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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 368-374, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4791

Acute Cigarette Smoke–Induced Connective Tissue Breakdown Requires both Neutrophils and Macrophage Metalloelastase in Mice

Andrew Churg, Katalin Zay, Selena Shay, Changshi Xie, Steven D. Shapiro, Robert Hendricks and Joanne L Wright

Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada; Roche Bioscience, Palo Alto, California; and Department of Respiratory Medicine, Brigham and Women's Hospital, Boston, Massachusetts

Address correspondence to: Andrew Churg, M.D., Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, V6T 2B5 Canada. E-mail: achurg{at}interchange.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cells/proteases responsible for the development of smoke-induced emphysema is an area of intense investigation. Mice with knockout of macrophage metalloelastase genes (MME-/-) do not develop emphysema after smoke exposure, but we also observed that neutrophils (PMN) in lavage appeared to be a requirement for acute connective tissue breakdown. In this study we exposed mice to cigarette smoke and examined lavage PMN, macrophages (MAC), desmosine (DES, a measure of elastin breakdown) and hydroxyproline (HP, a measure of collagen breakdown) 24 h afterwards. MME+/+ mice exposed to smoke showed elevations in PMN, DES, and HP, but no elevations were seen in MME-deficient mice. Both PMN influx and increased levels of DES/HP could be restored by administering MAC from MME+/+ mice to MME-deficient mice and then exposing them to smoke. RS113456, a metalloprotease inhibitor, also prevented PMN influx and connective tissue breakdown. Western blots against mouse {alpha}1-antitrypsin ({alpha}1AT) showed that {alpha}1AT was not protected in MME-deficient mice, nor by administration of RS113456. We conclude that, in mice, acute smoke-induced connective tissue breakdown, the precursor to emphysema, requires both PMN and MME, that PMN influx appears to be secondary to MAC activation, and that this process initially does not involve protection of {alpha}1AT from metalloprotease attack.

Abbreviations: {alpha}1 antitrypsin, {alpha}1AT • aminoethyl benezenesulfonyl fluoride, AEBSF • desmosine, DES • hydroxyproline, HP • macrophages, MAC • macrophage metalloelastase, MME • neutrophils, PMN • tris-buffered saline, TBS • tris-buffered saline with Tween 20, TBST


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is generally believed that the development of emphysema in cigarette smokers reflects a smoke-induced low level ongoing inflammatory process in the lower respiratory tract with a resulting relative excess of inflammatory cell–derived proteases that degrade the connective tissue of the lung, and a relative paucity of antiproteolytic defenses. This theory is often referred to the as protease–antiprotease hypothesis (1–3)

Although the protease–antiprotease hypothesis is widely accepted, there is considerable disagreement about the inflammatory cells/proteases that are involved. The traditional viewpoint has been that neutrophil (PMN)-derived proteases, especially neutrophil elastase, are the crucial effectors, and that cigarette smoke also oxidatively inactivates {alpha}1-antitrypsin ({alpha}1AT), the major antiproteolytic substance in the lung parenchyma (1–3). But this view has become increasingly controversial. Reports on the levels and functional status of {alpha}1AT in smokers are contradictory, with some authors finding no differences between smokers and control subjects, and others reporting evidence of decreased {alpha}1AT levels and/or decreased antiprotease activity (reviewed in Refs. 2–6). The idea that the PMN is the effector cell has also been challenged. Cigarette smoke consistently does produce an increase in lavage and tissue PMN (7–12), but studies examining histologic sections of lung have failed to find a correlation between PMN numbers and evidence of lung destruction (9, 12).

It is believed that protease-driven connective tissue breakdown is a requirement for the development of emphysema. Experimentally, neutrophil elastase instilled into the lung does indeed produce emphysema, as does induction of a PMN influx by instillation of formylmethionyl-l-leucylphenylalanine (13). But cigarette smoke also produces an increase in lavage and tissue macrophages (MAC), and correlations have been reported between tissue macrophage numbers and morphologic markers of tissue destruction (9, 12). Moreover, recent studies have greatly revised the traditional view that macrophage proteases show little elastolytic activity, and it is now recognized that a variety of macrophage-derived metalloproteases, including gelatinases A and B, matrilysin, and macrophage metalloelastase, all can degrade elastin (14–16). It has also become evident from human and experimental studies that emphysema is characterized not only by breakdown of elastin, but also by breakdown and resynthesis of collagen with scar formation, and macrophage-derived proteases can also degrade collagen (2, 4, 17, 18)

