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Published ahead of print on May 14, 2003, doi:10.1165/rcmb.2002-0235OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 562-570, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0235OC

Cigarette Smoke Prevents Apoptosis through Inhibition of Caspase Activation and Induces Necrosis

Julie A. Wickenden, Murray C. H. Clarke, Adriano G. Rossi, Irfan Rahman, Stephen P. Faux, Kenneth Donaldson and William MacNee

ELEGI/Colt, Phagocyte, and MRC Laboratories, Centre for Inflammation Research, University of Edinburgh Medical School; and Institute of Occupational Medicine, Roxburgh Place, Edinburgh, Scotland, United Kingdom

Address correspondence to: Prof. W. MacNee, ELEGI/Colt Research Laboratories, Wilkie Building, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK. E-mail: w.macnee{at}ed.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Emphysema is characterized by enlargement of the distal airspaces in the lungs due to destruction of alveolar walls. Alveolar endothelial and epithelial cell apoptosis induced by cigarette smoke is thought to be a possible mechanism for this cell loss. In contrast, our studies show that cigarette smoke condensate (CSC) induces necrosis in alveolar epithelial cells and human umbilical vein endothelial cells. Furthermore, study of the cell death pathway in a model system using Jurkat cells revealed that in addition to inducing necrosis, CSC inhibited apoptosis induced by staurosporine or Fas ligation, with both effects prevented by the antioxidants glutathione and dithiothreitol. Time course experiments revealed that CSC inhibited an early step in the caspase cascade, whereby caspase-3 was not activated. Moreover, cell-free reconstitution of the apoptosome in cytoplasmic extracts from CSC-treated cells, by addition of cytochrome-c and dATP, did not result in activation of caspases-3 or -9. Thus, smoke treatment may alter the levels of pro- and antiapoptogenic factors downstream of the mitochondria to inhibit active apoptosome formation. Therefore, unlike previous studies, cell death in response to cigarette smoke by necrosis and not apoptosis may be responsible for the loss of alveolar walls and inflammation observed in emphysema.

Abbreviations: calcium magnesium-free phosphate-buffered saline, CMF-PBS • chronic obstructive pulmonary disease, COPD • cigarette smoke condensate, CSC • dithiothreitol, DTT • glutathione, GSH • glutathione disulphide, GSSG • lactate dehydrogenase, LDH • human umbilical vein endothelial cells, HUVECs • poly (ADP-ribose) polymerase, PARP • reactive oxygen species, ROS • staurosporine, SS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cigarette smoke is a complex mixture of chemicals containing high levels of oxidants and is the major etiologic factor in the development of chronic obstructive pulmonary disease (COPD), of which emphysema is a major component. Emphysema is characterized by enlargement of distal airspaces due to destruction of alveolar wall endothelial cells, epithelial cells, and connective tissue resulting from both protease/antiprotease and oxidant/antioxidant imbalances (1, 2). Recently, it has been proposed that apoptosis of alveolar wall cells occurs in response to cigarette smoking, resulting in progressive cell loss and emphysema (3–7).

The study of cell death, including apoptosis, has attracted intense interest as the physiologic program for deletion of harmful or unwanted cells in vivo. A variety of newly characterized forms of cell death have highlighted the complexity, diversity, and redundancy that exist in a cell's ability to die (8). In general, there are two main forms of cell death: apoptosis and necrosis. Apoptosis is a well-defined programmed response that results in characteristic morphologic and biochemical changes, such as cell shrinkage and the condensation and fragmentation of nuclear material (9). Inappropriate apoptosis has been implicated in many pathologic conditions, such as neurodegenerative disorders and cancer (10). Necrosis, however, is regarded as a passive response to extremes of environmental stimuli, such as heat and ultraviolet light, and is characterized by cytoplasmic swelling, the rapid loss of plasma membrane integrity, and eventually cell lysis (11). It has been documented that the mode of cell death may be dependent on the cell type, the concentration of stimulus employed, and its environmental setting (12).

