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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 724-731, (2002)
© 2002 American Thoracic Society

Original Article

Increased p21CIP1/WAF1 and B Cell Lymphoma Leukemia-xL Expression and Reduced Apoptosis in Alveolar Macrophages from Smokers

Katsuyuki Tomita, Gaetano Caramori, Sam Lim, Kazuhiro Ito, Toyoyuki Hanazawa, Timothy Oates, Irena Chiselita, Elen Jazrawi, K. Fan Chung, Peter J. Barnes and Ian M. Adcock

Department of Thoracic Medicine, National Heart and Lung Institute at Imperial College School of Science, Technology and Medicine, London, United Kingdom

Correspondence and requests for reprints should be addressed to Ian M. Adcock, Ph.D., Department of Thoracic Medicine, National Heart and Lung Institute at Imperial College, Dovehouse Street, London SW3 6LY, UK. E-mail: ian.adcock{at}ic.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alveolar macrophages (AMs) are the predominant defense cells in the airway, and their numbers are increased in smokers and subjects with chronic obstructive pulmonary disease. This increase may result from increased recruitment, increased proliferation, or reduced cell death. Apoptosis regulates inflammatory cell survival, and p21CIP1/WAF1 is an important inhibitory regulator of cycle progression after oxidative stress. We have investigated whether chronic smoke exposure influences the expression and localization of cell cycle and apoptotic proteins in AM and bronchial epithelial cells in vivo. The increased numbers of AMs seen in smokers were only partially due to enhanced proliferation. p21CIP1/WAF1 protein expression was increased in AMs and biopsies isolated from smokers and was found predominantly within the cytoplasm. In addition, B cell lymphoma leukemia (Bcl)-xL, an antiapoptotic regulator, was also highly expressed in macrophages from smokers compared with nonsmokers and subjects with asthma. Hydrogen peroxide, an oxidative stress, induced cytoplasmic expression of p21CIP1/WAF1 and failed to induce apoptosis in an in vitro model. These results suggested that AM and bronchial epithelial cells from smokers, in contrast to those from normal subjects and subjects with asthma, have reduced cell death. Thus, oxidative stress induced by cigarette smoking may contribute to the chronicity of inflammation in the airway, through a reduction of apoptosis.

Key Words: p21CIP1/WAF1 • monocytes/macrophages • apoptosis • cigarette smoke • epithelial cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alveolar macrophages (AMs) are the predominant defense cells in the airway and are capable of phagocytosing inhaled particles. Inhaled particles in cigarette smoke lead to characteristic morphologic (1) and functional changes in AM (2, 3). Smokers have a marked increase in the number of AMs in bronchoalveolar lavage (BAL) fluid and within histologic sections (4). AMs are normally derived from blood monocytes, but some studies have suggested that AM may also replicate within the lungs (5, 6).

Inclusion bodies containing particles from cigarette smoke can be detected within the cytoplasm of AM for at least 2 years after smoking has ceased, suggesting prolonged survival (7). This may be due to enhanced proliferation or increased cell survival. Apoptosis regulates inflammatory cell survival, and thus, its reduction contributes to the chronicity of an inflammatory process. Apoptosis is controlled by suppressing or inducing proteins, such as B cell lymphoma leukemia-2 (Bcl-2) and p53. Bcl-2 and Bcl-xL prevent apoptosis induced by a wide range of agents, including oxidative stress (8–10).

Tobacco smoke is a potent stimulus of DNA damage by oxidant injury (11) and may therefore regulate the expression and activity of proteins associated with the cell cycle. Recent studies have shown that cyclins and cyclin-dependent kinase complexes have important regulatory roles during cell cycle progression (12, 13). Two distinct families of cyclin-dependent kinase inhibitors have been detected in mammalian cells. The Cip/Kip family includes the structurally related proteins p21, p27, and p57, all of which inhibit a variety of cyclin-dependent kinase activities in vitro. The INK4 family includes p15, p16, p18, and p19 and is not structurally related to the p21 family (12). p21CIP1/WAF1 is one of key proteins in regulating cell cycle control and is highly responsive to oxidative stress (14). Translocation of p21CIP1/WAF1 into the nucleus may occur through either a p53-dependent or -independent pathway. p21CIP1/WAF1 translocation via a p53-dependent pathway has been demonstrated in cells damaged by ionizing irradiation or genotoxic agents (15), whereas p21CIP1/WAF1 translocation through the p53-independent pathway occurs in cells undergoing terminal differentiation (16). Recently, it has been reported that cytoplasmic p21CIP1/WAF1 itself acts as an inhibitor of apoptosis (17).

