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Published ahead of print on January 9, 2003, doi:10.1164/rccm.200212-1396OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1083-1089, (2003)
© 2003 American Thoracic Society

Original Article

Macrophage Metalloelastase Mediates Acute Cigarette Smoke–induced Inflammation via Tumor Necrosis Factor-{alpha} Release

Andrew Churg, Rong D. Wang, Hsin Tai, Xiaoshan Wang, Changshi Xie, Jin Dai, Steven D. Shapiro and Joanne L. Wright

Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada; and Division of Respiratory Medicine, Brigham and Women's Hospital, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Andrew Churg, M.D., Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5. E-mail: achurg{at}interchange.ubc.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cells and proteases that mediate cigarette smoke–induced emphysema are controversial, with evidence favoring either neutrophils and neutrophil-derived serine proteases or macrophages and macrophage-derived metalloproteases as the important effectors. We recently reported that both macrophage metalloelastase (MMP-12) and neutrophils are required for acute cigarette smoke-induced connective tissue breakdown, the precursor of emphysema. Here we show how these disparate observations can be linked. Both wild-type (MMP-12 +/+) mice and mice lacking MMP-12 (MMP-12 -/-) demonstrated rapid increases in whole-lung nuclear factor-{kappa}B activation and gene expression of proinflammatory cytokines after cigarette smoke exposure, indicating that a lack of MMP-12 does not produce a global failure to upregulate inflammatory mediators. However, only MMP-12 +/+ mice demonstrated increased whole-lung tumor necrosis factor-{alpha} (TNF-{alpha}) protein or release of TNF-{alpha} from cultured alveolar macrophages exposed to smoke in vitro. Levels of whole-lung E-selectin, an endothelial activation marker, were increased in only MMP-12 +/+ mice. These findings suggest that, acutely, MMP-12 mediates smoke-induced inflammation by releasing TNF-{alpha} from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil-derived proteases. TNF-{alpha} release may be a general mechanism whereby metalloproteases drive cigarette smoke–induced inflammation.

Key Words: chronic obstructive pulmonary disease • tumor necrosis factor-{alpha} • cigarette smoke • macrophage metalloelastase • neutrophils • metalloproteases

In North America, between three- and seven-million people are diagnosed with chronic obstructive pulmonary disease each year, and chronic obstructive pulmonary disease is presently the fourth leading cause of death in the population (1). Most cases of chronic obstructive pulmonary disease are caused by cigarette smoke, and emphysema is the most important smoke-induced lesion. The protease-antiprotease hypothesis of smoke-induced emphysema postulates that smoke evokes a low level ongoing inflammatory response in the lower respiratory tract and that these inflammatory cells release proteases that overwhelm the local antiproteolytic defenses and degrade the connective tissue of the lung (2–4).

The protease–antiprotease hypothesis is generally accepted, but there is considerable disagreement about the inflammatory cells/proteases that are involved. The earliest formulation of this hypothesis was based on the observations that intratracheal instillation of elastase by itself caused emphysema and that individuals deficient in {alpha}-1-antitrypsin, the major antiprotease in the lower respiratory tract, developed early emphysema, leading to the idea that neutrophils (polymorphonuclear leukocyte [PMN]) and PMN-derived proteases, especially neutrophil elastase, were the crucial agents. This theory also suggested that cigarette smoke oxidatively inactivated {alpha}-1-antitrypsin, worsening the effects of neutrophil elastase (2–4). However, the role of PMN has become increasingly controversial. Although cigarette smoke does consistently produce an increase in lavage and tissue PMN (5–10), studies examining histologic sections of lung have failed to find a correlation between PMN numbers and evidence of lung destruction (7, 10). As well, mice lacking neutrophil elastase are only approximately 60% protected against smoke-induced emphysema (3).

