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Increased Granzyme A Expression in Type II Pneumocytes of Patients with Severe Chronic Obstructive Pulmonary Disease

¹ Nutrition and Toxicology Research Institute Maastricht, Department of Respiratory Medicine and ² Department of Pathology, University Hospital Maastricht, Maastricht, The Netherlands; ³ Department of Anatomy and Embryology, Maastricht University, Maastricht, The Netherlands; ⁴ Laboratory of Pneumology, Unit Lung Toxicology, K.U. Leuven, Leuven, Belgium; and ⁵ Centrum voor Integrale Revalidatie Orgaanfalen Horn, Horn, The Netherlands

Correspondence and requests for reprints should be addressed to Juanita H. J. Vernooy, Ph.D., Department of Respiratory Medicine, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. E-mail: j.vernooy{at}pul.unimaas.nl

	ABSTRACT

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Rationale: Chronic obstructive pulmonary disease (COPD) is associated with increased numbers of CD8⁺ cytotoxic T lymphocytes (CTLs) in the lung, but the functional activity of CTLs remains unknown. Granzyme A (GrA) and B (GrB) are serine proteases considered to be important effector molecules of CTLs and natural killer cells.

Objective: To investigate protein and mRNA expression of GrA and GrB in peripheral lung tissue from patients with COPD and control subjects with normal lung function.

Methods: Paraffin-embedded sections of surgical lung specimens from 22 patients with COPD (FEV₁, 22% predicted; GOLD stage 4) and 15 control subjects (FEV₁, 108% predicted) were immunostained for GrA and GrB, and semiquantified on a 3-point scale. Messenger RNA expression in total lung, specific cell types enriched for by laser capture microdissection, and freshly isolated primary cells were determined by reverse transcriptase–polymerase chain reaction.

Measurements and Main Results: GrA and GrB immunoreactivity was observed in CD8⁺ CTLs and CD57⁺ natural killer cells, but also in type II pneumocytes and alveolar macrophages in both groups. Bronchiolar epithelium stained positive for GrA, but negative for GrB. These observations were confirmed by reverse transcriptase–polymerase chain reaction on total lung, laser capture microdissection–enriched specific cell types and freshly isolated primary type II pneumocytes. The scores of GrA-expressing type II pneumocytes were significantly higher in patients with COPD versus control subjects.

Conclusions: GrA and GrB mRNA and protein are detectable in human lung tissue. GrA expression is increased in type II pneumocytes of patients with very severe COPD. These results indicate that GrA may be important in the development of COPD.

Key Words: CD8⁺ cytotoxic T lymphocyte • immunohistochemistry • laser capture microdissection • serine proteases • type II pneumocytes

AT A GLANCE COMMENTARY TOP ABSTRACT AT A GLANCE COMMENTARY METHODS RESULTS DISCUSSION REFERENCES   Scientific Knowledge on the Subject The significant correlation between the number of CD8+ cytotoxic T lymphocytes and the degree of airflow limitation in COPD indicates a role of these cells in the progression of COPD, but the mechanism by which lymphocytes contribute to the pathogenesis of COPD is unknown. What This Study Adds to the Field Granzyme A expression is increased in type II pneumocytes of patients with severe COPD and may be important in the development of this disease.

 
The development of airflow limitation in chronic obstructive pulmonary disease (COPD) is associated with cellular and structural changes in both peripheral and central airways, with a chronic inflammatory pattern that extends from the airways to the lung parenchyma and pulmonary arteries (1, 2). Although there is a distinct pattern of inflammation in each compartment, numerous studies showed a shift in the balance of the CD4⁺/CD8⁺ T-lymphocyte ratio in favor of the CD8⁺, with the CD8⁺ cytotoxic T lymphocytes (CTLs) infiltrating in the central and peripheral airways, the lung parenchyma, and the adventitial layer of the pulmonary arteries (3–7). The chemokine receptor CXCR3 and its ligand CXCL10 are suggested to be involved in the recruitment of the CTLs in peripheral airways of smokers (8). The significant correlation between the number of CTLs and both the emphysema index and the degree of airflow limitation in COPD indicates a role of these cells in the progression of COPD (3–5, 7, 9). To date, the mechanism by which lymphocytes contribute to the pathogenesis of COPD is unknown.