In light of these observations, an alternate formulation of the protease–antiprotease hypothesis has been proposed in which MAC are the crucial cells and macrophage-derived proteases, particularly metalloproteases, are the effector agents (1–3, 15, 19). It has been found that MAC from smoke-exposed animals or cultured MAC from lavage fluid of human smokers show increased elastolytic activity (20, 21), that there is upregulation of interstitial collagenase production in smoke-exposed guinea pigs (22), and increased levels of MMP-1, MMP-2, MMP-8, and MMP-9 in human lungs with emphysema compared with lungs without (23). Direct evidence supporting this view is the study of Hautamaki and coworkers (19), which reported that mice with knocked out genes for macrophage metalloelastase (MME-/- mice) did not develop increased airspace size after chronic smoke exposure.

If indeed MAC and MAC-derived proteases are responsible for matrix destruction in emphysema, a question still remains about the role of PMNs, which, as noted, are consistently elevated in human or animal smokers. It is interesting in this regard that mice lacking neutrophil elastase are 50–60% protected against smoke-induced emphysema (3), implying that PMNs play a part in this process. We have previously looked at this issue in an acute model (7) and found that there was a clear dose response and time response correlation between smoke-induced lavage PMN and elevated levels of lavage desmosine (DES, a measure of elastin breakdown) and hydroxyproline (HP, a measure of collagen breakdown), but no correlation between lavage MAC numbers and DES or HP. Further (7), we showed that suppressing PMN influx by pretreatment with anti-PMN antibody suppressed elevations in DES and HP. Similarly, administration of exogenous human {alpha}1AT, which in this circumstance appears to function as an anti-inflammatory as well as an antiproteolytic agent (24), also suppressed both PMN and DES/HP. This is not just a transient phenomenon: we have looked at lavage from animals exposed to daily smoke for varying times up to 1 mo and found the same correlated elevations in PMN, DES, and HP (J. Wright and A. Churg, unpublished data).

Thus, there appears to be evidence supporting a role for either PMN or MAC in the development of emphysema. In this paper we further examine the relationships between these effector agents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
MME-/- Mice
The MME-/- mice were originally created in line 129 stock (19). Because 129 strain mice are low TNF-{alpha} producers (25) and TNF-{alpha} is probably involved in acute smoke responses, they were bred back through five generations into C57 BL/6 stock. All MME-/- mice used in these experiments were C57 based. MME+/+ mouse littermate lines derived from the breeding process were used as controls and as sources of MME-competent macrophages.

Metalloprotease Inhibitor RS113456
The broad spectrum metalloprotease inhibitor RS113456 was obtained from Roche Bioscience (Palo Alto, CA) and was administered by gavage at a dose of 0.1 mg/g 1 h before smoke administration following the manufacturer's guidelines (details of the inhibitory profile of this compound are available in Ref. 26).

Smoke Exposure and Lavage Procedures
Experimental groups consisted of four 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 [27]). Control mice were sham smoked. At 24 h after smoke exposure, mice were killed by halothane overdose and the lungs removed from the chest cavity. A 20-gauge catheter was inserted into the trachea and the lungs lavaged six times with 1 ml of ice-cold saline for cell counts, or with distilled water for connective tissue degradation analysis. Water is used because concentration of salts during sample preparation for the high-performance liquid chromatography (HPLC) procedure interferes with the analysis of desmosine (28). Separate sets of animals were used for these experiments.

For inflammatory cell measurements, the saline lavage was centrifuged at 200 x g at 4°C for 10 min. The supernatants were decanted and the cell pellets were resuspended in 200 µl of saline. Total cell counts were performed in a hemacytometer and differential cell counts were performed on a 10-µl drop of the cell suspension heat fixed on a slide and stained with hematoxylin-eosin.