The most characterized effectors of apoptotic cell death are the caspases, a family of cysteine proteases that interact with each other in a hierarchical manner (13). One pathway involves induction of apoptosis by ligation of surface death receptors such as Fas and tumor necrosis factor. This so-called "extrinsic" pathway results in auto-activation of caspase-8, and the subsequent cleavage of procaspase-3 into its active subunits (14). The "intrinsic" mitochondrial pathway can be activated in response to stimuli such as ultraviolet light and oxidative stress, and results in the release of mitochondrial cytochrome-c, initiating formation of the apoptosome complex (15, 16). Consisting of APAF-1, cytochrome-c, and caspase-9, in the presence of dATP, formation of the apoptosome results in the autoactivation of caspase-9 and again, activation of the effector caspase-3 (17). Through cleavage of a distinct subset of cellular substrates, caspase-3 initiates many of the key changes witnessed during apoptosis, thus explaining how a diverse range of stimuli manifest identical phenotypic outcomes during cell death (13). It has been shown that interference with one or more of these stages may result in inhibition of the entire apoptotic process (18).

Reactive oxygen species (ROS) are molecules that have been implicated in mediating apoptotic processes. Depending on the concentration, ROS have been shown to both promote and inhibit apoptosis (12, 19). Studies have also shown that oxidants, including hydrogen peroxide (H2O2), inhibit the apoptotic process initiated by other stimuli (19, 20–22). Inhibition of the caspase cascade (19–23) and activation of poly (ADP-ribose) polymerase (PARP) (21, 22, 24) have been proposed as mechanisms for the oxidant-mediated inhibition of apoptosis.

The aim of the present study was to characterize cigarette smoke–mediated induction of cell death in epithelial and endothelial cells. An alveolar epithelial type II cell line (A549) and primary human umbilical vein endothelial cells (HUVECs) were chosen as surrogate cells to represent alveolar epithelial type II cells and pulmonary microvascular endothelial cells. Here we demonstrate that, in contrast to previous studies (3, 4, 25–28), cigarette smoke induced necrosis with no evidence of apoptosis, and in addition was able to inhibit apoptosis induced by staurosporine (SS). The cell death machinery is ubiquitous and highly conserved; thus, Jurkat T cells are commonly used to study the mechanisms of cell death, given that they undergo apoptosis readily and display classical apoptotic markers. Therefore, Jurkat cells were used as a model system to determine the effect of cigarette smoke condensate (CSC) on their well-characterized apoptotic pathway. Here we report that cigarette smoke exposure prevents caspase activation, thus inhibiting apoptosis, and instead promoting necrosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All chemicals were of analytical reagent grade and purchased from Sigma Chemical Co. (Poole, UK) unless stated otherwise.

Cell Culture
A549 cells (ECACC; Porton Down, UK) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (LabTech International, Ringmer, UK), 100 U/ml penicillin, 100 µg/ml streptomycin (P/S; Invitrogen, Paisley, UK) and 2 mM L-Glutamine (L-Glut; Invitrogen). A549 cells were seeded into 6-well plates at a density of 0.3 x 106 cells per well or 96 well plates at 9 x 104 cells per well for treatment. Cells were quiesced overnight in serum-free media and subsequently treated under serum-free conditions. HUVECs were grown in EBM-2 media (BioWhittaker, Verviers, Belgium) with supplements supplied by the manufacturer. HUVECs, between passages 4 and 6, were seeded into 24-well plates at a density of 5 x 104 cells per well or 96-well plates at 1 x 104 cells per well, and cultured overnight before treatment. Cells were washed once in calcium magnesium-free phosphate-buffered saline (CMF-PBS) before being exposed to varying concentrations of CSC in full media. Jurkat cells (ECACC) were grown in RPMI supplemented with 10% fetal bovine serum, P/S, and L-Glut and treated at a cell density of 1 x 106 cells/ml with either normal media, 2 µM SS (Calbiochem, Nottingham, UK), 100 ng/ml anti-Fas activating antibody (CH-11; Upstate Biotechnology, Lake Placid, NY), 10% CSC or a combination of 2 µM SS and 10% CSC or 100 ng/ml CH-11 and 10% CSC.

