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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 954-960, (2002)
© 2002 American Thoracic Society

Original Articles

Synthetic Serine Elastase Inhibitor Reduces Cigarette Smoke–induced Emphysema in Guinea Pigs

Joanne L. Wright, Stephen G. Farmer and Andrew Churg

Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada; and Department of Biosciences, AstraZeneca R&D, Lund, Sweden

Correspondence and requests for reprints should be addressed to J.L. Wright, M.D., Department of Pathology, University of British Columbia, Vancouver, BC, V6T 2B5 Canada. E-mail: jlwright{at}interchange.ubc.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To test whether a serine elastase inhibitor could prevent or reduce emphysema, we exposed guinea pigs to cigarette smoke acutely, or daily for 6 months, and treated some animals with the neutrophil elastase inhibitor ZD0892. Acute smoke exposure increased lavage neutrophils and increased desmosine and hydroxyproline, measures of elastin and collagen breakdown; all these measures were reduced by ZD0892. Long-term smoke exposure produced emphysema and increases in lavage neutrophils, desmosine, hydroxyproline, and plasma tumor necrosis factor {alpha} (TNF-{alpha}). ZD0892 treatment returned lavage neutrophils, desmosine, and hydroxyproline levels to control values, and decreased airspace enlargement by 45% and TNF-{alpha} by 30%. Animals exposed to smoke for 4 months and then to smoke plus ZD0892 for 2 months were not protected against emphysema. Mice exposed to smoke showed increases in gene expression of neutrophil chemoattractant macrophage inflammatory protein-2, macrophage chemoattractant protein-1, and TNF-{alpha} at 2 hours along with increased plasma TNF-{alpha}; ZD0892 prevented the increases in macrophage inflammatory protein-2 and macrophage chemoattractant protein-1 expression and reduced plasma TNF-{alpha} levels to baseline. These data demonstrate that a serine elastase inhibitor ameliorates the inflammatory and destructive effects of cigarette smoke, and that these effects are mediated in part by neutrophils and by smoke-driven TNF-{alpha} production.

Key Words: {alpha}1-antiprotease • neutrophil elastase • proteolysis • proteases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of pulmonary emphysema in cigarette smokers is believed to be mediated through destruction of the collagen and elastin in the alveolar walls by proteases secreted by smoke-evoked and activated inflammatory cells. Exactly which cell type(s) and which protease(s) are crucial to the development of emphysema is a matter of considerable debate. The traditional view has been that neutrophils, acting in part via the proteolytic activities of neutrophil elastase, are pivotal. More recent studies, however, and particularly experiments showing that mice lacking macrophage metalloelastase are protected from smoke-induced emphysema, have focused on the macrophage and macrophage-derived metalloproteases as the crucial mediators (see Shapiro [1] and DISCUSSION).

Our laboratory has established a guinea pig model of cigarette smoke-induced pulmonary emphysema (2). Animals exposed to daily smoke for a period of several months develop enlarged airspaces and altered pulmonary function, fulfilling the criteria established by the National Institutes of Health for animal emphysema (3). Of note, these animals always show bronchoalveolar lavage fluid neutrophilia, a well-documented finding in both animals and humans exposed to smoke.

In the present study, we adopted a pharmacological approach to investigating the cells and proteases that play a role in the development of emphysema by testing ZD0892, an orally active inhibitor of serine elastases, in guinea pigs exposed both acutely and chronically to cigarette smoke. This agent is highly selective, inhibiting human neutrophil elastase and, to a lesser degree, porcine pancreatic elastase, and has no significant affinity for other proteases including trypsin, thrombin, and various cysteine or metalloproteinases (4–6). ZD0892 has been shown to suppress the myocardial inflammatory infiltrate, necrosis, and fibrosis induced by the murine encephalomyocarditis virus (M variant) (7), and to reduce monocrotaline-induced pulmonary hypertension (8).

We postulated that chronic administration of ZD0892 would reduce or ameliorate the emphysematous lung destruction produced by cigarette smoke.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sources of Materials
Hartley strain male guinea pigs weighing 350 g and C57BL/6 mice weighing 25 g were obtained from Charles River (Montreal, PQ, Canada). ZD0892 was obtained from AstraZeneca (Lund, Sweden), and 2R1 research cigarettes were obtained from the University of Kentucky (Lexington, KY). The animal experimental protocols were approved by the University of British Columbia (Vancouver, BC, Canada).