A more recent theory has been that alveolar macrophages and macrophage-derived metalloproteases are really the important cells/proteases leading to emphysema in smokers (1–3, 11, 12). Correlations have been reported between macrophage numbers in histologic sections and morphologic markers of tissue destruction (7, 9). It has also become apparent that macrophage-derived metalloproteases, including gelatinase A (MMP-2), gelatinase B (MMP-9), matrilysin (MMP-7), and macrophage metalloelastase (MMP-12), can degrade elastin and collagen (11, 13, 14). Macrophages from smoke-exposed animals or cultured macrophages from lavage fluid of human smokers show increased elastolytic activity (15–17), and there are increased levels of MMP-2, MMP-9, and MT1-MMP in human lungs with emphysema compared with lungs without (18). Levels of MMP-1 (interstitial collagenase) are increased in guinea pigs exposed to smoke (19). This view has been supported by the study of Hautamaki and colleagues (12), which reported that mice with knocked out genes for MMP-12 (MMP-12 -/- mice) did not develop increased airspace size (emphysema) after chronic smoke exposure.

We recently showed that mice lacking tumor necrosis factor-{alpha} (TNF-{alpha}) receptors do not develop smoke-induced inflammation, indicating that TNF-{alpha} is crucial to the acute response to smoke (20). As well, we found that acute smoke-induced matrix breakdown, a necessary precursor of emphysema, actually requires both PMN and MMP-12 (5, 21). If PMNs are depleted in wild-type C57BL/6 mice using anti-PMN antibodies, matrix breakdown, measured as increased levels of bronchoalveolar lavage desmosine and hydroxyproline, is not seen (5). In MMP-12 -/- mice, neither PMN nor matrix breakdown is observed after smoke exposure, but both lavage PMN and matrix breakdown can be restored by intratracheal instillation of MMP-12 +/+ alveolar macrophages to MMP-12 -/- mice (21), implying that MMP-12 in some way drives neutrophil influx.

The hypotheses just described regarding metalloproteases and emphysema usually invoke the proteolytic action of MMP-12 on lung matrix to explain these events. We report here the novel observation that, at least acutely, this process instead runs through MMP-12–mediated liberation of TNF-{alpha}. This observation further provides a coherent theory that links the various discrepant observations described previously here.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
MMP-12 -/- mice were originally created in strain 129/J stock (12). Strain 129 mice are low TNF-{alpha} producers (22), and TNF-{alpha} is clearly involved in acute smoke responses (20); for this reason, the original MMP-12 -/- line was bred back through five generations into C57 BL/6 stock. All MMP-12 -/- mice used in these experiments were C57 based. MMP-12 +/+ mouse littermate lines derived from the breeding process (fifth generation backcrossed into C57 stock) were used as control subjects.

Smoke Exposure In Vivo
Experimental groups consisted of three mice. Each mouse was exposed to the whole smoke from four whole Kentucky 2R1 cigarettes (obtained from the University of Kentucky) using a standard smoking apparatus (described by us elsewhere [23]). Control mice were sham smoked. Smoke exposure took approximately 1 hour, and animals were killed at 2, 6, or 24 hours after the beginning of the smoke exposure.

Macrophage Culture and Smoke Exposure In Vitro
Untreated mice were killed and alveolar macrophages obtained by saline lavage; 96-well plates were filled with samples containing 4 x 105 alveolar macrophages. Each well was filled with macrophages from one mouse. The cells were allowed to adhere, and after 2 hours, nonadherent cells were removed together with the supernatant and one of the following was added: (1) 250 µl/well of RPMI 1640 culture medium (control) and (2) 250 µl/well of smoke-treated RPMI 1640 (stock solution 20-ml RPMI 1640 through which had been freshly bubbled the whole smoke of six 2R1 cigarettes). Plates were then incubated for 18 hours at 37°C in an air/6% CO2 incubator, and the supernatants were collected for TNF-{alpha} assay. For some experiments, the broad-spectrum metalloprotease inhibitor GM6001 (Chemicon, Temecula, CA) was added to the smoke-treatment groups. More details are provided in the online supplement.

TNF-{alpha} Assays
Production of TNF-{alpha} by lavaged and cultured alveolar macrophages was determined ELISA. The level of TNF-{alpha} production by the macrophages is relatively low; thus, to obtain a signal, the supernatant from four wells of cultured macrophages was combined. Because the ELISA kit is only intended for assaying TNF-{alpha} in fluids, whole-lung TNF-{alpha} was assayed using a modification of the L929 cell method of Levesque and colleagues (24). Details of the methods are provided in the online supplement.

Whole-Lung Western Blots for E-selectin
Excised lungs were homogenized and Western blots performed using goat anti–E-selectin. Details are provided in the online supplement.