Major effector molecules of CTLs as well as natural killer (NK) cells are the serine proteases granzyme A (GrA) and B (GrB) together with perforin, which are stored in cytotoxic granules (10, 11). Both GrA and GrB are involved in perforin-dependent processes leading to DNA damage and/or fragmentation, although their actions are phenotypically and kinetically distinct, implying that they function by alternative pathways (10, 11). Extracellularly, GrA and GrB can contribute to tissue destruction and remodeling because of their capability of degrading various extracellular matrix proteins (12, 13). In addition, extracellular GrA can induce secretion of IL-6 and IL-8 in lung fibroblasts and other cell types (14, 15), and directly activate certain cytokines (16).

In bronchoalveolar lavage fluid from patients with hypersensitivity pneumonitis, an inflammatory lung disease characterized by the presence of increased numbers of activated CTLs and NK cells, increased levels of GrA and GrB were measured (17). In addition, Nairn and colleagues showed that intracellular GrB levels in CD3⁺ and CD8⁺ bronchoalveolar lavage–derived T lymphocytes were significantly increased in patients with COPD versus control subjects (18). Furthermore, Chrysofakis and colleagues demonstrated elevated cytotoxic activity and increased perforin expression in CTLs isolated from induced sputum of patients with COPD as compared with asymptomatic smokers and nonsmoking subjects (19). To our knowledge, GrA and GrB expression patterns have not yet been analyzed in lung tissue from smokers with and without COPD. The aim of the current study is to investigate the protein and mRNA expression of GrA and GrB in peripheral lung tissue from patients with COPD and control subjects with normal lung function. Some of the results of these studies have been previously reported in the form of abstracts (20–23).

	METHODS

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Subjects
Lung tissue was obtained from 22 patients with very severe emphysema who underwent lung volume reduction surgery (LVRS; group COPD-I). All patients met the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (24) for the diagnosis of very severe COPD (GOLD 4), and were admitted to the pulmonary rehabilitation center Centrum voor Integrale Revalidate Orgaanfalen (CIRO) Horn (Horn, The Netherlands) for 6 weeks before undergoing surgery. In addition, lung tissue from three patients with GOLD-4 COPD who underwent lung transplantation at the University Hospital Gasthuisberg Leuven was collected for isolation of primary type II pneumocytes (group COPD-II). All patients with COPD were ex-smokers who had quit smoking for at least 6 months before the start of the study, with a previous history of at least 20 pack-years of smoking. All patients were prescribed combination therapy of inhaled corticosteroids and long-acting ₂-agonists, tiotropium/ipratropiumbromide and salbutamol, on demand. A history of respiratory diseases other than COPD as well as increased respiratory complaints or respiratory tract infection during the 4 weeks preceding the study were considered as criteria for exclusion.

Lung tissue from 15 control subjects with normal lung function (without cough and/or sputum production) was obtained from lung lobes resected for a solitary peripheral tumor. Five control subjects were current smokers and 10 were ex-smokers at the time of surgery. A smoking history of at least 10 pack-years was used as a criterion for inclusion.

The study was approved by the medical ethical committee of the University Hospital Maastricht and the Faculty of Medicine of K.U. Leuven/University Hospital Gasthuisberg Leuven. All subjects gave their informed consent in writing.

Pulmonary Function Tests
Spirometric measurements (FEV₁ and FVC) were performed by trained lung function technicians using a spirometer (Masterlab; Jaeger, Würzburg, Germany) before and 15 minutes after inhalation of a -agonist via a metered-dose inhaler. All values are expressed as percentages of predicted values. Residual volume (RV) was measured in a body plethysmograph (Viasys, Würzburg, Germany). Diffusing capacity of the lung for carbon monoxide (DL_CO) was assessed by the single-breath method using standard equipment (Masterlab; Jaeger) and expressed as percentage of predicted value.

Immunohistochemistry
Tissue blocks were taken from the subpleural area in the upper lobes (LVRS) or from macroscopic normal lung tissue in the subpleural area at appropriate distance from the tumor (tumor resection), fixed without inflation in 10% phosphate-buffered formalin, and embedded in paraffin wax. Sections (4 µm) were cut and processed for hematoxylin-and-eosin (H&E) staining or immunohistochemical analysis of GrA, GrB, CD8, and CD57.