Acute Experiments with Guinea Pigs
To test the ability of ZD0892 to inhibit the acute reaction to cigarette smoke, we utilized groups of five guinea pigs set up as follows: sham smoke; sham smoke plus ZD0892 (30-mg/kg experiment only); cigarette smoke only; and cigarette smoke plus ZD0892. ZD0892 was orally administered in a dose of 1, 3, 10, or 30 mg/kg mixed into a vehicle (stock solution of NaCl, carboxymethyl cellulose, benzyl alcohol, and Tween 80), and the various doses are identified in Figures 1–6 as S + nZ, where n indicates the dose of ZD0892 (1 mg/kg, etc.).



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  		Figure 1. Acute effects of ZD0892 in sham-smoked animals (control, C), animals exposed to the smoke of five cigarettes (S), and smoke-exposed animals given 1–30 mg of ZD0892 per kilogram (S + 1Z, S + 3Z, S + 10Z, and S + 30Z). (A) Total cell numbers in lavage fluid. Smoke exposure increased total cell numbers compared with control, and administration of ZD0892 at doses of 10 and 30 mg/kg reduced this increase significantly compared with the smoke-only group. The numbers of cells continued to be increased above control levels in all the groups receiving ZD0892. *p < 0.05 compared with control. (B) Total numbers of PMNs in lavage fluid. Smoke exposure increased total numbers of PMNs compared with control. Administration of ZD0892 in doses of 3, 10, and 30 mg/kg reduced the numbers of PMNs compared with the smoke-only group; the numbers of PMNs in groups receiving 10 and 30 mg/kg were reduced to control levels. *p < 0.05 compared with control. (C) Desmosine levels (expressed as pmol/ml) in lavage fluid. Smoke exposure significantly increased desmosine levels compared with control. Administration of ZD0892 at doses of 10 and 30 mg/kg reduced the desmosine level to that of control. *p < 0.05 compared with control. (D) Hydroxyproline levels (expressed as µg/ml) in lavage fluid. Smoke exposure significantly increased hydroxyproline levels compared with control. Administration of ZD0892 at doses of 10 and 30 mg/kg significantly reduced the level compared with the smoke-only group. The values in the group given 10 mg/kg continued to be greater than those found in the control animals, but values for the group given 30 mg/kg were reduced to control levels. *p < 0.05 compared with control.

 


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  		Figure 6. Levels of plasma TNF-{alpha} in guinea pigs exposed to the smoke of five cigarettes (S), and smoke-exposed animals given ZD0892 at 10 mg/kg for 6 months [S + ZD(6)] or smoke for 4 months followed by smoke plus ZD0892 at 10 mg/kg for 2 months [S + ZD(2)]. Smoke exposure more than doubled the plasma TNF-{alpha} level. Administration of ZD0892 for 6 months reduced this increase by 30% compared with the smoke-only group, but not when compared with control levels. Administration of ZD0892 for 2 months had no significant effect compared with the smoke-only group. *p < 0.05 compared with control.

 


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  		Figure 2. Gene expression of the inflammatory mediators MIP-2, MCP-1, and TNF-{alpha} in groups of mice (sham smoked [C] animals, animals exposed to the smoke of four cigarettes [S], and smoke-exposed animals given ZD0892 at 30 mg/kg [S + ZD]). Control values are normalized to 100 for ease of examination. Smoke increased the levels of all mediators by at least twofold. ZD0892 reduced the increase in MIP-2 but not to control levels and reduced the increase in MCP-1 to control levels but did not affect the level of TNF-{alpha}. *p < 0.05 compared with control.

 


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  		Figure 3. Levels of plasma TNF-{alpha} in groups of mice (sham-smoked [C] animals, animals exposed to the smoke of four cigarettes [S], and smoke-exposed animals given ZD0892 at 30 mg/kg [S + ZD]). Smoke exposure increased the plasma TNF-{alpha} level, and this was reduced to control levels by ZD0892. *p < 0.05 compared with control.