Nuclear Factor-{kappa}B Gel Shift Assay
Gel shift was performed on the pelleted nuclei from the homogenization procedure mentioned previously here using a single-stranded nuclear factor-{kappa}B (NF-{kappa}B) consensus oligonucleotide. Details are provided in the online supplement.

Expression of Inflammatory Mediators by Reverse Transcription-Polymerase Chain Reaction
Whole-lung RNA was extracted by the method of Chomczynski and Sacchi (25) and reverse transcription-polymerase chain reaction for TNF-{alpha}, macrophage inflammatory protein-2 (MIP-2), and macrophage attractant protein-1 (MCP-1) performed. Primer sequences were obtained from Davis and colleagues (26) and Zhao and colleagues (27). Details and primers are provided in the online supplement.

Statistical Analysis
Statistical analysis was performed by analysis of variance; p < 0.05 or less was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Because we have previously shown that mice lacking MMP-12 do not develop a neutrophil response after smoke exposure (21), we asked first whether such mice upregulated basic inflammatory drivers. NF-{kappa}B is a cytoplasmic protein aggregate that, when activated, translocates into the nucleus and binds to and activates the promoters of a variety of genes important in the acute inflammatory response. To determine whether smoke exposure leads to NF-{kappa}B translocation, gel shifts were performed on the nuclear pellets using an NF-{kappa}B consensus probe. Figure 1 compares NF-{kappa}B levels in MMP-12 +/+ and MMP-12 -/- mice 2 hours after smoke exposure. Both types of mice showed NF-{kappa}B translocation, and the increase was about the same magnitude.



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  		Figure 1. Whole-lung NF-{kappa}B gel shifts and densitometry at 2 hours after smoke exposure. Nuclear levels of NF-{kappa}B are upregulated to the same extent in MMP-12 +/+ and -/- mice. C1–C3 = control mice of each strain; S1–S3 = mice of each strain exposed to smoke. *p < 0.05 or less compared with nonsmoked animals of each strain.

 
Similarly, to evaluate whether smoke exposure induced gene expression of a variety of proinflammatory cytokines whose expression is ordinarily mediated by NF-{kappa}B activation, we performed whole-lung reverse transcription-polymerase chain reaction. Figure 2 shows ethidium bromide–stained gels and densitometry of reverse transcription-polymerase chain reaction products for the neutrophil chemoattractant MIP-2, the macrophage chemoattractant MCP-1, and TNF-{alpha} at 2 hours after smoke exposure. In control mice, the basal levels of gene expression were identical in MMP-12 +/+ and MMP-12 -/- animals. After smoke exposure, both strains of mice upregulated expression of TNF-{alpha} and MIP-2. MMP-12 +/+, but not MMP-12 -/-, mice also upregulated expression of MCP-1. By 6 hours after smoke exposure, levels of MCP-1 and TNF-{alpha} gene expression had returned to baseline, and MIP-2 gene expression levels were falling compared with the 2-hour time point in MMP-12 +/+ mice. However, expression levels were elevated for all three mediators in MMP-12 -/- mice (Figure 3) . At 24 hours after exposure, gene expression of MIP-2 remained elevated in MMP-12 -/- mice (data not shown). These findings indicate that lack of MMP-12 does not affect upregulation of proinflammatory cytokine gene expression.



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  		Figure 2. Whole-lung gene expression of proinflammatory mediators at 2 hours after smoke exposure. Ethidium bromide–stained gel showing reverse transcription-polymerase chain reaction products, and densitometry for neutrophil chemoattractant MIP-2 (upper right), macrophage chemoattractant MCP-1 (lower left), and TNF-{alpha} (lower right) from MMP-12 +/+ and -/- mice. Gene expression of TNF-{alpha} and MIP-2 is up-regulated in both MMP-12 +/+ and MMP-12 -/- mice; MCP-1 only in MMP-12 +/+ mice. Values are mean ± SD using data from three animals in each group (shown in ethidium bromide image). *p < 0.05 or less compared with non-smoked animals of each strain. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

 


