Background
Chronic obstructive pulmonary disease (COPD) is currently listed as the fifth leading cause of death in the world, and is also an important cause of chronic disability and permanent impairment, representing a major economic and social burden worldwide [
1,
2]. COPD is defined by the Global Initiative for chronic Obstructive Lung Disease (GOLD) as "a disease state characterized by airflow limitation that is not fully reversible, and that is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases" [
3]. Cigarette smoking is by far the most important risk factor for COPD. However, only a susceptible minority (± 15–20%) of tobacco smokers develops clinically significant COPD, suggesting that genetic factors (such as the rare hereditary deficiency of α1-antitrypsin) must modify each individual's risk. Therefore, although the major environmental risk factor for COPD – tobacco smoke – is well known since many years, the cellular and molecular mechanisms that are involved in the pathogenesis of COPD have not yet been fully elucidated.
In COPD, there is a chronic inflammation of the small airways and the lung parenchyma, leading to fixed narrowing of small airways and alveolar wall destruction (emphysema) [
4]. Multiple studies of lung specimens, bronchial biopsies and bronchoalveolar lavage fluid of patients with COPD have demonstrated that this chronic inflammation is characterized by increased numbers of alveolar macrophages, neutrophils and lymphocytes [
5]. Especially CD8
+ T-lymphocytes are increased in the peripheral airways and lungs of smokers with COPD as compared with asymptomatic smokers with normal lung function [
6,
7]. Moreover, the extent of emphysema in smokers has been related to the number of CD3
+ T-cells in the alveolar wall, and CD8
+ T-lymphocytes appeared to be the predominant cells in the alveolar wall of emphysematous lungs [
8,
9]. One of the important functions of CD8
+ T-cells is their cytolytic activity, inducing cell death by perforin mediated lysis and apoptosis by caspase activation [
10]. These data suggest an important role of CD8
+ T-lymphocytes in the pathogenesis of COPD and emphysema.
Recently, Hogg et al. further characterized the nature of the small airway obstruction in patients with COPD of increasing severity [
11]. Progression of COPD from mild disease (GOLD stage 1) to very severe COPD (GOLD stage 4) was associated with thickening of the airway wall and with an increased number of airways containing lymphocytes (not only CD8
+ T-cells, but also CD4
+ T-cells and B-cells). Interestingly, these T- and B-lymphocytes are not only increased in numbers, but are also organized into follicles, especially in patients with severe (GOLD stage 3) and very severe (GOLD stage 4) COPD [
11]. These data suggest that an adaptive immune response develops in COPD patients, but it is not known whether this response is elicited by the toxic particles and gases present in cigarette smoke, or develops in relation to the microbial colonization and infection known to occur in the later stages of COPD. Moreover, it is not known whether the adaptive immune response (including CD8
+ T-cells and B-cells) is required for the development of pulmonary emphysema. Recently, we characterized the time course of cigarette smoke-induced pulmonary inflammation in a murine model of COPD [
12]. In the present paper, we report the effects of subacute (5 weeks) or chronic (24 weeks) exposure to cigarette smoke in severe combined immunodeficiency (
scid) mice, which lack functional T-and B-cells, but exhibit normal differentiation and function of myeloid cells, antigen presenting cells and natural killer cells [
13,
14]. We analysed the inflammatory responses in both the bronchoalveolar lavage (BAL) and pulmonary compartment, and we performed morphometric analysis to quantify the extent of emphysema in
scid mice and wild type animals, exposed to cigarette smoke or control air for 6 months.
Methods
Animals
Homozygous male Fox Chase C.B17-scid mice (8 weeks of age) were obtained from M&B ((Møllegaard and Bomholtgård Breeding and Research Center A/S, Ry, Denmark). Wild type Balb/c mice were used as control mice, as suggested by M&B. All mice were housed in sterilized cages with filter tops and received sterilized food and water ad libitum. The local Ethics Committee for animal experimentation of Ghent University (Ghent, Belgium) approved all in vivo manipulations.