 


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  		Figure 4. Chronic effects of ZD0892 in animals exposed to the smoke of five cigarettes (S), and smoke-exposed animals given 10 mg/kg ZD0892 for 6 months [S + ZD(6)] or smoke for 4 months followed by smoke plus ZD0892 (10 mg/kg) for 2 months [S + ZD(2)]. (A) Total cell numbers in lavage fluid from sham-smoked animals (control, C). There were no significant differences between control and smoke-exposed animals. (B) Total numbers of PMNs in lavage fluid. Smoke exposure increased the total numbers of PMNs, whereas administration of ZD0892 for 6 or 2 months significantly reduced the numbers, compared with the smoke-only group, to those found in control animals. *p < 0.05 compared with control. (C) Desmosine levels (expressed as pmol/ml) in lavage fluid. Smoke exposure increased desmosine levels compared with control. Administration of ZD0892 for 6 or 2 months decreased desmosine levels, compared with the smoke-only group, to the level of control. *p < 0.05 compared with control. (D) Hydroxyproline levels (expressed as µg/ml) in lavage fluid. Smoke exposure increased hydroxyproline levels compared with control. Administration of ZD0892 for 6 or 2 months reduced levels to that of control. *p < 0.05 compared with control.

 


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  		Figure 5. Airspace size (µm) in guinea pigs exposed to the smoke of five cigarettes (S), and in smoke-exposed animals given ZD0892 at 10 mg/kg for 6 months [S + ZD(6)] or smoke for 4 months followed by smoke plus ZD0892 at 10 mg/kg for 2 months [S + ZD(2)]. Smoke exposure increased airspace size by 28%. Administration of ZD0892 for 6 months reduced this increase by 45% compared with the smoke-only group, but not to control levels. Administration of ZD0892 for 2 months had no significant effect compared with the smoke-only group. *p < 0.05 compared with control.

 
Animals received the first dose of ZD0892 on the afternoon of Day 1, and were exposed to the smoke of five cigarettes on the morning of Day 2. A final dose was administered on the morning of Day 3, and the animals were killed in the afternoon. Cigarette smoke exposure was performed according to our usual protocol, consisting of a vented nose-only chamber (9) into which 20 ml of cigarette smoke was injected at 1.5-minute intervals. Twenty-four hours after smoke exposure, the animals were killed, using an overdose of urethane anesthesia. The lungs were removed and lobes were lavaged with 20 ml of cold saline for cell count and differential, or with 20 ml of distilled water for subsequent analysis of hydroxyproline (HYP), a marker of collagen breakdown, and desmosine (DES), a marker of elastin breakdown, by high-performance liquid chromatography as previously described by us (10). Water is required for the latter procedures because concentrated salts interfere with high-performance liquid chromatography analysis.

Acute Experiments with Mice
To determine the effect of ZD0892 on the mediators of acute cigarette smoke-induced pulmonary inflammation, we exposed groups of three C57BL/6 mice to the smoke of four cigarettes or to sham smoke as control. Mice were used rather than guinea pigs because of the convenient availability of gene sequences for mice. One smoke-exposed group was treated 12 hours in advance with ZD0892 (30 mg/kg) in 1.0 ml of vehicle by gavage. Two hours after smoke inhalation, the mice were killed by exsanguination, and plasma was collected in EDTA tubes. RNA was immediately extracted from the lung tissue and reverse transcriptase-polymerase chain reaction was performed for macrophage chemoattractant protein-1 (MCP-1), the neutrophil chemoattractant macrophage inhibitory protein-2 (MIP-2), and tumor necrosis factor-{alpha} (TNF-{alpha}) as described by us (11). Primers were as listed in Table 1 (12, 13).


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  		TABLE 1. Primer sequences

 
Plasma TNF-{alpha} was assayed by a modification of the method of Levesque and coworkers (14). The assay can measure picogram concentrations of human, rat, and murine TNF-{alpha}. In brief, the method employs TNF-sensitive, actinomycin D-treated murine L929 fibroblasts to quantify TNF activity in supernatants derived from cell cultures or serum samples. The cells were maintained in culture with RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% (v/v) supplemented calf serum (HyClone Laboratories, Logan, UT). Recombinant mouse TNF-{alpha} (GIBCO) was used as a standard. After plating and incubating the fibroblasts, test samples were added to the wells, followed by actinomycin D, and the plates were incubated for 18 hours in a 37°C, 6% CO2 humidified incubator. The plates were then washed and a crystal violet–alcohol solution was added, followed by final washing, removal of solutions, and overnight drying. After addition of 100% methanol to the wells, each well was immediately read with a microtiter plate reader to determine absorbance at 595 nm.

Chronic Experiments with Guinea Pigs
To test the ability of ZD0892 to inhibit cigarette smoke-induced emphysema, we utilized groups of six guinea pigs set up for daily (5 days/week) treatment as follows:

  • Sham smoke, 6 months
  • Cigarette smoke only, 6 months
  • Cigarette smoke plus ZD0892, 6 months [identified as S + ZD(6)]
  • Cigarette smoke only, 4 months, followed by smoke plus ZD0892, 2 months [identified as S + ZD(2)]

with ZD0892 administered as a dose of 10 mg/kg twice daily.