Increased proliferation has been reported to be responsible for the increased numbers of macrophages seen in smokers (18, 19). The increased number of macrophages may also be due to enhanced recruitment. Expression of C-C chemokine receptor 2 (CCR2) is thought to be particularly important for monocyte/macrophage differentiation and recruitment to the airway (20–22).

We have previously reported that AMs from smokers are activated compared with those from normal nonsmokers with respect to interleukin-1–stimulated cytokine release (23). These data suggest that there is prolonged cellular activation of AM after chronic cigarette smoking. We have therefore investigated whether chronic cigarette smoke exposure influences the recruitment, proliferation, expression, and localization of cell cycle and apoptotic proteins in BAL macrophages and airway epithelial cells obtained from nonsmokers and smokers.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
The characteristics of the 10 healthy smokers, 6 normal subjects, and 7 subjects with asthma are summarized in Table 1 . All patients with asthma fulfilled the American Thoracic Society criteria for asthma (24). None of the patients with asthma had suffered a recent exacerbation and were free of acute upper respiratory tract infections. All normal subjects (except one) had normal lung function and negative skin prick tests to common allergens and were life-long nonsmokers.


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  		TABLE 1. Demographic characteristics of subjects

 
We also examined biopsies from three subjects with mild to moderate chronic obstructive pulmonary disease according to the American Thoracic Society criteria (24). Lung parenchyma was obtained from three subjects with chronic obstructive pulmonary disease undergoing lung resection for a solitary peripheral carcinoma, and sections were taken from the areas most distal to the carcinoma. None of the chronic obstructive pulmonary disease subjects had suffered a recent exacerbation and were free of acute upper respiratory tract infections. All chronic obstructive pulmonary disease subjects were nonatopic and had no past history of asthma or allergic rhinitis. The study had the approval of the local ethics committee, and written informed consent was obtained.

Fiberoptic Bronchoscopy, Bronchial Biopsies, and BAL Collection and Processing of BAL Macrophages
Bronchial biopsy collection, BAL, and isolation of BAL macrophages were performed by fiberoptic bronchoscopy as previously described (25). Four bronchial mucosal biopsy specimens were taken from segmental and subsegmental airways of the right lower and upper lobes using size 19 cupped forceps. Biopsy specimens for immunohistochemistry were gently extracted from the forceps and were processed for light microscopy. Two samples were embedded in Tissue Tek II OCT (Miles Scientific, Naperville, IL), frozen within 15 minutes in isopentane precooled in liquid nitrogen, and stored at -80°C. The best frozen sample was then oriented, and cryostat sections that were 6 µm thick were cut for immunohistochemical light microscopy analysis. Two sections at an interval of 100 µm were then appropriately stained using immunohistochemistry. Bronchial biopsies for immunohistochemistry were processed as described later. Bronchial biopsies for Western blot analysis were immediately placed on ice and processed as previously described (26, 27).

In some experiments, BAL macrophages were cultured for up to 3 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, benzylpenicillin (0.1 mg/ml), streptomycin sulfate (0.1 mg/ml), and L-glutamine. Cells for Western blot analysis were immediately placed on ice and processed as described later. Cytospin slides were prepared and dried for 30 minutes. Slides were wrapped in aluminum foil and stored at -70°C before immunostaining.

Western Blotting
Western blotting was performed as previously described (27), and the following antibodies were used at a 1:1,000 dilution: mouse or rabbit anti-human p21, Bcl-xL (Santa Cruz Biotechnology, Calne, UK), and Bcl-2 (R&D, Minneapolis, MN). Bands were visualized by enhanced chemiluminescence as recommended by the manufacturer (Amersham Pharmacia Biotech, Little Chalfont, UK) and quantified using a densitometer with Grab-It and GelWorks software (UVP, Cambridge, UK). The individual band optical density values for each lane of p21CIP1/WAF1, Bcl-2, and Bcl-xL were expressed as the ratio with the corresponding ß-actin optical density value of the same lane.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction for p21CIP1/WAF1
Total cellular RNA was isolated from AMs using the Qiagene RNase easy extraction kit (Rneasy Mini; Qiagene, Crawley, UK) according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction was performed as described previously (27) using 75 pM of the following primers: p21CIP1/WAF1 (F) 5'-CCTCTTCGGCCCAGTGGAC and (R) 3'-CCGTTTTCGACCCTGAGAG and GAPDH (F) 5'-CCACCCATGGCAAATTCCATGGCA and (R) 3'-TCTAGACGGCAGGTCAGGTCCACC for 35 cycles with an annealing temperature of 62°C. Bands were quantified using a densitometer with Grab-It and GelWorks software.