Preparation of CSC
CSC was prepared fresh at a concentration of 1 cigarette/ml in CMF-PBS. Whole smoke from a king-size medium tar filter-tipped cigarette was drawn into a glass syringe and passed over CMF-PBS in a tonometer with agitation (29, 30). The condensate was sterile filtered through a 0.22-mm syringe filter before use.

Determination of Apoptosis and Necrosis
Acridine orange/ethidium bromide staining was performed as previously described (31). Briefly, treated cells were stained with 4 µg/ml acridine orange and 4 µg/ml ethidium bromide and visualized by epifluoresence microscopy. Viable (normal, green nuclei), early apoptotic (condensed, green nuclei), late apoptotic (condensed, red nuclei), and necrotic (normal, red nuclei) cells were counted. For assessment of DNA fragmentation, 2 x 106 cells were lysed in 500 ml 7 M guanidine hydrochloride and applied to Wizard SV miniprep columns (Promega, Madison, WI). Columns were centrifuged at 10,000 x g for 2 min, column wash solution (9 mM Tris.Cl pH 7.4, 90 mM NaCl, 2.25 mM EDTA, 55% ethanol) was applied and the columns were re-centrifuged. The wash was repeated before eluting DNA with 50 ml TE/RNase and performing electrophoresis on a 1.8% agarose gel. The lactate dehydrogenase (LDH) assay (Roche, Lewes, UK) was performed on cells grown in 96-well plates. Treated cells were incubated at 37°C for times as indicated, plates centrifuged at 250 x g for 10 min and 100 µl of supernatant transferred to a fresh 96-well plate. The LDH assay was performed as per manufacturer's instructions. Jurkat cells were prepared by Cytospin (Shandon, Pittsburgh, PA), stained with DiffQuick (Dade Behring, Marburg, Germany), and viable, apoptotic and necrotic cells counted by brightfield microscopy (Olympus, London, UK). Two Cytospins were prepared for each treatment and at least 300 cells counted per slide.

Cell-Free Apoptosis Assay and Western Blotting
The assay was performed as previously described (32). Briefly, Jurkat or A549 cells, were incubated in cell extract buffer (CEB, 20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 100 µM PMSF, 10 µg/ml leupeptin, 2 µg/ml aprotinin) on ice for 10 min, before passing through a 25-G needle ten times and centrifuged at 10,000 x g for 15 min at 4°C. Supernatants were removed and stored at -70°C until used. Cell-free apoptosis was initiated by the addition of 10 µM cytochrome-c and 1 mM dATP to the extracts, followed by incubation at 37°C. Aliquots were removed at the time points indicated, 4x Laemmli buffer added, and the sample heated to 96°C for 5 min. Samples equivalent to 1 x 106 cells were analyzed by Western blot using a polyclonal anti–caspase-3 antibody, or a polyclonal anti–caspase-9 antibody (Pharmingen, Oxford, UK) recognizing both pro- and active forms. The signal was detected using a horseradish peroxidase–conjugated secondary antibody (Santa Cruz, Wembley, UK) and enhanced chemiluminesence (ECL; Amersham, Little Chalfont, UK).

Assessment of Recombinant Caspase Activity
Caspase-3 activity was determined using the caspase-3 assay kit (Calbiochem), as per manufacturer's instructions. Briefly, active caspase-3 (30 U) was placed into a half-volume 96-well plate before addition of inhibitor, 1, 5, 10% CSC, or 1 mM H2O2. The plate was incubated at 37°C for 1 h before addition of colorimetric caspase-3 substrate, DEVD-pNA. Caspase-3 activity was determined by measuring the change in absorbance at 405 nm after 2.5 h at 37°C.