Granzymes.
Deparaffinized sections were boiled in 10 mM citrate buffer (GrA) or 1 mM ethylenediaminetetraacetic acid (GrB) for antigen retrieval and treated with 5% bovine serum albumin in tris-buffered saline (TBS) to reduce background staining. Mouse monoclonal antibodies against GrA (GA6, M1791; Sanquin, Amsterdam, The Netherlands) and GrB (GA7, M1754; Sanquin) were used followed by biotin-conjugated rabbit anti-mouse IgG antibody (E-0413; Dako Cytomation, Glostrup, Denmark). After applying alkaline-phosphatase–labeled avidin-biotin complex (ABC-AP, K-0376; Dako Cytomation), enzymatic reactivity was visualized using the Red substrate kit I (SK-5100; Vector Laboratories, Burlingame, CA) and sections were counterstained with hematoxylin and mounted. Spleen tissue was used as a positive control. Negative controls for nonspecific binding by omitting the primary detecting antibodies or applying normal mouse IgG instead of the primary antibodies revealed no signal.

Quantification.
The slides were observed using a light microscope (Leica DMRB; Leica Microsystems, Rijswijk, The Netherlands) at a 20x magnification in blinded fashion. Immunostaining for GrA and GrB was examined in three cell types: lymphocytes, type II pneumocytes, and alveolar macrophages. To quantify GrA and GrB expression in each cell type, 5 to 10 fields of lung parenchyma were randomly selected for each section. GrA- and GrB-positive cells were scored semiquantitatively on an arbitrary 3-point scale by two independent observers unaware of the clinical data. This 3-point scale was defined for GrA- and GrB-positive lymphocytes as 1, 0–1%; 2, 1–5%; 3, 5–100% of total number of lymphocytes; for GrA- and GrB-positive pneumocytes as 1, 0–5%; 2, 5–30%; 3, 30–100% of total number of type II pneumocytes; and for GrA- and GrB-positive macrophages as 1, 0–30%; 2, 30–80%; 3, 80–100% of total number of macrophages. The data represent the mean of the two observers; the coefficient of variation between observers was less than 10%.

Double immunostaining.
To determine the lymphocyte subsets that stained positively for GrA or GrB, a validated protocol for sequential double staining was used. GrA and GrB proteins were detected as described above, and sections were then incubated with anti-CD8 (CTLs, NCL-CD8–4B11; Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) or anti-CD57 (NK cells, M1014; Dako Cytomation) antibody, followed by biotin-conjugated rabbit anti-mouse IgG antibody (E-0413; Dako Cytomation) and alkaline-phosphatase labeled avidin-biotin complex. Enzymatic reactivity was visualized using Blue substrate kit III (SK-5300; Vector Laboratories). To obtain a clear interpretation and to check for non–cross-reactivity after consecutive staining with two mouse primary antibodies, the results of single immunostaining were evaluated for all combinations of antigens and compared with those from double labeling. Staining controls were performed by omitting the primary antibody of either the first or the second incubation, or both, whereas secondary antibodies and both substrates were always applied. Double labeling was only visible when both primary antibodies were applied.

RNA Isolation from Total Lung
Total RNA from total lung homogenates (n = 5, COPD, and n = 5, control subjects) was isolated using the Totally RNA kit (Ambion, Austin, TX) and treated with DNase I (Promega, Madison, WI). Total RNA quantity and quality were evaluated by ultraviolet spectrophotometry and agarose gel electrophoresis. One microgram of total RNA was reverse transcribed into cDNA using the Reverse-iT 1st strand synthesis kit (ABgene, Epsome, UK).

Laser Capture Microdissection
Laser capture microdissection (LCM) was performed using the Pixcell II Laser Capture Microdissection system (Arcturus Engineering, Mountain View, CA). Lung tissue blocks from five patients with COPD and five control subjects were embedded in Tissue-Tek OCT compound (Sakura Finetek Europa BV, Zoeterwoude, The Netherlands) and snap-frozen in liquid nitrogen–cooled isopentane before storage at –80°C. Frozen sections (7 µm) on RNase-free uncoated glass slides were stained with hematoxylin, dehydrated, and placed onto the stage of the microdissector system. Lymphoid aggregates, type II pneumocytes, or alveolar macrophages (recognized by morphologic characteristics) were pulsed on separate CapSure macro LCM caps (Arcturus Engineering) (70–90 mW pulse power, 15-µm laser spot diameter, and 1-ms pulse duration). Caps were placed on extraction reservoirs and stored at –80°C. Total RNA was isolated using the Absolutely RNA nanoprep kit (Stratagen, La Jolla, CA). Isolated RNA was DNase I treated and reverse transcribed using Superscript III enzyme (Invitrogen, Breda, The Netherlands) according to the supplier's recommendations.