Desmosine and hydroxyproline analysis The water lavageate was lyophilized and hydrolyzed in HCl at 110°C for 48 h, and then analyzed for DES and HP on a Waters HPLC system (Waters Associates, Milford, MA) using our previously published protocol (28)

Whole lung Western blots for {alpha}1AT Excised lungs were perfused through the pulmonary artery with saline until the perfusate ran clear. The lungs were then homogenized in a solution containing 1 mM EDTA, 0.5 mM aminoethyl benezenesulfonyl fluoride (AEBSF), 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 µg/ml trypsin-chymotrypsin inhibitor, and 1 µg/ml pepstatin A. (all from Sigma) Samples were lyophilized and reconstituted in 2 ml distilled water, aliquoted, and kept at -80°C until used. Proteins were resolved on 10% polyacrylamide gel under reducing conditions using 0.75 mm gel thickness and loading 10 µl sample in each well. The sample volumes were calculated to produce equal protein content. After immobilization onto nitrocellulose membrane, the nonspecific binding sites were blocked with 5% skim milk in tris-buffered saline (TBS), pH 7.6, and the membranes were incubated overnight at 4°C with 1:1,000 dilution of goat anti-human {alpha}1AT antibody (cat:682021; ICN Biochemicals, Costa Mesa, CA) in TBS/5% skim milk. After three 10-min washes with TBS containing 0.05% Tween 20 (TBST), the membranes were incubated with 1:600 dilution of anti-goat IgG-horseradish peroxidase (Cat: sc-2020; Santa Cruz Biotechnology, Santa Cruz, CA) in TBS/5% skim milk for 1 h at room temperature. After three 10-min washes with TBST the membranes were visualized with an ECL kit (Cat:RPN 2,106; Amersham Pharmacia, Piscataway, NJ). The membranes were exposed to Kodak X-ray film (Kodak, Rochester, NY) and developed accordingly.

Reconstitution of the MME1/1 phenotype To reconstitute the MME+/+ phenotype in the lung, lavage was performed on normal MME+/+ animals to obtain MME+/+ macrophages. As controls, lavage was performed on MME-/- mice to obtain MME-/- macrophages. For each MME-/- mouse to be treated, lavaged cells from four donor MME+/+ or four donor MME-/- animals were concentrated by centrifugation, combined, and administered at a total dose of 1.5 x 106 MAC by intratracheal instillation. The cellular composition of the lavage from the MME+/+ animals was: neutrophils, 0.4 ± 0.1% (mean ± SD); lymphocytes, 1.5 ± 0.7%; and the remaining cells, macrophages. The cellular composition of the lavage from the MME-/- animals was: neutrophils, 0.3 ± 0.1%; lymphocytes, 1.8 ± 0.8%; and the remaining cells, macrophages. Two hours after MAC administration, the mice were exposed to smoke or sham smoked as above, and killed 24 h later.

Statistics
Statistical analysis was performed by analysis of variance. P < 0.05 or less was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1 shows the effects of administering smoke to MME-/- or MME+/+ mice. In the MME+/+ mice, smoke produced a sharp increase in lavage PMN at 24 h, an approximately 2-fold increase in lavage MAC, and 2- to 3-fold increases in lavage DES and HP. In contrast, MME-/- mice showed no significant increase in lavage PMN, DES, or HP, and a significant but small (25%) increase in lavage MAC.



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  		Figure 1. Effects of smoke on inflammation and connective tissue breakdown in MME-/- mice. MME-/- mice are largely protected from smoke effects. (A) Effects of cigarette smoke on lavage PMN. Smoke produces a PMN influx into the lavage fluid in MME+/+ mice, but has no effect in MME-/- mice. (B) Effects of cigarette smoke on lavage macrophages. Smoke produces a small increase in lavage MAC in both types of mice. (C) An increase in lavage DES is seen after smoke exposure in MME+/+ mice but not in MME-/- mice. (D) A similar phenomenon is seen for lavage HP. Solid bars, control; shaded bars, smoke. Values are mean ± SD. *P <= 0.05 compared with control.

 
Figure 2 shows the effects of reconstituting the MME-/- mice with MAC lavaged from MME+/+ or MME-/- mice. In these experiments, untreated MME-/- mice again showed no increase in PMN, DES, or HP. MME-/- mice reconstituted with MME+/+ macrophages showed a small increase in PMN in the control animals and a much larger increase in animals exposed to smoke; similarly, reconstituting the MME+/+ macrophage profile led to significant increases in lavage DES and HP in the MME-/- mice after smoke exposure. Reconstitution with MME-/- macrophages did not produce any increase in PMN, DES, or HP. These procedures did not affect numbers of lavageable macrophages (data not shown).