Measurement of Glutathione Levels
Four sets of glutathione (GSH) standards were prepared in KPE buffer (0.1 M phosphate buffer, 5 mM EDTA, pH 7.4), CSC added to a final concentration of 1, 5, or 10%, and solutions incubated with agitation for 1 h at 37°C. GSH levels were measured using a microplate assay adapted from the enzymatic method developed by Tietze and coworkers (33) and Vandeputte and colleagues (34). In brief, 1.67 U/ml GSH reductase and 0.2 mg/ml dithiobisnitrobenzoic acid were added to the GSH standards for 30 s to enable the conversion of GSSG to GSH before 0.2 mg/ml reduced ß-nicotinamide adenoside diphosphate (ß-NADPH) was added. The change in absorbance was measured over 2 min at 405 nm in a microplate reader (MRX).

Statistical Analysis
ANOVA was performed using the MiniTab package with Tukey's post-testing to determine significance between treatments. A P value < 0.05 was deemed significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CSC Induces Necrosis in Epithelial and Endothelial Cells
A significant increase in LDH release from A549 cells and HUVECs was seen in a dose-dependent manner after a 24-h exposure to CSC (Figure 1A). LDH release from A549 cells was also time-dependent (Figure 1B). In addition, acridine orange and ethidium bromide staining revealed that CSC induced necrosis, with no evidence of apoptosis in either A549 cells or HUVECs (Figures 2 and 3). The absence of apoptosis in response to CSC was further confirmed by a number of methods, including electron microscopy, oligonucleosomal DNA fragmentation, and chromatin condensation assessed by Hoechst 33342, over a range of doses and time points (data not shown). In contrast, however, apoptosis was induced by staurosporine in both cell types (Figures 2E and 3E). Interestingly, A549 cells co-cultured with SS and CSC together did not die by apoptosis, but instead underwent necrosis (Figure 4), thereby implicating a potential for a direct inhibitory effect of CSC on the apoptotic machinery. These results clearly demonstrate that epithelial and endothelial cells undergo necrosis, not apoptosis, in response to CSC.



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  		Figure 1. CSC induces necrosis in A549 alveolar epithelial cells and HUVECs. (A) A549 cells (open bars) and HUVECs (closed bars) were exposed to 1–10% CSC for 24 h and the percentage of LDH released into the culture media was determined, compared with a total lysis control (1% Triton X-100). (B) Cytotoxicity to A549 cells in response to 10% CSC (closed squares) was time-dependent; control media (closed diamonds). Results are mean of three experiments ± SEM. ***P < 0.001 compared with control.

 


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  		Figure 2. CSC induces necrosis in A549 alveolar epithelial cells with no evidence of apoptosis. A549 cells were exposed to media alone (A), 1% CSC (B), 5% CSC (C), 10% CSC (D), 2 µM SS (E), or 5 mM H2O2 (F) for up to 24 h. Acridine orange and ethidium bromide staining was performed, and the percentage of viable (white bars), apoptotic (gray bars), and necrotic (black bars) cells was determined (G, 24 h shown). Note classical apoptotic nuclei in E (arrows) and absence of this morphology in B, C, and D. Results are mean of three experiments ± SEM. ***P < 0.001 compared with control. Magnification: x36.

 


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  		Figure 3. CSC induces necrosis in HUVECs with no evidence of apoptosis. HUVECs were exposed to media alone (A), 1% CSC (B), 5% CSC (C), 10% CSC (D), 2 µM SS (E), or 5 mM H2O2 (F) for up to 24 h. Acridine orange and ethidium bromide staining was performed and the percentage of viable (white bars), apoptotic (gray bars), and necrotic (black bars) cells was determined (G, 24 h shown). Note classical apoptotic nuclei in E (arrows) and absence of this morphology in B, C, and D. Results are mean of three experiments ± SEM. ***P < 0.001 compared with control. Magnification: x36.