Human Type II Pneumocyte Isolation and Stimulation
Type II pneumocytes were isolated from explant lung tissue dissected free of pleura, visible vessels, and bronchi (n = 3, COPD) as described previously by Hoet and colleagues (25, 26). An average of 0.7 ± 0.3 x 10⁶ cells were isolated per gram of tissue, and cell viability assessed by trypan blue exclusion was 97 ± 0.5%. Isolated cells were plated in 24-well plates at a density of 5 x 10⁵ cells/well in 1 ml Waymouth's medium 752/1 containing 10% fetal calf serum, 1% glutamine, 1% penicillin–streptomycin solution, and 0.5% fungizone (Gibco Life Technologies, Merelbeke, Belgium). After 48 hours of culture, 80 to 100% confluence was reached, and cultures were incubated for 24 hours in Waymouth's medium in the following ways: (1) without any additions, (2) in 0.1% dimethyl sulfoxide (DMSO), (3) in 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Bornem, Belgium) in 0.1% DMSO, or (4) in 1 µM dexamethasone (Sigma-Aldrich) in 0.1% DMSO. RNA was isolated from freshly isolated type II pneumocytes or (un-)stimulated primary cell cultures using the RNeasy microkit including a DNase I treatment (Qiagen, Valencia, CA). One microgram of total RNA was reverse transcribed into cDNA using the Reverse-iT 1st strand synthesis kit (ABgene).

Polymerase Chain Reaction
Polymerase chain reaction (PCR) detection of GrA, GrB, -actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed according to standard protocols. In addition, expression of surfactant protein C (SP-C) and CD68 was determined to confirm enrichment for type II pneumocytes and alveolar macrophages, respectively. Primers are listed in Table 1. For standardization of the samples, PCR analyses for two housekeeping genes (-actin and GAPDH) were performed on serial dilutions of total lung cDNA. Because the amplified products for these two housekeeping genes showed a very strong correlation (R = 0.819, p < 0.01), only results of -actin are shown.

	RESULTS

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Clinical Findings
Details concerning age, sex, smoking history, and lung function of the patients with COPD and the control subjects from whom lung tissue was obtained at resection are summarized in Table 2. The groups (COPD-I and Control) were similar with regard to age, sex, and body mass index. As expected from the selection criteria, patients with COPD had a markedly reduced value of FEV₁ (% predicted), FEV₁/FVC ratio and diffusing capacity (DL_CO % predicted), and increased RV (% predicted) as compared with control subjects. There was no significant difference in the smoking history (pack-years) between the two groups, but all subjects with COPD had been ex-smokers for at least 6 months (inclusion criterion for LVRS), whereas the control group consisted of 10 ex-smokers and 5 current smokers. The group COPD-II consisted of three patients with severe COPD who underwent lung transplantation (LT). Lung tissue from LT subjects was used to isolate and purify primary type II pneumocytes for in vitro cell stimulation experiments.

GrA and GrB Immunohistochemistry
Immunohistochemical staining with an anti-GrA or anti-GrB antibody revealed positive staining for these serine proteases in all patients with GOLD-4 COPD and in 12 of 15 subjects without COPD. A positive granular immunostaining for GrA and GrB was found in lymphocytes infiltrated in the alveolar walls and interstitial aggregates (Figure 1). Because both CD8⁺ CTLs and CD57⁺ NK cells are known to express GrA and GrB, sequential double staining of selected specimens was performed. Granular staining for GrA was noted in both CTLs and NK cells, and the same was true for GrB. Figure 2 illustrates the colocalization of GrA with CD57 (Figure 2A) and GrB with CD8 (Figure 2B).