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  		Figure 2. Effects of reconstituting MME+/+ macrophage phenotype. In these experiments, MAC were lavaged from MME+/+ or MME-/- mice, collected by centrifugation, and administered by intratracheal instillation to MME-/- mice. Two hours later the animals were smoked or sham-smoked and killed 24 h afterwards. Administration of MME+/+ macrophages restored the smoke-induced increase in lavage PMN (A), DES (B), and HP (C) ordinarily seen in MME+/+ mice. Administration of MME-/- macrophages has no effect. Solid bars, control; shaded bars, smoke. Values are mean ± SD. *P <= 0.05 compared with control.

 
Figure 3 shows the effects of the broad spectrum metalloprotease inhibitor RS113456. RS113456 prevented the smoke-mediated increases in lavage PMN, DES, and HP in MME+/+ mice; it did not, however, affect smoke-mediated increases in MAC.



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  		Figure 3. Effects of the broad spectrum metalloprotease inhibitor RS113456. RS113456 prevents smoke induced lavage PMN influx (A), does not affect smoke induced increases in MAC (B), and prevents smoke induced increases in DES (C) and HP (D). Values are mean ± SD. *P <= 0.05 compared with control.

 
Because {alpha}1AT is a substrate for MME, we examined the question of whether the lack of MME in MME-/- mice, or inhibition of metalloproteases with RS113456, acts to protect {alpha}1AT (Figure 4) . The signals shown come from whole lungs first vascular-perfused with saline to eliminate false increases in {alpha}1AT levels from serum. Using Western blots there was in fact an increase in whole lung {alpha}1AT at 24 h after smoke in MME+/+ mice, and this was reduced to control values by RS113456. No significant changes in {alpha}1AT levels were seen in MME-/- mice (see Discussion). As a further comparison, levels of {alpha}1AT were compared by Western blot in untreated MME+/+ and MME-/- animals. These levels were not different (data not shown).



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  		Figure 4. Western blots and densitometry against mouse lung {alpha}1AT. In MME+/+ smoke-exposed animals there is an increase in {alpha}1AT levels compared with control, and this is prevented by RS113456. No changes are seen in MME-/- mice. There is no evidence that smoke decreases {alpha}1AT levels. Blots show three animals in each treatment group. Values are mean ± SD. *P <= 0.05 compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we have performed a series of experiments designed to look at the role of MME in smoke-induced connective tissue breakdown, and to attempt to determine whether differing data that support a role for MAC/ MME versus PMN/neutrophil elastase can be reconciled. The gold standard measurement of smoke effects in this context is, of course, the development of emphysema; but, because emphysema requires months to develop, evaluating acute smoke effects requires an alternate approach. The protease–antiprotease hypothesis is based on the notion that connective tissue breakdown is crucial to the development of emphysema (1–3), and thus measurements of lavage DES and HP provide a useful acute endpoint. However, it must be realized that the exact correlation between acute (or even chronic) connective tissue breakdown (measured as matrix fragments in lavage) and the eventual degree/type/location of emphysema is not known. In addition, our data by definition do not look at long-term effects. Thus, some caution is required in extrapolating our acute results to actual emphysema.

In our previous study we showed a clear correlation between PMN numbers in lavage and levels of DES and HP (7). Our fundamental observations in this set of experiments are that MME is also required for smoke-induced connective tissue breakdown, and that this MME can be endogenous, or can be supplied by reconstituting MME-/- mice with MAC from MME+/+ mice. More general support for a role for metalloproteases is provided by the observation that a metalloprotease inhibitor, RS113456, prevents connective tissue breakdown. This latter experiment must be interpreted with care, however, because we have shown elsewhere that RS113456 has anti-inflammatory properties probably related to inhibition of PMN migration, and thus it is possible that inhibition of neutrophil proteolytic effects as opposed to macrophage proteolytic effects are operative here (24). On the other hand, if the anti-inflammatory properties of RS113456 are involved, then these experiments provide further proof that suppressing PMN influx suppresses connective tissue breakdown.

