric analysis were taken by 200× magnification. For patient characteristics see Supplemental Table S1 (Supporting Information). (A) Immunohistochemical staining using a SP-A -specific antibody. Red-brown color indicates positivity. The alveolar wall lining cells stain mainly positive. High-power field (1000×) using five different planes of focus were combined to create an extended depth of field stack-image. (B) Morphometric quantification of SP-A immunoreactivity in control lung tissue (n ) 7), Stages II-III (n ) 7) and Stage IV COPD (n ) 6). (C) Immunohistochemical double staining using SP-A and TTF-1 antibodies. The red-brown color shows double positivity in the alveolar (left) and bronciolar (right) epithelial cells. (D) Morphometric quantification of SP-A immunihistochemistry in lung tissue of control (n ) 5) and AAT deficiency (n ) 7).

Figure 4. Relative intensity of SP-A by Western blot analysis in sputum of nonsmokers (n ) 10), healthy smokers (n ) 11) and patients with Stage II COPD (n ) 11). For patient characteristics see Supplemental Table S5 (Supporting Information).

In sarcoidosis, most studies have reported that SP-A level is not increased in the serum or BAL fluid.11,12,45,48,49 Therefore, IPF tissue was selected as a disease control group to this study. In the IPF lung, 2-DE, immunohistochemistry and morphometry showed that, generally, SP-A levels were not elevated. The variability in Western blot analysis of the lung specimens of IPF patients may be due to the fact that lung fibrosis is a patchy

Figure 5. SP-B, SP-C and SP-D determined by Western blot analysis; (A) SP-B in control (n ) 4) and COPD Stage II-III (n ) 4) lung tissues. (B) SP-C in control (n ) 4) and COPD Stage II-III (n ) 4) lung tissues. (C) SP-C in control (n ) 4) and COPD Stage II-III (n ) 4) lung tissues. For patient characteristics see Supplemental Table S2 (Supporting Information). Journal of Proteome Research • Vol. 7, No. 12, 2008 5129

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Ohlmeier et al. in some smokers may be a reflection of early cell/lung damage even though there is no apparent decline in the lung function parameters. Whether this is the case needs to be confirmed in larger longitudinal investigations. Since SP-A levels were elevated in the lungs of patients with Stage IV COPD and AAT deficiency years after smoking cessation, the elevation is more likely related to emphysema- and bronchitisrelated changes than smoking alone. The finding that SP-A levels were elevated in the induced sputum of smokers with Stage II COPD in comparison to healthy smokers supports this assumption. SP-A could also be detected in some nonsmokers, suggesting that factors not related to smoking/COPD might influence the SP-A levels in the lung or lung secretions. SP-A levels have been shown to depend on the genotype and age of the patient.35,38 Corticosteroid treatment has been widely used in the treatment of infants with respiratory distress syndrome and surfactant deficiency.50 In the present study, patients with very severe COPD (Stage IV) had received corticosteroid therapy. However, elevation of SP-A level was also observed at earlier COPD stages (Stage II-III), indicating corticosteroid-independent upregulation. To explain the possible variability of SP-A expression in the human lung and lung diseases, further studies will be needed not only on smoking, passive smoking and smoking cessation, but also on the genotype, phenotype, disease severity, infections and other diseases.

Conclusions The level of SP-A protein is elevated not only in the COPD lung but also in the sputum specimens of these patients. This suggests that SP-A might represent a helpful biomarker in the early detection of COPD and other related disorders. The clinical significance of this finding still awaits future investigations.

Figure 6. SP-A in control and IPF lung tissues. (A) Twodimensional gel electrophoresis (2-DE) shows SP-A expression in control (n ) 3) and IPF lungs (n ) 3). For patient characteristics see Supplemental Table S4 (Supporting Information). (B) Morphometric quantification of SP-A immunoreactivity in control lung (n ) 6) and IPF lung tissue (n ) 9). For patient characteristics, see Table S3 (Supporting Information). (C) Relative intensity of SP-A by Western blot analysis in control lung (n ) 3) and patients with IPF lung tissue (n ) 3). For patient characteristics see, Table S4 (Supporting Information).

disease. Overall, these observations suggest that elevation of SP-A is related specifically to emphysematous lung injury, but not to pulmonary fibrosis. Increased SP-A levels observed in some healthy smokers point to a relationship between the level of SP-A and smoking. We are not aware of studies that would show whether cigarette smoke or its constituents affect SP-A expression. In our unpublished observations, a marginal elevation of SP-A level in cultured human lung adenocarcinoma epithelial cells (NCI-H441) could be observed by cigarette smoke, but only transiently and by a low smoke concentration. It is therefore more likely that high SP-A levels 5130

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Acknowledgment. We are grateful for the patients who consented to participate in our study. We thank Anitra Ahonen, Tiina Marjomaa and Eeva-Liisa Stefanius for excellent laboratory assistance. This study was supported by the Funding of Helsinki University Hospital (EVO), Finnish Antituberculosis Association Foundation, the Finnish Medical Foundation, the Jalmari and Rauha Ahokas Foundation, and Yrjo¨ Jahnsson Foundation. Supporting Information Available: Additional Materials and Methods include detailed information about the methods and characteristics of the patients; example of a typical mass spectrum identifying SP-A. This material is available free of charge via the Internet at http://pubs. acs.org. References (1) 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 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. (2) Willemse, B. W.; ten Hacken, N. H.; Rutgers, B; Lesman-Leegte, I. G.; Postma, D. S.; Timens, W. Effect of 1-year smoking cessation on airway inflammation in COPD and asymptomatic smokers. Eur. Respir. J. 2005, 26, 835–845. (3) Barnes, P. J. Chronic obstructive pulmonary disease: a growing but neglected global epidemic. PLoS Med. 2007, 4, e112.

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