 


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  		Figure 4. CSC prevents apoptosis and induces necrosis in A549 cells. A549 cells were incubated with either media alone, or media containing CSC, 2 µM SS, 5 mM H2O2, or a combination of SS and CSC for 24 h. The percentage of viable (white bars), apoptotic (gray bars), and necrotic (black bars) cells was determined following acridine orange and ethidium bromide staining. Results expressed as the mean of three experiments ± SEM. ***P < 0.001 compared with control. 1% CSC/SS and 5% CSC/SS were significantly different from smoke alone treatments (P < 0.001).

 
CSC Prevents SS- and CH-11–Induced Apoptosis, Resulting in Necrosis
The Jurkat cell is ubiquitously used to study the "core" molecular machinery of cell death, thus this model was employed to elucidate the effect of CSC on the caspase pathway in particular. Jurkat cells treated simultaneously with SS or CH-11 underwent the classical morphologic and biochemical changes indicative of apoptosis, such as chromosomal condensation and oligonucleosomal DNA fragmentation, or laddering (Figures 5A–5C). However, as observed with A549 cells, apoptosis did not occur when CSC was added to the SS- or CH-11–treated cells or after CSC treatment alone (Figures 5D–5G). Morphologically, the predominant form of cell death in the presence of CSC was necrosis. This necrotic mode of cell death was underscored by the concurrent release of LDH from the cells treated with CSC or a combination of SS and CSC over 24 h (Figure 6). A low level of LDH release was observed from cells incubated with SS alone and this was attributed to secondary necrosis given the absence of phagocytic clearance. These results indicate that cigarette smoke prevents apoptosis induced by SS or CH-11, and instead promotes necrosis.



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  		Figure 5. CSC switches apoptosis induced by SS or CH-11 to necrosis. Jurkat cells were treated with SS, CH.11, 10% CSC, or a combination as indicated. After a 6-h exposure (D) or 9-h exposure (E), death was assessed by morphology and the percentage of normal (A, white bars), apoptotic (B, gray bars), and necrotic (C, black bars) cells was determined. Results represent mean of three experiments ± SEM, where at least 300 cells were counted per slide. ***P < 0.001 compared with control. Apoptosis was confirmed by the presence of oligonucleosomal DNA fragments, or so-called "ladders" (F, G).

 


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  		Figure 6. CSC induces LDH release from Jurkat cells in a time-dependent manner. Cells were treated with control media (diamonds), 2 µM SS (squares), 10% CSC (triangles), or a combination of SS and CSC (open circles), and the level of released LDH measured over 24 h was determined. Results are expressed as percentage LDH release compared with a total lysis control. Mean of three experiments performed in triplicate ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with untreated cells.

 
To ascertain more accurately the stage of apoptosis that was inhibited, Jurkat cells were treated with SS and 10% CSC was added at hourly intervals, after which the cells were coincubated for the remainder of the experiment. A higher ratio of apoptotic cells to necrotic cells was seen as the time between SS and CSC treatment increased (Figure 7). This suggests that CSC may impede an early phase in the apoptotic pathway, as CSC only affects apoptosis when present in the early stages of the process.



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  		Figure 7. Cigarette smoke can switch SS-induced apoptosis to necrosis when added after SS treatment. Jurkat cells were treated with 2 µM SS, followed by addition of 10% CSC at hourly intervals. Six hours after initial SS treatment cytospins were prepared and the percentage of normal (white bars), apoptotic (gray bars), and necrotic (black bars) cells was determined (A). Apoptosis was confirmed by the presence of oligonucleosomal DNA "ladders" (B). Results are mean of three experiments ± SEM, where at least 300 cells were counted per slide. ***P < 0.001 compared with control.