Immunohistochemistry and Immunocytochemistry of p21CIP1/WAF1, p27, p53, Proliferating-Cell Nuclear Antigen, and CCR2
Immunohistochemistry and single-cell immunocytochemistry of proteins were performed as previously described (23) using mouse or goat anti-human p21CIP1/WAF1, p27, p53, proliferating-cell nuclear antigen (PCNA), and CCR2 antibody (dilution 1:100; Santa Cruz Biotechnology) in phosphate-buffered saline/0.1% bovine serum albumin. Stained cells were examined under confocal laser scanning microscope (Leica, Heidelberg, Germany) equipped with a 488/568-nm dual-band argon ion laser.

Immunoreactive cells in bronchial biopsies were quantified in the area 100 µm beneath the epithelial basement membrane in several nonoverlapping high-power fields until all of the available area was covered. The final result, expressed as the number of positive cells per square millimeter, was calculated as the average of all of the cellular counts performed in both slides of each biopsy. Immunoreactive cells were also quantified in the well-preserved epithelium (constituted by columnar and basal epithelial cells) and expressed as number of immunoreactive cells per millimeter of epithelial length. A mean ± SD of 0.750 ± 0.350 mm of epithelium was analyzed in smokers and control subjects; 300 BAL macrophages and cultured cells from each sample were examined for immunostaining, and the number of positive cells or nuclei was counted. For confocal microscopy, sections were stained with p21CIP/WAF (1:50 dilution; Santa Cruz) and visualized using a rhodamine-conjugated secondary antibody. Nuclei were visualized using Vectashield mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK) staining and the cover glass sealed with mounting medium (distyrene/plasticiser/xylene [DPX]). Slides were viewed using confocal microscopy. Confocal scanning laser microscopy images were collected with a Leica confocal microscope equipped with a 488/514-nm dual band argon ion laser. An oil-immersion objective was used. Images were collected using TCSNT software (Leica). Proteins were counted as nuclear if the intensity of staining within the nucleus was clearly greater than that seen with control antibodies. Values are given as mean ± SD. Counting was performed by a colleague in a blinded manner.

Cell Culture
A549 and BEAS-2B cells were cultured as previously described (28) and treated with hydrogen peroxide (H2O2), diamide, or ozone in 0.5% fetal calf serum for various times as indicated in the results.

Measurement of Apoptosis and Cell Cycle Progression
A549 cells were examined for evidence of apoptotic bodies using Hoechst 33,258 dye (bisbenzimide; Sigma) and by fluorescence-activated cell sorter analysis using forward and side scatter and by internuclear content of propidium iodide-stained DNA fluorescence. For fluorescent analysis of DNA unwinding, the amount of unwound double-stranded DNA was detected by binding to Hoechst 33,258 and measuring the fluorescence (29). Cell cycle progression was measured by fluorescence-activated cell sorter analysis using 5-bromo-2'-deoxyuridine and propidium iodide–stained cells.

Data Analysis
Group data were expressed as mean ± SD or median and range, except when stated, where appropriate. Comparisons between data from three groups were tested using the Mann-Whitney U test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Smokers Have Increased Numbers of BAL Macrophages
Viability of AM was more than 90%, as determined by 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye exclusion assay, and cell composition was more than 95% macrophages, as determined by morphology. The numbers of macrophages were found to be increased in BAL samples from smokers (4.0 ± 0.8 x 106 cells) compared with normal nonsmokers (1.0 ± 0.3 x 106 cells, p < 0.05). These macrophages were also more mature, as confirmed by their greater size (68 ± 9% versus 2 ± 3% greater than 30 µM, p < 0.05). There was no difference in CCR2 expression in either group, as measured by immunocytochemistry (88 ± 6% versus 91 ± 6%; Figures 1A and 1B) . We found enhanced numbers of PCNA-positive cells in AMs from smokers (5 ± 2% versus 2 ± 1%, p < 0.05) (Figures 1C and 1D).