Smoke exposure
Mice (n = 8 per group) were exposed to cigarette smoke (CS), as described previously [
12]. Briefly, groups of 8 mice were exposed to the tobacco smoke of 5 cigarettes (Reference Cigarette 2R4F without filter, University of Kentucky, Lexington, KY) four times a day with 30 minutes smoke-free intervals, 5 days a week for 5 and 24 weeks (1 and 6 months). During the first week of the experiment, mice were exposed to the smoke of only one cigarette a day to acclimatize them to the higher dose during the second week. The smoke:air ratio used in this study was 1:6. The control group was exposed to air. Carboxyhemoglobin levels in serum of smoke-exposed mice was 8.3 ± 1.4 % vs 1.0 ± 0.2 % in air-exposed mice (n = 7).
Bronchoalveolar lavage (BAL)
24 hours after the last smoke exposure mice were weighed and sacrificed with an overdose of pentobarbital (Sanofi, Libourne, France) and a tracheal cannula was inserted. 1 ml of Hank's balanced salt solution (HBSS), free of ionized calcium and magnesium but supplemented with 0.05 mM sodium EDTA, was instilled 4 times via the tracheal cannula and recovered by gentle manual aspiration. The 4 lavage fractions were centrifuged, the cell pellet was washed twice and finally resuspended in 1 ml of HBSS. A total cell count was performed in a Bürcker chamber and the differential cell counts (on at least 400 cells) were performed on cytocentrifuged preparations using standard morphologic criteria after May-Grünwald-Giemsa staining. Flow cytometric analysis of BAL-cells was also performed.
Preparation of lung single cell suspensions
Following BAL, the pulmonary and systemic circulation was rinsed. The left lung was used for histology, the right lung for the preparation of a cell suspension as detailed previously [
15]. Briefly, the lung was thoroughly minced, digested, subjected to red blood cell lysis, passed through a 50 μm cell strainer, and kept on ice until labelling. Cell counting was performed with a Z2 Beckman-Coulter particle counter (Beckman-Coulter, Ghent, Belgium).
Labelling of BAL-cells and lung single cell suspensions for flow cytometry
Cells were pre-incubated with Fc-receptor blocking antibody (anti-CD16/CD32, clone 2.4G2) to reduce non-specific binding. Monoclonal antibodies used to identify mouse DC populations were: biotinylated anti-CD11c (N418) and phycoerythrin (PE)-conjugated anti-I-Ek (14-4-4S). The following antibodies were used to stain mouse T-cell subpopulations: fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (L3T4), FITC-conjugated anti-CD8 (Ly-2), and biotinylated anti-CD3 (145-2C11) monoclonal antibodies. The additional marker used for activation was PE-conjugated anti-CD69 (H1.2F3). Antibodies used to characterize B-lymphocytes were: PE-conjugated anti-CD19 (1D3) together with anti-CD11c. Biotinylated anti-CD11c and anti-CD3 were revealed by incubation with streptavidine-allophycocyanine (SAv-APC). All monoclonal antibodies were obtained from BD Pharmingen (Erembodegem, Belgium), except anti-CD11c (N418 hybridoma, a gift from Prof. M. Moser, Brussels Free University, Belgium).
As a last step before analysis, cells were incubated with 7-amino-actinomycin (7-AAD or viaprobe, BD Pharmingen) for dead cell exclusion. All labelling reactions were performed on ice in FACS-EDTA buffer.
Flow cytometry data acquisition was performed on a dual-laser FACS Vantage™ flow cytometer running CELLQuest™ software (Becton Dickinson, Mountain View, California). FlowJo software
http://www.Treestar.com was used for data analysis.
Histology
The left lung was fixated by gentle infusion of fixative (4% paraformaldehyde) through the tracheal cannula [
12]. After excision, the lung was immersed in fresh fixative during 2 h. The lung lobe was embedded in paraffin and cut in 3 μm transversal sections. Lung tissue samples were stained with hematoxylin and eosin, and examined by light microscopy for histological sections. For each animal, 10 fields at a magnification of 200× were captured randomly from the 4 different zones of the left lung (upper, middle upper, middle basal and basal zone) by lab technicians using a Zeiss KS400 image analyzer platform (KS400, Zeiss, Oberkochen, Germany).