However, what is particularly intriguing, combining these findings and our previous findings as described in the Introduction (7), is that merely having normal levels of MME is by itself not enough. Acute connective tissue breakdown only appears when there are corresponding elevations in lavage PMN (Figures 1–3; see also figures in Ref. 7). These observations lead to a number of possible hypotheses concerning the mechanism of smoke-induced connective tissue breakdown, which for convenience can be formulated as follows:

  1. Smoke -> PMN -> release of neutrophil elastase (or other serine proteases) -> matrix attack -> lavage DES/HP. This theory would explain the fact the lavage DES/HP levels appear to track lavage PMN numbers in a dose- and time-response fashion, but would not explain the lack of acute connective tissue breakdown or long-term emphysema (19) in MME-/- mice.
  2. Smoke -> MAC -> MME (or other MMP in other species) -> matrix attack -> lavage DES/HP. This theory would explain the lack of connective tissue breakdown or emphysema in MME-/- mice, but would not explain why lavage DES/HP follows PMN numbers, nor the lack of connective breakdown in MME+/+ animals treated with anti-PMN antibodies to deplete PMN (7).
  3. Smoke -> MAC -> MME (or other MMP in other species) -> matrix attack -> DES/HP -> lavage PMN. It has been shown a number of years ago that the collagen and elastin fragments produced by proteolytic attack act as both neutrophil and macrophage chemoattractants (29–32). Shapiro (3) has suggested that this might explain persisting macrophage recruitment in smokers. We propose that it might also explain PMN influx, but with PMN influx as a completely secondary event. This theory is attractive but founders on the observation that there is no evidence for elevated matrix fragments when PMN influx is suppressed; thus, acutely, MAC by themselves appear incapable of generating detectable matrix destruction, at least in mice.
  4. Smoke -> MAC -> MME -> PMN influx -> matrix attack -> lavage DES/HP. This sequence fits the available data, but the connection between MME and PMN is obscure. Smoke does elicit production of PMN and MAC chemoattractants such as IL-8 (murine MIP-2 or KC) and MCP-1 from cultured respiratory epithelial cells, but, at least in tissue culture, these effects do not appear to require the presence of MME (33).

It is interesting in this regard that increased macrophage numbers are not required to initiate connective tissue breakdown. In the present experiments we used doses of four cigarettes and found an approximate doubling of lavage MAC numbers in the MME+/+ animals. Lavage MAC numbers appear to be a dose-response effect, because we almost never see an increase in lavage MAC with two cigarettes, we find it sometimes with three cigarettes, and observe an increase at least 80% of the time with four cigarettes (7; A. Churg and J. Wright, unpublished data). Nonetheless, we still found an increase in lavage PMN, DES, and HP with exposure to two cigarettes (7), implying that it is smoke-induced MAC activation rather than mere increases in numbers of cells that is crucial to initiating the acute inflammatory response.

It should also be noted that our results on cell counts appear to differ from those of Hautamaki and coworkers (19), because they reported equal numbers of PMN in MME+/+ and MME-/- mice exposed to smoke. This difference may reflect the method of counting cells: Hautamaki and colleagues counted cells in the tissue in histologic sections, whereas we used lavage.

Another possible connection between PMN- and MAC-derived proteases can be seen in the data of Liu and coworkers (34) on a mouse model of bullous pemphigoid. In that model, blister formation required both neutrophil elastase and MMP-9 (gelatinase B), a major neutrophil MMP. If either protease was knocked out by genetic manipulation, blister formation did not occur, but could be re-established by reconstituting the wild phenotype. This process appears to proceed through an MMP-9–mediated attack on {alpha}1AT, as evidenced by the appearance of breakdown bands on Western blot for {alpha}1AT in the wild-type but not in the knockout mice.