At the end of each experimental time, the guinea pigs were killed by an overdose of anesthesia, followed by exsanguination. Plasma was obtained from EDTA-anticoagulated blood, and used for TNF-{alpha} assay as described above. The lungs were removed and individual lobes were lavaged with 20 ml of cold saline for cell count and differential, or with 20 ml of distilled water for subsequent analysis of HYP and DES by high-performance liquid chromatography. For morphometric analysis, a lobe of lung was inflated with formalin for 24 hours at a constant pressure of 25 cm H2O, after which it was serially sectioned in a sagittal plane; a random slice was submitted for paraffin embedding, sectioning, and staining with hematoxylin and eosin.

To measure airspace size, we utilized a standard morphometric grid, with a total line length of 1.02 mm at x200 magnification, and 42 points. Twenty random sites were counted for each section, and mean linear intercept (Lm) was determined from the summative values of all sites (15).

All statistical procedures were performed with the SYSTAT system (16), using analysis of variance, and corrected as appropriate by the Bonferroni method.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Acute Experiments with Guinea Pigs
To ascertain the effect of administration of ZD0892, the first set of acute experiments used a ZD0892 dose of 30 mg/kg. There was no difference in lavage total cell count or numbers of polymorphonuclear cells (PMNs) in lavage, or in the desmosine and hydroxyproline levels, between the control group and the control plus ZD0892 group. We therefore eliminated the control plus ZD0892 group from the remainder of the protocols.

The data from acute smoke exposure are illustrated in Figure 1. Smoke exposure increased the total cells by approximately twofold (p < 0.001), and numbers of PMNs by about 10-fold (p < 0.001) in the lavage fluid. In conjunction with the cellular increases, lavage DES was increased, usually by about 2.5 times (p = 0.002), whereas HYP was slightly less than tripled (p < 0.001) compared with the sham-exposed control group. Administration of ZD0892 at doses of 10 or 30 mg/kg reduced the numbers of total lavage cells (compared with smoke only, p < 0.001); numbers of lavage PMNs were progressively reduced by doses of 3 mg/kg (p < 0.01), 10 mg/kg (p < 0.001), and 30 mg/kg (p = 0.002). Lavage DES was significantly decreased from the smoke-exposed level only by the 30-mg/kg dose (p = 0.004), whereas HYP was decreased by doses of 10 mg/kg (p = 0.02) and 30 mg/kg (p = 0.05).

Acute Experiments with Mice
The mRNA data for acute inflammatory mediators in C57BL/6 mice are illustrated in Figure 2. Smoke more than doubled gene transcription of MIP-2, MCP-1, and TNF-{alpha} at 2 hours (all values significantly different from control, p < 0.001). Administration of ZD0892 reduced the level of MIP-2 by about 75% compared with the smoke-only level (p < 0.01), but not compared with the control level (p < 0.01), and reduced MCP-1 to control levels. By contrast, the levels of TNF-{alpha} gene expression were not altered by administration of ZD0892. Acute exposure of mice to cigarette smoke was associated with an approximate doubling of plasma TNF-{alpha} (p < 0.001); administration of ZD0892 completely abrogated this increase (Figure 3).

Chronic Experiments with Guinea Pigs
Lavage cells. There were no significant differences in the total numbers of lavage cells in the smoke-exposed groups at 6 months compared with the control group (Figure 4A), with a large degree of variability from animal to animal in the numbers of macrophages (data not shown). PMNs (Figure 4B) were significantly and markedly increased in the smoke-only group compared with the control group (p < 0.001); however, they were reduced to control levels in the S + ZD(6) group and in the S + ZD(2) group [difference from smoke only, p = 0.01 and p = 0.02, S + ZD(6) and S + ZD(2), respectively]. Numbers of eosinophils and lymphocytes in the lavage fluid did not change with smoke exposure [(mean ± SD, eosinophils) x 104: control, 23.6 ± 12.9; smoke, 12.3 ± 6.9; S + ZD(6), 6.6 ± 7.0; S + ZD(2), 11.0 ± 11.5; (mean ± SD, lymphocytes) x 104: control, 2.1 ± 1.1; smoke, 3.8 ± 1.8; S + ZD(6), 1.6 ± 1.3; S + ZD(2), 3.3 ± 2.4].