 
CSC-Induced Necrosis Is Not Mediated by Oxidative Stress
Inhibition of apoptosis and induction of necrosis has been demonstrated in response to oxidants such as H2O2 (20–22), which is present in cigarette smoke (35). Therefore, to determine whether the oxidant component of cigarette smoke was mediating inhibition of apoptosis, SS-exposed Jurkat cells were treated with CSC in the presence of various antioxidants and evaluated by morphology and DNA laddering. No effect was seen on the necrosis-inducing ability of CSC in the presence of the antioxidant mannitol. However, GSH and dithiothreitol (DTT) prevented necrosis induced by CSC, with cells remaining viable, whereas cells cocultured with SS and CSC underwent apoptosis rather than necrosis (Table 1 and Figure 8).


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  		TABLE 1 The antioxidants DTT and GSH prevent CSC-induced necrosis and inhibition of apoptosis

 


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  		Figure 8. Activation of caspase-3 and subsequent DNA "laddering" does not occur in Jurkats treated with CSC or SS/CSC. Cells were treated with 2 µM SS and/or 10% CSC in the presence of the thiol antioxidants DTT or GSH, or mannitol. Activation of caspase-3 was determined by Western blot for loss of the pro-form and the resultant DNA "laddering" was determined by agarose gel electrophoresis. Representative of three experiments.

 
An alternative role for GSH in vivo is in the detoxification of electrophilic compounds by direct conjugation via the thiol group. A dose-dependent decrease in measurable GSH was observed after incubation with CSC (results expressed as percentage of control: 1% CSC, 80.66 ± 4.4; 5% CSC, 17.97 ± 5.15, P < 0.001; 10% CSC, 7.11 ± 0.44, P < 0.001). The assay employed measures both reduced (GSH) and oxidized (GSSG) glutathione. If oxidation had taken place, then no decrease in total GSH would have occurred. Thus, this data implies that cigarette smoke components may conjugate with GSH.

In addition, unlike in response to oxidants (21, 22, 24), the presence of a PARP inhibitor, 3-aminobenzinamide, did not alter the relative levels of apoptosis and necrosis in response to SS and CSC (data not shown), implying that depletion of ATP as a result of PARP activation is not involved in CSC-mediated cell death. These data suggest that oxidative stress induced by CSC is not responsible for inhibition of apoptosis and induction of necrosis.

CSC Inhibits Caspase-3 Cleavage but Not Activity
Because it was established that CSC affected an early stage of apoptosis, caspase activation was investigated. Caspases can be inhibited by direct modification; however, CSC had no direct effect on recombinant caspase-3 activity (data not shown). Caspases exist as an inactive proform that is cleaved to yield active subunits; thus, activation can be monitored by Western blot. Assessment of caspase-3 activation in Jurkat cells revealed cleavage of the proform after SS treatment, but not after CSC or coculture with SS and CSC (Figure 8), indicating that the caspase pathway may be halted in the presence of CSC. Furthermore, when cells were treated with SS and CSC in the presence of DTT or GSH, caspase-3 cleavage occurred, whereas mannitol was again not effective (Figure 8). Thus, GSH and DTT are able to "quench" or antagonize components in CSC that prevent caspase-3 activation.

Cell-Free Reconstitution of the Apoptosome Reveals CSC-Mediated Inhibition of Apoptosome Formation
To ascertain how CSC affected the caspase pathway, an active apoptosome was reconstituted in a cell-free system with cytochrome-c and dATP, in either the presence of CSC, or in extracts prepared from cells treated with CSC. No effect on activation of caspase-3 was observed when CSC was added to the lysates directly (data not shown). However, in extracts from Jurkats exposed to CSC for 2 h before preparation, neither caspase-3 nor caspase-9 activation occurred, in contrast to the progressive processing to the active form seen in lysates from untreated cells (Figures 9A–9D). This caspase inhibition was not dependent on PI3-kinase activation, due to the lack of effect of the inhibitor LY294002 (data not shown). Moreover, caspase-3 was not cleaved after reconstitution of the apoptosome in lysates from A549 cells treated with CSC (Figure 9E). Thus, cigarette smoke prevents apoptosis by inhibiting the formation of an active apoptosome complex and the subsequent activation of caspase-9 and -3.