Quantification of emphysema
Emphysema is a structural disorder characterized by destruction of the alveolar walls and enlargement of the alveolar spaces. We determined enlargement of alveolar spaces by quantifying the mean linear intercept (L
m) and destruction of alveolar walls by measuring the destructive index (DI) in air- and CS-exposed mice (n = 7 for air-exposed
Balb/c and
scid mice and CS-exposed
Balb/c mice; n = 6 for CS-exposed
scid mice), as described previously [
12,
16,
17].
Quantification of airspace enlargement was determined after 6 months smoke exposure by measuring the mean linear intercept (L
m), using image analysis software (Image J 1.33). Only sections without cutting artefacts, compression or hilar structures (airway or blood vessel with a diameter larger than 50 μm) were used in the analysis. The L
m was measured by placing a 100 × 100 μm grid over each field. The total length of each line of the grid divided by the number of alveolar intercepts gives the average distance between alveolated surfaces, or the L
m [
16].
The destruction of alveolar walls was quantified by the destructive index (DI) [
17]. A grid with 42 points that were at the center of hairline crosses was superimposed on the lung field. Structures lying under these points were classified as normal (N) or destroyed (D) alveolar and/or duct spaces. Points falling over other structures, such as duct walls, alveolar walls, etc. did not enter into the calculations. The DI was calculated from the formula: DI = D / (D + N) × 100.
Two investigators – blinded to the exposure status and the genotype of the mice – independently measured DI and Lm (interobserver correlation of r = 0.85, p < 0.001).
Morphometric quantification of lymphoid follicles
To evaluate the presence of lymphoid follicles in lung tissue after 6 months smoke exposure (n = 7 for air-exposed Balb/c and scid mice and CS-exposed Balb/c mice; n = 6 for CS-exposed scid mice), lung sections obtained from formalin-fixed, paraffin-embedded lung lobes were subjected to the following immunohistological stainings: (A) B220 staining and (B) CD3/B220 doublestaining. (A) Sections were stained with anti-B220-biotin (BD Pharmingen) after Boehringer blocking (with triton) (DakoCytomation, Heverlee, Belgium). Then slides were incubated with streptavidin horseradish peroxidase and colored with diaminobenzidine (DAB) (both obtained from DakoCytomation). (B) At first, sections were incubated with Boehringer blocking reagent with triton and primary antibody anti-CD3, followed by goat-anti-rabbit biotin (both obtained from DakoCytomation). Then, slides were incubated with streptavidin horseradish peroxidase and colored with DAB. In a second step, sections were stained with anti-B220-biotin after Boehringer blocking (with triton). Finally, slides were incubated with streptavidin alkaline phosphatase (DakoCytomation) and colored with Vector blue (Vector Laboratories, Inc., Burlingame, CA). Lymphoid follicles were counted in the tissue area surrounding the airways (airway perimeter 0–2000 μm). Results were expressed as counts relative to the numbers of airways per lung section.
Measurement of immunoglobulins (Ig)
After 1 and 6 months CS-exposure, IgG and IgM concentrations in serum were determined using a commercially available ELISA kit (R&D Systems Europe, Abingdon, UK).
Measurement of cytokines and chemokines
After 6 months CS-exposure, Macrophage Inflammatory Protein-3α (MIP-3α) and KC (mouse IL-8) protein levels were determined in BAL-fluid using commercially available ELISA kits (R&D Systems Europe, Abingdon, UK).