Desmosine and Hydroxyproline
Desmosine and hydroxyproline are illustrated in Figures 4C and 4D, respectively. Lavage desmosine was approximately doubled in the smoke-exposed animals (p < 0.001) compared with control animals. Administration of ZD0892 for 6 months reduced the desmosine level to control values, as did administration of ZD0892 for only the last 2 months. Hydroxyproline levels were approximately doubled in the smoke-only group compared with the control group (p < 0.01); administration of ZD0892 for 6 months or only the last 2 months reduced the levels to control values.

Airspace Size
Airspace size data are illustrated in Figure 5. Airspace size was increased by 28% after smoke exposure for 6 months (p < 0.001); this is typical of the level of increase in Lm seen in small animal models of smoke exposure (2). Administration of ZD0892 for 6 months reduced this increase by about 45% [smoke only versus S + ZD(6), p < 0.02], but airspace size was still significantly greater than control levels [control versus S + ZD(6), p < 0.001]. Administration of ZD0892 for only the last 2 months of the experiment resulted in increased airspace size, identical to that seen in the smoke-only group.

Plasma TNF-{alpha}
Plasma TNF-{alpha} data are illustrated in Figure 6. Chronic smoke exposure resulted in a 2.2-fold increase in plasma TNF-{alpha}. Treatment for 6 months with ZD reduced smoke-mediated increases in plasma TNF-{alpha} by 30% (p < 0.04 compared with the smoke-only group). Administration of ZD for only the last 2 months of a 6-month smoke exposure reduced TNF-{alpha} slightly but not significantly compared with smoke-only exposure.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The protease–antiprotease hypothesis of smoke-induced emphysema posits that there is an imbalance between the proteolytic enzymes released by inflammatory cells and the presence/activity of proteolytic inhibitors. The results reported in the present study present additional support for the protease–antiprotease hypothesis by showing that decreasing neutrophil influx decreases evidence of connective tissue breakdown and ameliorates cigarette smoke-induced emphysema. However, the mechanism behind decreased neutrophil influx may or may not be directly related to inhibition of neutrophil elastase.

As noted above, the role of neutrophils in smoke-induced emphysema is controversial. Neutrophils have been consistently documented in the lavage fluid, alveolar walls, and airspaces in human smokers (17–20), and this finding is recapitulated in animal models (21–23). A series of studies from Cavarra and colleagues (24–26) have demonstrated correlations between neutrophil influx into the lungs and elastolytic activity resulting in emphysema. We (21) showed that levels of lavage DES and HYP correlated well with lavage neutrophil numbers, but not with macrophage numbers, after acute smoke exposure in mice. Although the animal model studies suggest a relationship between the presence of neutrophils and matrix destruction, correlations between neutrophils and indices of emphysema in human lungs have been inconsistent. Retamales and coworkers (22) found a significant increase in a wide variety of inflammatory cells, including PMNs and macrophages, in both alveolar walls and airspaces in severe, compared with mild, emphysema, as determined by computed tomography. In contrast, Eidelman and coworkers (18) failed to find any correlation between neutrophil numbers in histologic sections and morphologic evidence of matrix breakdown as assessed by the destructive index, and both Ludwig and coworkers (20) and Finkelstein and coworkers (19) found an inverse relationship between numbers of neutrophils and emphysema.

The importance of the macrophage in cigarette smoke-induced lung disease, at least in animals, has been emphasized by the finding that macrophage metalloelastase-deficient mice failed to develop emphysematous airspace enlargement after cigarette smoke exposure (27), suggesting that macrophage elastases may be important in altering the proteolysis–antiproteolysis balance (3, 27–30). Macrophage-derived metalloproteases including gelatinases A and B, matrilysin, and macrophage metalloelastase all can degrade elastin (31–33) and macrophages from smoke-exposed animals or cultured macrophages from human lavage fluid of human smokers have been demonstrated to produce increased elastolytic activity (34, 35). Furthermore, increased levels of matrix metalloproteinase (MMP)-1, MMP-2, MMP-8, and MMP-9 have been reported in human lungs with emphysema compared with lungs without emphysema (36, 37).

Support for a role for macrophages also comes from the studies by Finkelstein and coworkers (19), who demonstrated positive correlations with alveolar macrophages and T lymphocytes and emphysematous destruction in histologic sections of human lungs. As noted above, Retamales and colleagues (22) found positive correlations between tissue and airspace macrophages in tissue sections. Metalloproteinases are also known to inactivate {alpha}1-antiprotease (38), thus potentially adding to proteolytic imbalance. However, macrophages may also act indirectly: in one report, we (39) showed that alveolar macrophages, and specifically macrophage metalloelastase, appeared to be driving neutrophil influx after smoke exposure.