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  		Figure 3. Whole-lung gene expression of proinflammatory mediators at 6 hours after smoke exposure. Ethidium bromide–stained gel showing reverse transcription-polymerase chain reaction products, and densitometry for neutrophil chemoattractant MIP-2 (upper right), macrophage chemoattractant MCP-1 (lower left), and TNF-{alpha} (lower right) and from MMP-12 +/+ and -/- mice. Gene expression of MCP-1 and TNF-{alpha} inflammatory mediators has returned to baseline, and MIP-2 is decreased compared with the 2-hour time point in MMP-12 +/+ mice. Expression is elevated for all three mediators in MMP-12 -/- mice. Values are mean ± SD using data from three animals in each group (shown in ethidium bromide image). *p < 0.05 or less compared with nonsmoked animals of each strain. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

 
Actual release of TNF-{alpha} protein is crucial to mounting an acute response to a number of infectious and inflammatory agents. To evaluate TNF-{alpha} protein production in vivo, whole-lung TNF-{alpha} was assayed using L929 cells (Figure 4) ; this assay measures biologically active TNF-{alpha}. In MMP-12 +/+ mice, TNF-{alpha} levels were increased by 2 hours after smoke exposure and remained elevated at 6 and 24 hours. In MMP-12 -/- mice, no elevations were observed at any time point.



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  		Figure 4. Whole-lung TNF-{alpha} protein levels. In MMP-12 +/+ mice (top), there is a sustained increase in TNF-{alpha} over 24 hours after smoke exposure; in MMP-12 -/- mice (bottom), smoke has no effect at any time. Values are mean ± SD. *p < 0.05 or less compared with nonsmoked animals.

 
Because many inflammatory processes in the lung proceed through activation of alveolar macrophages and release of TNF-{alpha} (28), alveolar macrophages were lavaged from untreated animals, cultured, and exposed to smoke as described in the METHODS section. In macrophages not exposed to cigarette smoke, no TNF-{alpha} release could be detected (detection limit with the ELISA kit is 23.4 pg/ml). In MMP-12 +/+ mice, TNF-{alpha} levels were elevated after smoke exposure, whereas macrophages from MMP -/- mice did not produce detectable TNF-{alpha}. The metalloprotease inhibitor GM6001 prevented TNF-{alpha} release after smoke exposure in macrophages from MMP +/+ mice in a dose–response fashion (Figure 5) .



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  		Figure 5. Effects of in vitro smoke exposure on TNF-{alpha} production by cultured alveolar macrophages from MMP-12 +/+ mice. The marker for control (nonsmoke exposed) macrophages indicates that TNF-{alpha} production was below detection limits. Macrophages from MMP-12 +/+ mice showed a marked increase in TNF-{alpha} production after smoke exposure, and the metalloprotease inhibitor GM6001 at concentrations of 25 or 100 µM (Smoke+GM25, Smoke+GM100) progressively decreased the smoke effect. TNF-{alpha} production by macrophages from MMP-12 -/- mice was below detection limits after smoke exposure (data not shown). Values are mean ± SD of three data points. *p < 0.05 or less compared with control macrophages.

 
Activation of vascular endothelial cells is crucial to producing an inflammatory cell influx in the lung. Because one effect of TNF-{alpha} is endothelial activation, whole-lung levels of E-selectin, a very specific marker of endothelial activation, were evaluated by Western blot (Figure 6) . In MMP-12 +/+ mice, there was a rapid increase in E-selectin by 2 hours after smoke exposure, and this remained elevated through 24 hours. In MMP-12 -/- mice, there was no effect of smoke.



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  		Figure 6. Western blots of whole lung E-selectin levels after smoke exposure. Each treatment group shows data from three mice. In MMP-12 +/+ mice (left), there is a marked increase in E-selectin levels at 6 and 24 hours after smoke; in MMP-12 -/- mice (right), there is no effect of smoke. Values are mean ± SD using three mice in each group. *p < 0.05 or less compared with nonsmoked control of each strain.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As noted previously here, using this model, we have previously shown that a neutrophil influx is required for cigarette smoke-induced matrix breakdown and that the neutrophil influx is dependent on the presence of MMP-12 (5, 21). In this article, we have attempted to explain the mechanism behind these observations. The striking result in our study is that MMP-12 -/- mice show impaired TNF-{alpha} release after smoke exposure. Whole-lung TNF-{alpha} protein was not increased in the MMP-12 -/- mice, but was rapidly increased in the MMP-12 +/+ mice after a single smoke exposure. Similarly, cultured alveolar macrophages from MMP-12 -/- mice fail to produce detectable TNF-{alpha} after in vitro smoke exposure, whereas a marked increase in TNF-{alpha} production was seen with macrophages from MMP-12 +/+ mice. The broad-spectrum metalloprotease inhibitor GM6001 inhibited the effects of smoke on in vitro macrophage TNF-{alpha} release, confirming that release proceeds through a metalloprotease-mediated mechanism.