After 6 months CS-exposure, Interleukin-6 (IL-6), Interleukin-10 (IL-10), Monocyte Chemotactic Protein-1 (MCP-1), Interferon-γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α) and Interleukin-12 (IL-12p70) in BAL-fluid were determined by FACS using the Cytometric Bead Array (CBA – Mouse inflammation kit; BD Biosciences, San Diego, CA, USA), following the manufacturers instructions. A mixture of 6 capture bead populations, each with distinct fluorescence intensities and coated with antibodies specific for IL-6, IL-10, MCP-1, IFN-γ, TNF-α and IL-12p70, were prepared. The CBA capture beads were incubated together with PE-conjugated detection antibodies, test samples or standards, to form sandwich complexes. Following acquisition of sample data using the flow cytometer (FACSCalibur™ flow cytometer, BD Biosciences), the sample results were generated in graphical and tabular format using the CBA Analysis Software (BD Biosciences, San Diego, CA, USA).
Semiquantitative RT-PCR analysis
Total lung RNA was extracted using the RNeasy Midi kit (Qiagen, Hilden, Germany), with an additional DNAse-step. Reverse transcription was performed at 48°C for 30 min followed by 12 min incubation at 95°C for denaturation of RNA-DNA heteroduplexes, and a DNA-amplification with 50 cycles of 95°C for 15 sec and 60°C for 1 min. RT-PCR was performed starting from 10 ng of total RNA, using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, USA). After 1 month CS-exposure, expression of matrix metalloproteinase-12 (MMP-12) mRNA, perforin mRNA, granzyme B mRNA and Macrophage Inflammatory Protein-3α (MIP-3α) mRNA relative to hypoxanthine guanine phophoribosyl transferase (hprt) mRNA, was analysed with the Assays-on-Demand™ Gene Expression Products (Applied Biosystems, USA).
Statistical analysis
Reported values are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed with Sigma Stat software (SPSS 11.0 Inc, Chicago, IL, USA) using non-parametric tests (Kruskall-Wallis, Mann-Whitney U). P-values under 0.05 were considered as significant.
Discussion
In this study we show for the first time that chronic cigarette smoke (CS)-exposure for six months (i) increases the number of innate inflammatory cells in BAL and lungs of both wild type and scid mice; (ii) increases the number of peribronchial lymphoid follicles in wild type mice in contrast to scid mice; and, most importantly, (iii) induces pulmonary emphysema not only in wild type animals, but also in scid mice, despite the complete absence of B- and T-lymphocytes in the latter.
A potential role for T-lymphocytes in the development of pulmonary emphysema in human smokers has been suggested, since cytotoxic CD8
+ T-cells are increased in the lungs of smokers and have the capacity to cause (perforin- or granzym B-mediated) cytolysis and apoptosis (by Fas-Fas ligand interaction or by caspase activation) of alveolar epithelial cells [
6,
7,
9,
10,
19‐
21]. However, the observed correlation between increased T-lymphocytes in the alveolar wall of COPD patients and the extent of emphysema, does not prove a causal relationship. Moreover, considerable variation in the counts of CD8
+ T-cells has been described in bronchial biopsies of smokers with chronic bronchitis [
22].
Therefore, to examine whether the adaptive immune response, consisting of B- and T-lymphocytes, is actually causing pulmonary emphysema in response to CS, we investigated the effects of subacute and chronic CS-exposure in wild type
Balb/c mice and in
scid mice, which lack functional B- and T-cells [
13,
23]. Chronic CS-exposure induced emphysema in both wild type and
scid mice. Since
scid mice have a normal innate immune system, including the epithelial barrier, monocytes/macrophages, neutrophils and highly functional NK cells [
24], these experimental findings underline the important role of innate immunity in the development of pulmonary emphysema. After one month CS-exposure,
scid mice had less neutrophils and macrophages in BAL compared to wild type animals. At six months, however, the number of BAL neutrophils, macrophages and dendritic cells – specialized antigen presenting cells, linking the innate and the adaptive immune system [
25] – were even higher in CS-exposed
scid mice. We speculate that the higher number of neutrophils, macrophages and dendritic cells may indicate a compensatory mechanism for the lack of B- and T-lymphocytes in
scid mice. Cigarette smoke may activate lung resident cells, including epithelial cells and macrophages, which then release inflammatory mediators, including cytokines (eg TNF-α and KC (mouse IL-8)) and chemokines (eg MIP-3α and MCP-1) [
26‐
29]. We demonstrated increased protein levels of TNF-α, KC, MCP-1 and MIP-3α in BAL-fluid upon CS-exposure, thereby attracting neutrophils, macrophages and dendrititc cells to the lungs. Inflammatory cells might contribute to the development of pulmonary emphysema, since they have the capacity to secrete many elastolytic enzymes, such as neutrophil elastase (by neutrophils) and MMP-12 (by macrophages and dendritic cells). Both proteases have been reported to be important in COPD [
4,
30,
31].