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  		Figure 1. Immunocytochemical analysis of CCR2 (A and B) and PCNA (C and D) expression in AM from normal nonsmokers (A and C) and smokers (B and D). Results are representative of six subjects in each group. A graphic representation of the quantification of CCR2 expression is shown beneath and is reported as mean ± SD (n = 6).

 
Expression of p21CIP1/WAF1 Protein
Western blot analysis showed an approximately tenfold higher expression of p21CIP1/WAF1 in smokers (0.25 ± 0.26) as compared with normal subjects (0.01 ± 0.03, p < 0.01) and steroid-naive asthmatic subjects (0.02 ± 0.03, p < 0.05) (Figure 2A) . There was a significant correlation between p21CIP1/WAF1 expression and pack years (r2 = 0.72, p < 0.0001). There was a tendency to reduce the expression of p21CIP1/WAF1 in AM isolated from smokers cultured in Dulbecco's modified Eagle's medium for 3 days (data not shown).



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  		Figure 2. Enhanced p21CIP1/WAF1 protein and mRNA expression in AM from smokers. Whole-cell lysates of AM from 6 normal subjects (N), 10 smokers (Sm), and 7 subjects with asthma (As) were subjected to Western blot analysis for p21CIP1/WAF1 (A). The left panel (A) shows representative Western blotting for N, Sm, and As subjects. The right panel (A) shows the p21CIP1/WAF1/ß-actin ratio in the three groups. Reverse transcription-polymerase chain reaction was used to assess the expression of p21CIP1/WAF1 in AM from N, Sm, and As subjects (B). The right panel (B) shows the p21CIP1/WAF1/GAPDH cDNA ratio in the three groups. Results are expressed as mean ± SD. *p < 0.05; **p < 0.01 compared with N subjects.

 
Expression of p21CIP1/WAF1 mRNA
To investigate whether p21CIP1/WAF1 was upregulated at the transcriptional level in AM from smokers, reverse transcription-polymerase chain reaction was performed (Figure 2B). The average increase in p21CIP1/WAF1 cDNA expression normalized with GAPDH cDNA in AM from smokers (1.04 ± 0.18) compared with those from nonsmokers (0.62 ± 0.12) and patients with asthma (0.41 ± 0.11) was 1.68- and 2.54-fold, respectively. This suggests that enhanced p21CIP1/WAF1 protein expression is due, at least in part, to elevated steady-state mRNA expression either as a result of increased transcription or increased mRNA stability.

Subcellular Localization of p21CIP1/WAF1
It has recently been reported that the intracellular localization decides the distinct function of p21CIP1/WAF1, namely induction of growth arrest when present in the nucleus and as an inhibitor of apoptosis when present in the cytoplasm (17). A negative control without the addition of anti-p21CIP1/WAF1 antibody gave no autofluorescence in AM (Figure 3A) . When viewed with a red fluorescence configuration, AM from smokers had a stronger signal than either of the two nonsmoker groups (compare Figure 3B with Figures 3C and 3D). In AM from smokers, all of the cells showed clear cytoplasmic staining for p21CIP1/WAF1 in contrast to the reduced cytoplasmic levels seen in AM from normal subjects (27 ± 8) and subjects with asthma (31 ± 10) (Figure 3).



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  		Figure 3. p21CIP1/WAF1 expression in AM isolated from smokers. Representative immunohistochemical staining for p21CIP1/WAF1 in AM examined by confocal microscopy. Isotype control antibody gave no autofluorescence of rhodamine red at 570/590 nm (A). After permeabilization and staining by rhodamine red–labeled anti-p21 antibody, AM from a smoker (B), a normal nonsmoker (C), and a subject with asthma (D) gave positive fluorescent staining (internal scale bar = 10 µm). The percentage of cells expressing cytoplasmic p21CIP/WAF is shown graphically in the three groups in the lower panel. Results are expressed as mean ± SD (n = 6).

 
Subcellular Localization of p27 and p53
p27, another member of the Cip/Kip family, showed no difference in expression between the three groups. Positive signals for p27 were demonstrated predominantly in cytoplasm in all groups (Figures 4B and 4D) . In AM from normal subjects but not the other groups, there was weak scattered distribution of p27 within the nucleus (Figure 4C). Because the expression of p21CIP1/WAF1 may occur through either a p53-dependent or -independent pathway, we also investigated the expression of p53. AM from subjects with asthma showed more intense staining for cytoplasmic p53 than those from the other groups (Figure 4H compared with Figures 4F and 4G). However, there was no difference in p53 expression between smokers and nonsmokers.