It is also important to note that, in our previous study (21), both strains of mice showed increased numbers of lavage macrophages at 24 hours after smoke exposure, at which point MMP-12 +/+ animals also had a marked lavage neutrophilia, whereas neutrophils in MMP-12 -/- animals were not different from control subjects. These observations serve to emphasize the idea that it is macrophage activation (i.e., secretion of MMP-12 and then release of TNF-{alpha}) rather than macrophage number that is responsible for the acute inflammatory response to smoke. This idea is also implied in the original study of Hautamaki and colleagues (12); when they administered MCP-1 to MMP-12 -/- mice by intratracheal instillation, macrophages accumulated in the lung, but the animals did not develop emphysema over 6 months.

TNF-{alpha} is normally synthesized as a 26-kD precursor (pro-TNF{alpha}), which is stored in a membrane-bound form ready for rapid release. With the appropriate stimulus, for example, bacterial LPS, the membrane-bound form is converted to a 17-kD biologically active mature form that is usually released from the cell (29–31). Pro–TNF-{alpha} is ordinarily converted to active TNF-{alpha} by a membrane-bound metalloprotease called TNF-{alpha} converting enzyme (TACE; ADAM-17) (30), but other matrix metalloproteases, including MMP-3 (stromelysin), MMP-7 (matrilysin), MMP-1 (interstitial collagenase), and MMP-2 and MMP-9 (gelatinases A and B) also have greater or lesser degrees of TNF-{alpha} converting activity (30). In vitro, MMP-12 has been shown to release TNF-{alpha} from a synthetic pro–TNF-{alpha} fusion protein (32).

However, apart from TACE, most of the data that support a role for metalloprotease-mediated TNF-{alpha} release have been generated in vitro using artificial fusion proteins, and little is known about the importance of this process in intact biologic systems. Haro and colleagues (33) recently showed that MMP-7 was required for release of TNF-{alpha} from peritoneal macrophages in an organ culture model of intervertebral disc resorption. Our current observations suggest that, at least in mice, TNF-{alpha} release from macrophages after smoke exposure depends not only on TACE, but also on the presence of MMP-12.

The ELISA assay that we used specifically detects the 17-kD form of TNF-{alpha}; thus, these data suggest that MMP-12 may have significant TNF-{alpha}–converting activity in intact biologic systems, as well as in synthetic fusion proteins. Alternately, it is possible that there is a defect in macrophage TNF-{alpha} production in these animals, although this appears much less likely as whole-lung TNF-{alpha} levels are the same in control MMP-12 +/+ and -/- mice. We also considered the possibility that TACE and MMP-12 are the same protein in mice, in which case a knockout of MMP-12 might remove all TNF-{alpha} converting activity, but examination of sequences in GenBank indicates that TACE and MMP-12 are in fact quite distinct. Because of the sensitivity limitations of the ELISA assay, it is entirely possible that cigarette smoke exposure does lead to some TACE-mediated TNF-{alpha} release, but at a level that is too low to detect. However, this phenomenon would not change our basic conclusion that MMP-12 is crucial to the release of biologically effective amounts of TNF-{alpha} after smoke exposure. Whatever the mechanism, these findings in addition support an increasing body of evidence that metalloproteases have important roles in modulating the activity of a variety of cytokines and growth factors, including monocyte chemoattractant protein, transforming growth factor-ß, insulin-like growth factors, and epidermal and fibroblast growth factors (34).

The observation that levels of E-selectin, a specific marker of endothelial activation, were increased in MMP-12 +/+ mice but not in MMP-12 -/- mice after smoke exposure indicates that TNF-{alpha} is serving here, as in many other inflammatory processes (28, 35), to activate endothelial cells so that they initiate the sequence of molecular changes necessary to cause PMN adhesion and diapedesis through the vessels and into the lung; conversely, in the absence of MMP-12, TNF-{alpha} is not released after smoke exposure, endothelial cells are not activated, and no increase in lavage PMN is observed. To determine whether other TNF-{alpha}–activated vascular adhesion molecules were also upregulated, we examined whole-lung vascular cell adhesion molecule-1 (VCAM-1) by Western blot and again found increases in VCAM-1 in MMP-12 +/+ but not MMP-12 -/- mice (data not shown). Our results correspond to human data, as E-selectin and VCAM-1 have been shown by immunochemistry to be upregulated in the vascular endothelium in cigarette smokers (36).