View larger version (97K): [in this window] [in a new window]   Figure 1. Immunohistochemistry for granzyme A (GrA) and GrB in lymphocytes. Human lung surgical specimens were formalin fixed and paraffin embedded. Sections were processed for immunohistochemistry using anti-human GrA and GrB antibodies and developed in red with the avidin-biotin complex technique. (A) Granular staining for GrA in interstitial lymphoid aggregates in a subject with very severe chronic obstructive pulmonary disease (COPD). (B) Granular staining for GrA in lymphocytes located in alveolar walls in a subject with very severe COPD. Original magnification, x400.

View larger version (71K): [in this window] [in a new window]   Figure 2. A double-labeling experiment for GrA and GrB in lymphocytes. Paraffin sections from a patient with very severe COPD were processed for immunohistochemistry using anti-human GrA, GrB, CD8, and CD57 antibodies with granzymes developed in red and CD markers in blue. (A) Granular staining for GrA in CD57+ natural killer cells. (B) Granular staining for GrB in CD8+ T lymphocytes. Arrowheads indicate double-labeled cells. Original magnification, x630.

View larger version (78K): [in this window] [in a new window]   Figure 3. Immunohistochemistry for GrA and GrB in type II pneumocytes, bronchiolar epithelium, alveolar macrophages, and pulmonary vessels. Paraffin sections were processed for immunohistochemistry using anti-human GrA (A–E) and GrB (F–J) antibodies and developed in red with the ABC technique. (A) Strong staining for GrA in type II pneumocytes in areas with interstitial fibrosis. (B) GrA staining in bronchiolar epithelium. (C) Diffuse staining for GrA in alveolar macrophages. (D) Absence of GrA staining in pulmonary vessels. (E) No specific staining for GrA when primary antibody was omitted. (F) Moderate staining for GrB in type II pneumocytes. (G) No staining for GrB in bronchiolar epithelium. (H) Diffuse staining for GrB in alveolar macrophages. (I) Pulmonary vessels stained negative for GrB. (J) No specific staining for GrB when primary antibody was omitted. All photographs represent staining for GrA and GrB in patients with very severe COPD. Comparable staining pattern and intensity were observed in control subjects with normal lung function. Arrowheads indicate cells of interest. Original magnification, x630 (A, C, F, H) and x400 (B, D, E, G, I, J).

View larger version (13K): [in this window] [in a new window]   Figure 4. Quantification of GrA-positive (GranA+) type II pneumocytes in patients with very severe COPD and control subjects. The results represent semiquantitative scoring from two independent observers on a 3-point scale. * p < 0.05 versus control.

View larger version (21K): [in this window] [in a new window]   Figure 5. GrA and GrB mRNA expression determined by reverse transcriptase–polymerase chain reaction (RT-PCR) in laser capture microdissection–enriched lymphoid aggregates, type II pneumocytes, and alveolar macrophages from two representative patients with very severe COPD and two representative control subjects.

View larger version (34K): [in this window] [in a new window]   Figure 6. GrA, GrB, and surfactant protein (SP)–C mRNA expression determined by RT-PCR in freshly isolated type II pneumocytes (biological duplicates) as well as after culture for 48 hours and stimulation for 24 hours with 1 µM dexamethasone (DEXA), 100 ng/ml phorbol 12-myristate 13-acetate (PMA), or solvent (0.1% dimethyl sulfoxide [DMSO]). Data from one representative patient with very severe COPD are shown.

	DISCUSSION

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Another potentially interesting observation is that, in addition to CD8⁺ CTLs and CD57⁺ NK cells, resident lung cells also appeared to express GrA and GrB. It was surprising to observe intense staining for GrA in the type II pneumocytes in lungs from patients with very severe COPD, which were resected at LVRS, especially those located in peribronchial and perivascular areas and in alveolar walls with interstitial fibrosis. In addition, bronchiolar epithelial cells displayed GrA expression consistently in all subjects examined. Alveolar macrophages showed moderate staining for GrA, whereas tissue macrophages containing anthracosis pigment were completely negative. Quantifying GrA expression, we found a twofold increase in the number of GrA-expressing type II pneumocytes in patients with COPD as compared with control subjects with normal lung function, whereas the scores for GrA in the other cell types examined were not different between the groups. These data indicate that not only lymphocytes but also type II pneumocytes may participate in the immunologic response in advanced COPD. The latter was also suggested by Vlachaki and colleagues, who demonstrated altered production of SP-A by type II pneumocytes from patients with COPD versus "healthy" smokers (28).