Recently, James Hogg et al. characterized the nature of the small airway obstruction in patients with COPD [
11,
32]. Interestingly, the progression of COPD was associated with the infiltration of the airway wall by innate and adaptive immune cells that formed lymphoid follicles [
11]. Although the cause of the appearance of lymphoid follicles in the later stages of COPD (GOLD stage 3 and 4) is not known, it was speculated that this adaptive immune response develops in response to the microbial colonization and infection known to occur in patients with severe COPD. However, whether these lymphoid follicles are required to develop emphysema in response to CS, is currently not known. Our experimental findings in mice pointed out that (i) chronic CS-exposure indeed induced peribronchial lymphoid follicles in wild type
Balb/c mice, but that (ii) pulmonary emphysema still developed even in the absence of any lymphoid follicles, as is the case in the
scid mice. This does not exclude the possibility that the lymphoid follicles could accelerate the decline in lung function in COPD patients or aggravate the extent of CS-induced emphysema in humans. Nevertheless, in this murine model of COPD, the wild type animals did not suffer from more severe emphysema despite the presence of lymphoid follicles.
A possible limitation of the study could be the occurrence of "leakiness" in
scid mice, which leads to the production of some functional B- and T-cells [
33]. However, after chronic exposure to air or CS, only one
scid mouse out of 13 had serum IgG levels exceeding 20 μg/ml, indicating leakiness. Lastly, the CS-induced emphysema in
scid mice could theoretically be attributed to the presence of the NK-cell, since an enhanced activity of these innate immune cells has been described in these animals [
24]. To test this hypothesis, we determined the expression levels of the 2 major cytolytic effector proteins of NK-cells, namely perforin, a pore-forming protein, and granzyme B, a member of the serine proteinase family [
10]. We found elevated RNA levels of granzyme B in
scid mice compared to wild type animals, but these levels of granzyme B did not augment upon CS-exposure in both strains of mice. Moreover, after chronic CS-exposure, the mRNA levels of perforin did not increase in wild type and
scid mice, arguing indirectly against the hypothesis that the NK-cell could drive the CS-induced emphysema in
scid mice (data not shown).
Acknowledgements
We dedicate this paper to the memory of our promotor and mentor, the late Prof. Dr. Romain Pauwels.
The authors thank G. Barbier, E. Castrique, I. De Borle, K. De Saedeleer, P. De Gryze, M. Mouton, A. Neesen, and C. Snauwaert for their technical contribution to this work.
This work was supported by the Fund for Scientific Research in Flanders (FWO Vlaanderen, Research Project G.0011.03) and the Ghent University Bijzonder Onderzoeksfonds/Geconcerteerde OnderzoeksActie (BOF/GOA 01251504). Ingel Demedts is holder of a doctoral research fellowship of the fund of Scientific Research in Flanders (FWO Vlaanderen). Kurt Tournoy is a senior clinical investigator supported by the fund of Scientific Research in Flanders (FWO Vlaanderen).
Authors' contributions
AD carried out the design and coordination of the study, performed the quantification of emphysema and lymphoid follicles, carried out the RT-PCR analysis, performed the data and statistical analysis, helped to interpret the data and drafted the manuscript. TM helped to interpret the data and critically revised the manuscript. KB participated in the RT-PCR analysis. ID performed the Cytometric Bead Array analysis and helped to draft the manuscript. KT critically revised the manuscript. GJ critically revised the manuscript. GB participated in the design and coordination of the study, helped to interpret the data and drafted the manuscript. All authors read and approved the final manuscript.