In this study, smoke rapidly activated NF-{kappa}B and upregulated gene expression of TNF-{alpha}, the neutrophil chemoattractant MIP-2, and the macrophage chemoattractant MCP-1, and this occurred in both strains of mice, indicating that the defect in MMP-12 -/- mice is not a global failure to drive an inflammatory response. There were some differences in the timing of gene upregulation: MCP-1 was upregulated later in the MMP-12 -/- mice compared with the MMP-12 +/+ mice, and MIP-2 upregulation was more prolonged in the MMP-12 -/- mice, suggesting that a normal feedback mechanism (presumably related to TNF-{alpha} protein release) that downregulates the inflammatory response is lacking.

We have not investigated the source and mechanism behind MIP-2 and MCP-1 production in this study. Activated macrophages can produce these mediators (28, 35), but they are also secreted by alveolar and bronchial epithelial cells in monolayer tissue culture systems after cigarette smoke exposure (37, 38), apparently in the absence of TNF-{alpha} production, although this question has not been directly examined. The fact that there is equal or greater gene expression of MIP-2 and MCP-1 in our MMP-12 -/- compared with MMP-12 +/+ mice supports a direct effect of smoke, rather than a TNF-{alpha}–mediated effect of smoke, in chemoattractant chemokine production and NF-{kappa}B activation.

As noted previously here, in addition to MMP-12, a variety of other metalloproteases appear to be produced in the lung in excess amounts in humans and animals exposed to cigarette smoke. It is interesting in this regard that Joos and colleagues (39) have recently reported that polymorphisms in the genes for MMP-1 and MMP-12 are associated with the rate of decline in function in human smokers. Other types of metalloproteases as well as MMP-12 at least potentially have in vivo TNF-{alpha} converting activity, and the observations of Joos and colleagues might be reflecting differences in TNF-{alpha} release.

We propose that one pathway by which smoke produces alveolar inflammation, matrix breakdown, and eventual emphysema is through activation of macrophages to secrete MMP-12, and possibly other metalloproteases, which in turn release TNF-{alpha} from macrophages, causing activation of vascular endothelial cells with subsequent neutrophil influx and connective tissue breakdown produced by neutrophil proteases (Figure 7) . This hypothesis potentially unifies the disparate and often contradictory observations regarding which cells and proteases mediate cigarette smoke-induced parenchymal destruction, as described previously here, as it shows how macrophage-derived and PMN-derived proteases are playing a conjoint role.



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  		Figure 7. Cartoon showing proposed model of the steps involved in acute smoke-mediated inflammation and matrix breakdown. In this scheme, cigarette smoke causes release of MMP-12 from macrophages, and MMP-12 acts as a TNF{alpha} converting enzyme, releasing active TNF-{alpha}. TNF-{alpha} then activates endothelial cells with adhesion of PMN to the endothelial cells and subsequent entry of PMN into the lung. PMN-derived proteases (most importantly, neutrophil elastase) are the actual agents of alveolar wall matrix breakdown and eventual emphysema.

 
It should be noted that our experiments are very acute, and although we know that acute smoke exposures produce matrix breakdown in MMP-12 +/+ but not MMP-12 -/- mice (21), extrapolating these results to long-term exposures and actual emphysema needs to be done with caution. We did observe consistently elevated levels of serum TNF-{alpha} in guinea pigs exposed to cigarette smoke for 6 months (40), suggesting that persistent TNF-{alpha} production may be important in the eventual development of emphysema. Further studies are required to determine whether the same mechanisms of TNF-{alpha} release apply over the long term.


   FOOTNOTES
 
Supported by grant MOP 42,539 from the Canadian Institutes of Health Research (A.C.) and PO1 HL29594 (S.D.S.) from the National Institutes of Health

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form December 1, 2002; accepted in final form January 8, 2003


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 METHODS
 RESULTS
 DISCUSSION
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