Expression of GrB was most pronounced in lymphocytes, but was also present in alveolar macrophages and type II pneumocytes. Quantifying GrB expression did not show any significant differences between subjects with or without COPD, although the infiltration of GrB-positive lymphocytes tended to be increased in the lung parenchyma of control subjects. Future work is required to establish whether the inflammatory events mediated by GrB-positive lymphocytes are biologically relevant to (ex-) smokers who will eventually develop COPD.

To our knowledge, this is the first study reporting GrA and GrB expression in other cell types than CD8⁺ CTLs and CD57⁺ NK cells. To confirm the data obtained by immunohistologic staining, GrA and GrB mRNA expression was analyzed in total lung and in specific cell types enriched by LCM. By performing PCR analysis on LCM-enriched cells, GrA and GrB mRNA expression was demonstrated in lymphoid aggregates and type II pneumocytes from subjects with and without COPD. To our surprise, no positive signal for GrA and GrB could be detected in alveolar macrophages enriched by LCM from patients with COPD, which showed a moderate staining for GrA and GrB protein. In contrast, LCM-enriched alveolar macrophages from the control group did display mRNA expression for both GrA and GrB. Whether this is due to altered mRNA stability of GrA and GrB in alveolar macrophages of patients with severe COPD remains for further study.

Increased GrA and GrB levels and/or activities have been demonstrated in various lung disorders characterized by inflammation, such as hypersensitivity pneumonitis (17), acute respiratory distress syndrome (29), atopic asthma (30), cytomegalovirus pneumonitis (31), and COPD (this study, and References 18 and 27), but the pathogenetic mechanisms of GrA and GrB in chronic inflammatory lung disorders are not yet elucidated. The tryptase GrA, the most abundantly expressed protease in cytotoxic granules (10), is known to stimulate the production of different inflammatory mediators (tumor necrosis factor [TNF]-, IL-6, and IL-8), which are important in coordinating the lung inflammatory and immune responses (14, 15). Because TNF-, IL-6, and IL-8 are known to be increased in the lungs of patients with COPD, it can be speculated that GrA may contribute, at least in part, to their up-regulation. Being an IL-1–converting enzyme (16), GrA could further amplify the existing local inflammatory response characterizing COPD.

Furthermore, GrA and GrB are known to initiate many of the perforin-dependent events that lead to apoptosis in cells targeted for destruction by cytotoxic cells (10, 11). Recent studies demonstrated increased cytotoxicity of CTLs and an association between CTLs and apoptosis of alveolar cells in emphysema (6, 19). Because of their expression in lymphocytes and type II pneumocytes located within the alveolar walls, it is tempting to speculate that GrA and GrB are involved in alveolar cell apoptosis that may (partially) account for the loss of alveolar wall structures in COPD.

In addition to their inflammatory and apoptotic properties, GrA and GrB have been shown to exert proteolytic action on proteoglycans and collagen, one of the main constituents of the extracellular matrix (12, 13). GrA and GrB may therefore also participate in airway remodeling and tissue destruction in COPD as well as in migration of inflammatory cells into the alveolar wall and space. Taken together, GrA and GrB are potent proteases that may contribute to the chronic state of COPD in various ways, but their targets and precise functions remain to be determined.