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  		Figure 9. CSC treatment inhibits apoptosome formation and subsequent activation of the caspase pathway. Jurkat cells were incubated for 2 h with either control media (A, C) or 10% CSC (B, D). Cytoplasmic extracts were prepared and reconstitution of the apoptosome was initiated by addition of 10 mM cytochrome-c and 1 mM dATP. Western blotting for caspase-3 (A, B), or caspase-9 (C, D) indicated that apoptosome formation and activation of the caspase cascade did not occur in CSC-treated lysates. Similarly, CSC treatment inhibited apoptosome formation and caspase-3 activation in identically prepared A549 cell lysates (E). Result is representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cigarette smoking is the major etiologic factor in the pathogenesis of emphysema. The gas phase of cigarette smoke contains many free radicals and oxidant molecules, with the potential to generate additional oxidant molecules via redox cycling, leading to an increased oxidative burden in the lungs of smokers (35). Therefore, cigarette smoke inhalation leads to a depletion of antioxidants, release of inflammatory mediators, and an increase in epithelial permeability (36–39). Recent evidence has suggested that apoptosis of lung cells may be a factor in cigarette smoke–induced emphysema (3–5, 7, 40). In contrast, our studies show that CSC does not induce apoptosis and in fact induces necrosis in the alveolar epithelial type II cell line (A549) and primary endothelial cells (HUVECs). Interestingly, CSC also prevented apoptosis induced by SS in A549 cells.

Oxidative stress is responsible for many of the effects of cigarette smoking. Given that oxidants, such as hydrogen peroxide, have been shown to inhibit apoptosis and induce necrosis (19–22) we hypothesized that CSC may function in a similar manner. Jurkat cells, which undergo apoptosis readily and display easily identifiable markers, treated with CSC underwent necrosis. Moreover, Jurkat cells cocultured with a combination of SS or CH-11 and CSC also underwent necrosis, comparable to A549 cells, with no evidence of apoptosis. Therefore it appeared that CSC was inhibiting apoptosis and promoting necrosis.

An investigation into the role of oxidants demonstrated that GSH and DTT protected against CSC-induced necrosis, and prevented the inhibition of apoptosis usually seen during SS and CSC coculture. However, the antioxidant mannitol was ineffective. GSH is an important antioxidant in vivo; however, it also fulfils other vital roles such as regulation of immune function, signal transduction, metabolism, and the detoxification of electrophilic compounds (41–45). Detoxification occurs via the thiol group under the control of GSH–S-transferases, although conjugation is also observed in the absence of the enzyme (43, 45). A dose-dependent decrease in measurable GSH, in the absence of GSSG formation, after incubation with CSC was observed, indicating that GSH may form conjugates with the many electrophilic compounds present in cigarette smoke. Considering that the thiol compounds GSH and DTT were both able to prevent CSC-induced necrosis and CSC-mediated inhibition of apoptosis, whereas mannitol was not, indicates that electrophilic compounds, not oxidants, may be responsible for these effects.

To elucidate the mechanism of apoptosis inhibition, the caspase pathway was studied in more detail. Western blot analysis revealed that caspase-3 activation did not occur in cells treated with CSC. Moreover, treatment with GSH and DTT, and not mannitol, prevented the inhibitory effect of CSC on caspase activation. Caspases contain a central thiol group that is essential for function and prone to oxidation, alkylation, and s-nitrosylation (46). However, direct addition of CSC to recombinant caspase-3 had no effect on the ability of the enzyme to cleave its substrate.