Because MME also has significant activity against {alpha}1AT (3, 14), the same process might apply to our model. For this reason, we performed Western blots against {alpha}1AT, either using the metalloprotease inhibitor RS113456, or using MME-/- mice. Smoke in fact produced a small increase rather than a decrease in {alpha}1AT in MME+/+ animals. In some senses, this finding is not surprising, because {alpha}1AT is an acute phase reactant and smoke evokes an inflammatory reaction in the lungs of MME+/+ mice. There was no evidence that MME-/- mice had higher levels of {alpha}1AT, and {alpha}1AT levels did not change with smoke in these animals, an observation consistent with the lack of inflammation in their lungs. Of interest, RS113456 did prevent the smoke-mediated increase in {alpha}1AT in MME+/+ mice, presumably because it prevents the inflammatory reaction. However, these experiments make it clear that, acutely, potentiation of the role of neutrophil elastase through MMP-mediated removal of the protective agent, {alpha}1AT (the process that appears to be important in the bullous pemphigoid model) is not evident with smoke.

There are other forms of evidence that MMP and PMN interact to produce injury (reviewed in Refs. 3, 35, 36). Warner and coworkers (35) reported that, in a model of immune complex–mediated lung disease, MMP-/- animals showed ~ 50% of the PMN levels of MME+/+ mice, along with reduced lung injury. The authors concluded that MME-/- animals are partially protected against neutrophil influx. They suggested a number of possible mechanisms, including a decrease in the amounts of chemotactic agents produced (for which there is some evidence in the study of Hautamaki and coworkers [19]); modulation of release of proinflammatory and anti-inflammatory cytokines; and decreases in the ability of inflammatory cells to migrate through vessel walls. In this regard the same group (36) reported that stromelysin-deficient mice also showed decreased PMN infiltration in their model, whereas gelatinase B–deficient mice did not. Lastly, we (24) recently reported that both {alpha}1AT and RS113456 markedly reduced or eliminated the PMN response to silica, a potent inducer of PMN chemoattractants, suggesting that some combination of serine and metalloproteases may be important in controlling PMN influx.

This study examines early events in response to cigarette smoke exposure. The changes observed, including MME-dependent neutrophil recruitment and matrix degradation, are probably sufficient, over the long term, to cause emphysema. We believe that these results help explain seemingly contradictory requirements for neutrophils, macrophages, and their respective proteases. Additional changes, set up by these acute events, likely occur with long-term cigarette smoking. This has been made very clear by a recent study by Retemales and colleagues (37), who demonstrated continued intense inflammation and presumed matrix destruction in patients with end-stage emphysema who had not smoked cigarettes for years.

Putting findings from this study in the context of others leads to the following proposed sequence of events. Acute exposure to cigarette smoke leads to macrophage activation, which somehow via a macrophage MMP-dependent process leads to neutrophil recruitment, with consequent elastin and collagen degradation. Subacutely, macrophages also accumulate, and both neutrophils and macrophages and their respective proteases interact, resulting in accelerated matrix destruction and emphysema.


   Acknowledgments
 
This study was supported by grant MOP 42539 from the Canadian Institutes of Health Research and Bayer Blood Partnership.