There are a number of important limitations to our study. We analyzed GrA and GrB expression in lung tissue from patients with very severe disease selected for LVRS, who represent a subgroup of patients with COPD suffering prevalently from emphysema, and lung tissue from asymptomatic subjects with normal lung function. These groups, representing two extremes, were carefully matched for sex, age, body mass index, and smoking status (pack-years smoked), which have been recognized as potential confounders (32–35). A limitation to the control group was the fact that these patients had primary lung cancer adjacent to the macroscopic normal lung tissue studied, raising the concern that gene expression in the normal lung tissue may have been influenced by the adjacent tumor. This may be especially relevant when studying expression of granzymes, because tumors have the capability to suppress the apoptosis-driven caspase pathways where GrA and GrB are likely to be involved (36). In addition, activation of CTLs and NK cells is known to be affected in lung cancer (37). Although only those specimens without any histologic abnormalities (apart from different stages of emphysema and alveolar macrophages with smokers' pigment) were selected, we cannot exclude that our data are biased by the presence of the tumor. Future studies are therefore required to further assess GrA and GrB expression in normal lung tissue without malignancy of (never, current, ex-) smokers, and compare these data with additional data in the intermediate GOLD classes. In this respect, recently reported data of Boorsma and colleagues showed that the number of GrB-positive CTLs per millimeter squared of airway mucosa was significantly correlated with loss of lung function (FEV₁% predicted) but not with pack-years (38). A second limitation concerns the use of the LCM technique, which is an excellent procedure to isolate (not purify) specific cell types from tissue sections. Given the limited number of frozen lung tissue specimens available, our sample size for the LCM analysis was relatively small. In addition, a disadvantage of LCM is the small number of targeted cells that can be isolated before the RNA in the section becomes degraded. These aspects of LCM combined with an amplification method like PCR may introduce significant errors of the cell source of the presented markers. We addressed these limitations by checking gene expression of cell-specific markers CD68 for macrophages and SP-C for type II pneumocytes (lymphoid aggregates were recognized by general morphology). Furthermore, we isolated primary type II pneumocytes of patients with COPD and were able to demonstrate gene expression of both GrA and GrB in freshly isolated as well as cultured cells, hereby further confirming our data on GrA and GrB expression obtained by immunostaining and LCM. A third limitation to the present study is that we did not measure GrA and GrB activity in lung homogenates, which is important because the function of granzymes is dependent on its serine protease activity. Because Tremblay and colleagues showed that GrA and GrB activity is not inhibited by the three main serine protease inhibitors present in lung (17), we speculate that granzymes detected by immunostaining in peripheral lung from subjects with and without COPD may be biologically active. Last, GrA and GrB are involved in killing invading microorganisms and a viral or bacterial infection may promote the expression of GrA and GrB. All lung specimens examined showed no histologic evidence for a viral or bacterial infection and systemic C-reactive protein levels of all subjects were within the normal range. Therefore, it is very likely that subjects examined did not have a viral or bacterial infection (locally or systemically), but this cannot be excluded with 100% certainty.

In summary, our study provides the first evidence that CD8⁺ CTLs and CD57⁺ NK cells infiltrating the lung tissue express GrA and GrB, and these cells are therefore considered to be activated. Analysis of other cell types demonstrated expression of GrA and GrB protein and mRNA in type II pneumocytes and alveolar macrophages. The scores of GrA-expressing type II pneumocytes were significantly higher in patients with very severe COPD as compared with control subjects with normal lung function. Detailed investigation will be required to confirm these findings in subjects at risk and in patients with COPD suffering from mild to moderate disease to determine GrA and GrB activity levels in COPD, and to elucidate the actions of these serine proteases in lung, which may be critical in understanding the role of CTLs in the pathogenesis of COPD.

	Acknowledgments

 
The authors thank Coby van Run and Jack Cleutjens (Department of Pathology, University Hospital Maastricht, Maastricht, The Netherlands) for technical assistance with the immunohistochemical staining; the thoracic surgeons in the University Hospital Gasthuisberg Leuven (Leuven, Belgium) for providing help in collecting human lung tissue; Nadja Drummen (Department of Respiratory Medicine, University Hospital Maastricht, Maastricht, The Netherlands) for technical assistance with the PCR analyses on primary type II pneumocytes; Dr. A.D.M. Kester (Department of Methodology and Statistics, Maastricht University, Maastricht, The Netherlands); and Prof. Dr. A.M.W.J. Schols (Department of Respiratory Medicine, University Hospital Maastricht, Maastricht, The Netherlands) for expert assistance with the statistical evaluation of the data.

	FOOTNOTES

 
^* These authors contributed equally to this article.

Originally Published in Press as DOI: 10.1164/rccm.200602-169OC on November 30, 2006

Conflict of Interest Statement: J.H.J.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.J.v.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.P.v.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.H.E.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.H.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.J.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.F.M.W. is a member of the scientific advisory boards for GlaxoSmithKline (GSK), Boehringer Ingelheim, AstraZeneca (AZ), and Numico, and received lecture fees from GSK, AZ, and Boehringer Ingelheim. He received research grants between 2002 and 2004 from GSK, AZ, Boehringer Ingelheim, Centocor, and Numico.

Received in original form February 6, 2006; accepted in final form November 30, 2006

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