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  		Figure 4. p27 and p53 proteins are expressed predominantly within the cytoplasm of AM. Representative immunohistochemical staining for p27 and p53 in AM examined by confocal microscopy. Negative control for rhodamine red signal at 570/590 nm (A and E). After permeabilization and staining by rhodamine red–labeled anti-p27 antibody, AM from a smoker (B), a normal nonsmoker (C), and a subject with asthma (D) all expressed positive fluorescent staining. After permeabilization and staining by rhodamine red–labeled anti-p53 antibody, AM from a smoker (F), a normal nonsmoker (G), and a subject with asthma (H) all expressed positive fluorescent staining (internal scale bar = 10 µm).

 
Expression of Bcl-2 and Bcl-xL Proteins
We also examined the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL in AMs by Western blot analysis (Figure 5) . Bcl-xL, but not Bcl-2, was expressed to a much greater extent in AM from smokers (0.26 ± 0.19) compared with normal subjects (0.03 ± 0.02, p < 0.01) and steroid-naive asthmatic subjects (0.03 ± 0.02, p < 0.01).



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  		Figure 5. Increased Bcl-xL expression in AMs isolated from smoking subjects (Sm). The left panel shows representative Western blotting of normal subjects (N), Sm, and subjects with asthma (As). The right panel shows the Bcl-xL/ß-actin ratio in AMs from three groups. Results are expressed as mean ± SD. **p < 0.01, compared with N subjects. Panels i–iv show a representative Western blot indicating no change in Bcl-2 expression in any of the three groups examined.

 
Expression of p21CIP1/WAF1 in Bronchial Epithelial Cells
We have examined whether the expression of p21CIP1/WAF1 was altered in other cells within the airway. Bronchial biopsies from smokers showed a much greater expression of p21CIP1/WAF1 in epithelial cells than that seen in epithelial cells from normal nonsmokers (Figures 6A and 6B) . In all cases, the enhanced expression of p21CIP1/WAF1 was localized predominantly to the cytoplasm (Figures 6A and 6B).



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  		Figure 6. Immunohistochemical detection and confocal analysis of p21CIP1/WAF1 expression in bronchial biopsies from normal nonsmokers (A) and smokers (B). Results are representative of at least five subjects from each group.

 
H2O2 Induces p21CIP1/WAF1 Cytoplasmic Localization and No Cell Death
H2O2 (200 µM) treatment of A549 cells induced a significant increase in cytoplasmic p21CIP1/WAF1 expression in contrast to the nuclear expression of p21CIP1/WAF1 seen after stimulation of the cells with the thiol oxidant diamide (500 µM; Figure 7A) . The induced p21CIP1/WAF1 was inhibited by pretreatment of cells with the antioxidant N-acetyl cysteine (2 mM; Figure 7B).



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  		Figure 7. Immunocytochemical detection of H2O2 (200 µM)-induced cytoplasmic expression of p21CIP/WAF in A549 cells (A). Diamide (500 µM), a thiol oxidant, induced nuclear expression of p21CIP/WAF. Pretreatment of cells with N-acetyl cysteine (2 mM) inhibited the H2O2-induced expression of p21CIP/WAF (B). Results are representative of three separate experiments. Diamide, in contrast to H2O2, induced significant apoptosis as measured by Hoechst 33,258 staining (C) and fluorescent analysis of DNA unwinding (D). Results are expressed as mean ± SEM of at least three independent observations. (E) Fluorescence-activated cell sorter analysis of propidium iodide and 5-bromo-2'-deoxyuridine– labeled cells indicated that both H2O2 and diamide induced a small increase in proliferating cells. Results are expressed as mean ± SEM of at least five experiments.

 
We examined these cells for markers of apoptosis and found very few apoptotic bodies and no change in the number of apoptotic cells at 4 hours after H2O2 treatment (Figure 7C). In contrast, diamide induced abundant nuclear bubbling and a tenfold increase in the number of cells undergoing apoptosis (Figure 7C). These data were confirmed using fluorescent analysis of DNA unwinding, which showed enhanced apoptosis after diamide, but not H2O2, treatment in these cells (Figure 7D).