Received in original form November 28, 2001

Received in final form March 21, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Snider, G. L., J. Kleinerman, W. Thurlbeck, and Z. Bengali. 1985. The definition of emphysema. Am. Rev. Respir. Dis. 132:182–185.[Medline]
  2. Senior, R. M., and N. R. Anthonisen. 1998. Chronic obstructive pulmonary disease (COPD). Am. J. Respir. Crit. Care Med. 157(Suppl.):S139–S147.
  3. Shapiro, S. D. 2000. Evolving concepts in the pathogenesis of chronic obstructive pulmonary disease. Clin. Chest Med. 21:621–632.[Medline]
  4. Snider, G. L., E. Lucey, and P. J. Stone. 1994. Pitfalls in antiprotease therapy of emphysema. Am. J. Respir. Crit. Care Med. 150(Suppl.):S131–S137.
  5. Evans, M. D., and W. A. Pryor. 1994. Cigarette smoking, emphysema, and damage to {alpha}1-proteinase inhibitor. Am. J. Physiol. 266:L593–L611.[Abstract/Free Full Text]
  6. Ogushi, R., R. Hubbard, C. Vogelmeier, G. Fells, and R. G. Crystal. 1991. Risk factors for emphysema: cigarette smoking is associated with a reduction in the association rate constant of lung {alpha}1-antitrypsin for neutrophil elastase. J. Clin. Invest. 87:1060–1065.
  7. Dhami, R., B. Gilks, C. Xie, K. Zay, J. Wright, and A. Churg. 2000. Acute cigarette smoke–induced connective tissue breakdown is mediated by neutrophils and prevented by {alpha}-1-antitrypsin. Am. J. Respir. Cell Mol. Biol. 22:244–252.[Abstract/Free Full Text]
  8. Hunninghake, G., and R. G. Crystal. 1983. Cigarette smoking and lung destruction. Am. Rev. Respir. Dis. 128:833–838.[Medline]
  9. Finkelstein, R., R. Fraser, H. Ghezzo, and M. Cosio. 1995. Alveolar inflammation and its relation to emphysema in smokers. Am. J. Respir. Crit. Care Med. 152:1666–1672.[Abstract]
  10. Ludwig, P., B. Schwartz, J. Hoidal, and D. Niewoehner. 1985. Cigarette smoking causes accumulation of polymorphonuclear leukocytes in the alveolar septum. Am Rev Respir. Dis 131:828–830.[Medline]
  11. Janoff, A. 1985. Elastases and Emphysema: current assessment of the protease-antiprotease hypothesis. Am. Rev. Respir. Dis. 132:417–433.[Medline]
  12. Eidelman, D., M. Saetta, H. Ghezzo, N. Wang, J. Hoidal, M. King, and M. Cosio. 1990. Cellularity of the alveolar walls in smokers and its relation to alveolar destruction. Am. Rev. Respir. Dis. 141:1547–1552.[Medline]
  13. Cavarra, E., P. A. Martorana, F. Bambelli, M. de Santi, P. van Even, and G. Lungarella. 1996. Neutrophil recruitment into the lungs is associated with increased lung elastase burden, decreased lung elastin, and emphysema in alpha-1-proteinase inhibitor–deficient mice. Lab. Invest. 75:273–280.[Medline]
  14. Senior, R., N. Connolly, J. Cury, H. Welgus, and E. Campbell. 1989. Elastin degradation by human alveolar macrophages. Am. Rev. Respir. Dis. 139: 1251–1256.[Medline]
  15. Shapiro, S. D. 1994. Elastolytic metalloproteinases produced by human mononuclear phagocytes. Am. J. Respir. Crit. Care Med. 150:S160–S164.
  16. Senior, R. M., G. L. Griffin, C. J. Fliszar, S. D. Shapiro, G. I. Goldberg, and H. G. Welgus. 1991. Human 92- and 72-kilodalton type IV collagenases are elastases. J. Biol. Chem. 266:7870–7875.[Abstract/Free Full Text]
  17. Cardoso, W. V., H. S. Sekhon, D. M. Hyde, and W. M. Thurlbeck. 1993. Collagen and elastin in human pulmonary emphysema. Am. Rev. Respir. Dis. 147:975–981.[Medline]
  18. Wright, J. L., and A. Churg. 1995. Smoke-induced emphysema in the guinea pig is associated with morphometric evidence of collagen breakdown and repair. Am. J. Physiol. 268:L17–L20.[Abstract/Free Full Text]
  19. Hautamaki, R., D. Kobayashi, R. Senior, and S. Shapiro. 1997. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277:2002–2004.[Abstract/Free Full Text]
  20. Sansores, R., R. Abboud, C. Becceril, M. Montano, C. Ramos, B. Vanda, and M. Selman. 1997. Effect of exposure of guinea pigs to cigarette smoke on elastolytic activity of pulmonary macrophages. Chest 112:214–219.[Abstract/Free Full Text]