The effect of CSC on caspase activation was studied further by reconstitution of the apoptosome, in cytoplasmic extracts of Jurkat cells, on addition of cytochrome-c and dATP. The occurrence of caspase-3 cleavage, as determined by Western blot, was used as a positive indicator of apoptosome formation and caspase-9 activation. Initially, CSC was incubated with the extracts before addition of cytochrome-c and dATP, whereby caspase-3 cleavage was observed, indicating that CSC had no direct effect on formation of an active apoptosome. Interestingly, in extracts from Jurkat cells treated with CSC for 2 h before preparation, caspase-3 activation was prevented. Moreover, caspase-9 activation did not occur, indicating CSC treatment prevented the formation of a functional apoptosome. Importantly, apoptosome formation and caspase-3 cleavage was also prevented by CSC treatment of A549 lysates, thus underscoring this effect on the cell death machinery to be more general and not Jurkat-specific. Numerous inducible regulators of apoptosis exist, presenting the possibility that CSC exposure may mediate an alteration of the intracellular balance between pro- and antiapoptogenic factors.

Although Jurkat cells were used as a model here, the involvement of T cells in the development of emphysema must not be overlooked. Increased numbers of T cells are observed in the lungs of emphysema sufferers, and their presence is correlated with increased lung destruction (47). Necrosis of T cells present in the lung may also contribute to the progression of emphysema by increasing local tissue damage by release of intracellular contents.

The data presented here are in contradiction to some previous studies (3, 4, 25–28). However, a number of these have used TUNEL nick-end labeling to identify apoptotic cells (26, 28), a method that merely detects DNA strand breaks. Cigarette smoke exposure results in oxidant-induced DNA strand breaks (48), and so these studies may have inadvertently identified cells with oxidant-induced DNA damage as apoptotic. Moreover, caspase activation was either not involved (4), or not studied (27), in "cigarette smoke–induced apoptosis". Characterization of cell death is becoming increasingly complex. A number of alternative forms of cell death have been identified with many of the "classical" markers of apoptosis evident. However, in some cases cell death is independent of caspase activation (8). For this reason, it is becoming increasingly necessary to characterize cell death by a number of methods. This presents a plausible explanation as to why previous studies have purported to observe apoptosis in response to smoke exposure. Additionally, no standardized protocol for the production of CSC exists; each procedure can isolate a slightly different spectrum of components. Nevertheless, we believe that our method of exposing cells to CSC replicates the situation in a smoker's lung, whereby passing the smoke over the buffer in the tonometer system more accurately mimics smoke filling the airspace and exposing the lung lining fluid.

The key finding of this study is that cigarette smoke induces necrosis in alveolar type II cells, endothelial cells, and Jurkat cells. Moreover, CSC inhibited caspase activation and apoptosis in A549 and Jurkat cells. From these findings, we suggest a mechanism whereby cigarette smoke may induce emphysema. Although initially thought to be due to apoptosis (3–5, 7, 40), emphysema may in fact result from loss of alveolar tissue by necrosis of lung epithelial and endothelial cells. In these experiments, the effects of CSC could be prevented by the presence of extracellular thiol compounds such as GSH, which is native to the lung and forms one of the most important lung antioxidant defenses (49). Therefore, it could be deduced that the risk of necrosis in response to cigarette smoking is insignificant. However, in situations of acute smoking, GSH has been shown to be decreased in lung lining fluid with a subsequent rebound to levels higher than those of nonsmokers (36, 38, 50, 51). During this window of antioxidant depletion, the cells of the lung are likely to be susceptible to necrotic cell death as a consequence of additional cigarette smoke exposure. Moreover, local tissue damage may be amplified by the subsequent release of intracellular enzymes and lysosomal contents, resulting in recruitment of inflammatory cells to the site of injury and further necrosis of surrounding tissue. This scenario is supported by Retamales and coworkers (52), who observed increased inflammation in increased severity of emphysema. Thus, although much interest has been generated on the involvement of apoptosis, our studies emphasize the role of necrosis and the subsequent proinflammatory responses as more likely candidates in the pathogenesis of emphysema.


   Acknowledgments
 
The authors thank Drs. Peter Henriksen and Jean-Michel Sallenave for supply of HUVECs, Drs. Ellen Drost and Peter Gilmour for helpful advice and comments on the manuscript, and The Colt Foundation for financial support.

Received in original form November 6, 2002

Received in final form May 7, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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