Analysis of cell cycle progression indicated that both H2O2 and diamide increased to a similar extent the number of cells in the proliferative (G2/M) phase (Figure 7E). This is in contrast to the G1 arrest associated with p21CIP1/WAF1 nuclear overexpression (17, 30).

In BEAS-2B cells, H2O2 (200 µM) induced cytoplasmic localization of p21CIP/WAF in contrast to the predominant nuclear staining seen in control and ozone-treated cells (Figure 8A) . In BEAS-2B cells, 24-hour treatment with ozone (300 parts per billion) induced an increase in apoptosis (15 ± 1% versus 4 ± 0.5%, p < 0.05) with no change in proliferating cells (Figure 8B). In contrast, stimulation of BEAS-2B cells with H2O2 (200 µM) produced similar effects to those seen in A549 cells in that there was no change in apoptotic cells compared with medium-treated cells (Figure 8B). However, in comparison to A549 cells, the number of cells in G2/M increased markedly (Figure 8B).



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  		Figure 8. Immunocytochemical detection of H2O2 (200 µM)-induced cytoplasmic expression of p21CIP/WAF in BEAS-2B cells (A). Ozone (300 parts per billion) maintained nuclear expression of p21CIP/WAF. Results are representative of three separate experiments. (B) Fluorescence-activated cell sorter analysis showing the effects of medium (control), H2O2, and ozone treatment on cell cycle progression in BEAS-2B cells (left panel). The effects of treatments on the percentage of apoptotic cells are shown in the right panel. Results are expressed as mean ± SEM of at least three experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has demonstrated an increased number of AM in BAL samples from smokers. These cells express high levels of p21CIP1/WAF1 predominantly within the cytoplasm. Similarly, the antiapoptotic protein Bcl-xL was also increased in AM from smokers. However, the expression of p27 and p53 in AM was not affected by cigarette smoking. Using a model system in a human alveolar epithelial cell line (A549), we showed that oxidative stress induced cytoplasmic p21CIP1/WAF1 expression in a redox-dependent manner, resulting in little or no apoptosis and slightly enhanced cell proliferation. These effects of H2O2 on proliferation and apoptosis were confirmed in the SV-40–transformed human bronchial epithelial cell line (BEAS-2B).

The increased numbers could not be accounted for completely by increased proliferation within the airways. In fact, the number of PCNA-positive cells is increased only 2.5-fold in smokers, despite their 4-fold increase in cell number. These data are in agreement with earlier work from Bitterman and colleagues (18). This earlier article showed a two or fourfold increase in macrophage proliferation in smokers compared with normal subjects depending on the method used to detect proliferation. Interestingly, when these authors examined in vitro replication of these cells in methylcellulose, they found a sixfold increase in cell numbers in smokers compared with nonsmokers, suggesting that increased replication could not account for all of the increase in cell numbers seen.

We studied cell migration by only one marker, the expression of the CCR2 receptor. The percentage of cells expressing this receptor (approximately 90%) was not increased in smokers. This may not be necessary to enhance recruitment or migration of blood monocytes if the concentration of monocytic chemokines for monocytes is increased in the alveolar space of smokers. In addition, other chemokine receptors not investigated in this study might be responsible for the migration of blood monocytes.

p21CIP1/WAF1 has been shown to bind to cyclin–cyclin-depend- ent kinase complexes, preventing phosphorylation of retinoblastoma protein. When this occurs, the E2F pathway is blocked, and the cell cycle is arrested at the G1/S interface (30). p21CIP1/WAF1 exists in a quaternary complex with a cyclin, a cyclin-dependent kinase, and the PCNA (31). p21CIP1/WAF1 controls cyclin-dependent kinase activity and thereby affects the cell cycle. On the other hand, PCNA functions in both DNA replication and repair. p21CIP1/WAF1 blocks the ability of PCNA to activate DNA polymerase {delta}, the principal replicative DNA polymerase (30). This suggests that p21CIP1/WAF1 may be a key regulator of DNA replication, DNA repair, and the cell cycle machinery.