  21. Finlay, G. A., L. R. O'Driscoll, K. J. Russel, E. M. D'Arcy, J. B. Masterson, M. X. Fitzgerald, and C. M. O'Connor. 1997. Matrix metalloproteinase expression and production by alveolar macrophages in emphysema. Am. J. Respir. Crit. Care Med. 156:240–247.[Abstract/Free Full Text]
  22. Selman, M., M. Montano, C. Ramos, B. Vanda, C. Becerril, F. Delgado, R. Sanores, R. Barrios, and A. Pardo. 1996. Tobacco smoke-induced lung emphysema in guinea pigs is associated with increased interstitial collagenase. Am J Physiol. 271:L734–L739.[Abstract/Free Full Text]
  23. Ohnishi, K., M. Takagi, Y. Kurokawa, S. Satomi, and Y. Konttinen. 1998. Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema. Lab Invest. 78:1077–1086.[Medline]
  24. Churg, A., J. Dai, K. Zay, A. Karsan, R. Hendricks, C. Yee, R. Martin, R. MacKenzie, C. Xie, L. Zhang, S. Shapiro, and J. L. Wright. 2001. Alpha-1-antitrypsin and a broad spectrum metalloprotease inhibitor, RS113456, have similar acute anti-inflammatory effects. Lab Invest. 81:1119–1131.[Medline]
  25. Brass, D. M., G. W. Hoyle, H. G. Poovey, J. Y. Liu, and A. R. Brody. 1999. Reduced tumor necrosis factor-alpha and transforming factor beta1 expression in the lungs of inbred mice that fail to develop fibroproliferative lesions consequent to asbestos exposure. Am. J. Pathol. 154:853–862.[Abstract/Free Full Text]
  26. Abbruzzese, T. A., R. J. Guzman, R. L. Martin, C. Yee, C. K. Karins, and R. L. Dalman. 1998. Matrix metalloproteinase inhibition limits arterial enlargement in a rodent arteriovenous fistula model. Surgery 124:328–335.[Medline]
  27. Sekhon, H., J. L. Wright, and A. Churg. 1994. Cigarette smoke causes rapid cell proliferation in small airways and associated pulmonary arteries. Am. J. Physiol. 267:L557–L563.[Abstract/Free Full Text]
  28. Li, K., B Keeling, and A Churg. 1996. Mineral dusts cause collagen and elastin breakdown in the rat lung: a potential mechanism of dust-induced emphysema. Am. J. Respir. Crit. Care Med. 153:644–649.[Abstract]
  29. Senior, R. M., G. L. Griffin, and R. P. Mecham. 1980. Chemotactic activity of elastin-derived peptides. J. Clin. Invest. 66:859–862.
  30. Senior, R. M., G. L. Griffin, R. P. Mecham, D. S. Wrenn, K. U. Prasad, and D. W. Urry. 1984. Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes. J. Cell Biol. 99:870–874.[Abstract/Free Full Text]
  31. Hunninghake, G. W., J. M. Davidson, S. Rennard, S. Szapiel, J. E. Gadek, and R. G. Crystal. 1981. Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema. Science 212:925–927[Abstract/Free Full Text]
  32. Riley, D. J., R. A. Berg, R. A. Soltys, J. S. Kerr, H. N. Gueds, S. F. Curren, and D. L. Laskin. 1988. Neutrophil response following intratracheal instillation of collagen peptides into rat lungs. Exp. Lung Res. 14:549–563.[Medline]
  33. Masubuchi, T., S. Koyama, E. Sato, A. Takamizawa, K. L. Kubo, M. Sekiguchi, S. Nagai, and T. Izumi. 1998. Smoke extract stimulates lung epithelial cells to release neutrophil and monocyte chemotactic activity. Am. J. Pathol. 153:1903–1912.[Abstract/Free Full Text]
  34. Liu, Z., X. Zhou, S. D. Shapiro, J. M. Shipley, S. S. Twining, L. A. Diaz, R. M. Senior, and Z. Werb. 2000. The serpin {alpha}1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Cell 109:647–655.
  35. Warner, R. L., C. S. Lewis, L. Beltran, E. M. Younkin, J. Varani, and K. J. Johnson. 2001. The role of metalloelastase in immune complex induced acute lung injury. Am. J. Pathol. 158:2139–2144[Abstract/Free Full Text]
  36. Warner, R. L., L. Beltran, E. M. Younkin, C. S. Lewis, S. J. Weiss, J. Varani, and K. J. Johnson. 2001. Role of stromelysin 1 and gelatinase B in experimental acute lung injury. Am. J. Respir. Cell Mol. Biol. 24:537–544.[Abstract/Free Full Text]
  37. Retamales, I., M. Elliott, B. Meshi, H. O. Coxson, P. D. Pare, F. C. Sciurba, R. M. Robers, S. Hayashi, and J. C. Hogg. 2001. Amplification of inflammation in emphysema and its association with latent adenoviral infection. Am. J. Respir. Crit. Care Med. 164:469–473.[Abstract/Free Full Text]



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