The presence of p21CIP1/WAF1 and p53 in the cytoplasm was a surprising finding, as it has been generally recognized that p21CIP1/WAF1 and p53 activation are localized to the nucleus in most various cells, such as fibroblasts and epithelial cells. However, it has been recently reported that cytoplasmic p21CIP1/WAF1 itself acts as an inhibitor of apoptosis (17). When the C terminal of p21CIP1/WAF1 is cleaved by caspase-3/CPP32, the p21CIP1/WAF1 fragment fails to arrest the cells at G1, and having lost its nuclear localization sequence moves into the cytoplasm (32). However, this is unlikely to be the case here because we found only a single p21CIP1/WAF1 band by Western blotting. Alternatively, a novel p21CIP1/WAF1-interacting protein, Cip1 interacting zinc finger protein, may regulate the cellular localization of p21CIP1/WAF1 by an oxidant-stimulated process (33). It has recently been reported that cytoplasmic p21CIP1/WAF1 forms a complex with the apoptosis signal-regulating kinase 1, thereby inhibiting the stress-activated mitogen-activated protein kinase cascade and resulting in inhibition of apoptosis (17). This inhibition of AM apoptosis may explain the finding that cigarette smoking induces a marked increase in the number of AM, despite minimal replication.

The Bcl-2 family also plays a role in regulating apoptosis (34, 35). Bcl-2 and Bcl-xL enhance the survival of several cell types and prevent apoptosis induced by a wide range of agents, including oxidative stress (8–10). Bcl-xL controls a common pathway for apoptosis mediated by nitric oxide and oxidants (36). We show here that AM from smokers expressed higher levels of Bcl-xL compared with nonsmokers. This further suggests that AM from smokers may be prevented from undergoing apoptosis.

It is possible that oxidative stress induced by cigarette smoke may contribute to the prolonged induction of p21CIP1/WAF1. It has previously been reported that oxidant-induced DNA damage prolongs the expression of p21CIP1/WAF1 (37). It is also possible that cytokines, such as interferon-{gamma}, transforming growth factors-ßs, or epidermal growth factor may affect the persistent expression of p21CIP1/WAF1; however, this tends to result in G1 arrest (38–40). The reason that some phagocytosed particles can be detected for 2 years in AM from smoker despite smoking cessation (7) may be due to prolonged expression of p21CIP1/WAF1 protein, resulting in enhanced cell survival as a result of the reduction in apoptosis.

Oxidative stress has long been thought of as a trigger for apoptotic cell death; indeed, our study shows this to be the case with the membrane-permeable thiol-specific oxidizing agent diamide. We have found differential effects of diamide and H2O2 on apoptosis in common with some other studies (41, 42). These differential effects may relate to the ability of H2O2, but not diamide, to nitrosylate proteins. Recent evidence, however, suggests a more complex role for oxidants in the regulation of apoptosis (reviewed in 43). Thus, it is becoming clear that H2O2 can suppress caspase activation and activity (44) either through a nitrosylation-dependent process or through a Bcl-2–dependent mechanism (43). In addition, glutathione depletion far from being detrimental has been reported to be protective against apoptosis (45).

Our data have also demonstrated a two or threefold increase in p21CIP1/WAF1 mRNA. Previous data suggest that the redox-mediated increase in p21CIP1/WAF1 mRNA is controlled through a posttranscriptional mechanism resulting from an increase in p21CIP1/WAF1 mRNA stability (46). This increase in stability could be a consequence, of the presence in the 3' untranslated region of the p21CIP1/WAF1 mRNA of several (A + U)-rich sequences including their AUUUA elements, which have been indicated as possible cis-acting regulatory signals of rapid mRNA turnover (47). Thus, increased p21CIP1/WAF1 expression in AM from smokers may be attributed from transcriptional as well as posttranscriptional mechanisms.

In conclusion, this study showed a marked induction of p21CIP1/WAF1 and Bcl-xL in AM from smokers. The distribution of p21CIP1/WAF1 in the cytoplasm and the increased Bcl-xL suggest that AM from smokers may have a prolonged life span due to an inhibition of apoptosis. These data further suggest that antioxidants may have an important therapeutic role in the treatment of cigarette smoking–induced respiratory diseases. Further studies will be directed at determining whether smoking inhibits AM apoptosis in vitro. Cigarette smoke–induced p21CIP1/WAF1 expression and decreased cell death in AMs and epithelial cells not only have implications for airway inflammatory diseases but may also play an important role in lung cancer.


   Acknowledgments
 
Supported by the Clinical Research Committee, Royal Brompton Hospital (London, UK), Glaxo-Wellcome (Stevenage, UK), and the International Union against Cancer.

Received in original form April 2, 2001; accepted in final form March 26, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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