The present article reinforces the idea that neutrophils are important in the development of emphysema, but whether the crucial effect of ZD0892 is really due to neutrophil elastase inhibition is less clear. As shown in Figures 2 and 3, smoke acutely upregulates the production of proinflammatory and chemoattractant mediators and these mediators are presumably the driving force for inflammatory cell influx into the lung. Because elastin and collagen fragments generated by proteolytic attack are in themselves chemoattractants (40, 41), it is possible that the initial acute inflammatory response to smoke is sustained by protease-mediated matrix breakdown and that inhibition of neutrophil elastase by ZD0892 shuts off this process.

Alternatively, the TNF-{alpha} plasma and gene expression data suggest that ZD0892 might in fact act as an antiinflammatory agent, because ZD0892 reduces chemoattractant (but not TNF-{alpha}) gene expression and reduces plasma TNF-{alpha} both acutely and, with less efficacy, chronically. It is intriguing that in the chronically exposed animals there is a rough correlation between the levels of TNF-{alpha} reduction and protection against airspace size enlargement, suggesting a relationship between the two. The role of TNF-{alpha} in smoke-induced chronic obstructive pulmonary disease is somewhat controversial (reviewed by Churg and coworkers [11]), but we have shown that mice with knocked-out TNF-{alpha} receptors and mice that are low-level TNF-{alpha} producers (strain 129 mice) are protected against acute increases in lavage PMNs, DES, and HYP and gene expression of MIP-2 and MCP-1 after smoke exposure (11). As well, Lucey and coworkers (42) have reported that TNF-{alpha} receptor knockout mice are partially protected against elastase-induced emphysema.

How ZD0892 might decrease TNF-{alpha} release is also uncertain, but there is increasing evidence that a variety of antiproteases, including secretory leukocyte protease inhibitor, {alpha}1-antiprotease, and RS113456, a broad-spectrum antimetalloprotease, appear to have direct antiinflammatory effects in models of exposure to cigarette smoke (21), silica-induced inflammation (23), and an immune complex model of lung injury (23, 43). These anti-inflammatory effects are driven at least in part by antiprotease-mediated inhibition of NF-{kappa}B activation (23, 43), and there appears to be direct inhibition of neutrophil influx. Our current data suggest that this effect might reflect suppression of TNF-{alpha} production and thus suppression of endothelial activation.

Under this hypothesis, matrix attack in the current smoking model is secondary to neutrophils leaving the vessels and reaching the lung, and protection is provided by keeping them out of the lung parenchyma, although we still cannot rule out an effect solely related to inhibition of neutrophil elastase. It is interesting in this regard that ZD0892 also reduces inflammation in a murine myocarditis model (7). Further prevention of proteolytic attack may prevent degradation of antiproteases and thus increase the antiproteolytic screen of the lower respiratory tract. For example, in a bullous pemphigoid model, deletion of genes for either neutrophil elastase or MMP-9 prevents skin blisters by preventing proteolytic breakdown of endogenous {alpha}1-antiprotease (44).

An additional important question concerns why we observe only partial protection against smoke-mediated increases in airspace size and TNF-{alpha} levels when using 6 months of continuous ZD0892 treatment. Although this might be a dose effect (i.e., a higher dose would have been completely protective), this explanation seems unlikely because neutrophil infiltration, DES, and HYP were all returned to baseline levels by the dose of ZD0892 used (10 mg/kg, twice daily). It is, of course, possible that although ZD0892 appears to completely prevent elevations in PMNs, DES, and HYP, in reality small elevations in all these measures are actually present but cannot be detected statistically, given the sample size. It is also possible that other species of proteases that are not inhibited by ZD0892, possibly metalloproteases as described above, are activated during cigarette smoke-induced inflammation. However, if this were true we would expect to see elevations in lavage DES and HYP at the 6-month sample point despite reduction in PMN numbers to baseline. Last, these findings raise the possibility that proteolytic attack is only one component in the development of emphysema, and that other processes that lead to matrix damage without matrix breakdown allow matrix stretching. Whatever the process, it clearly is initiated early in the course of smoke-induced emphysema, because exposure to smoke for 4 months and then to smoke plus ZD0892 for 2 months did not provide any degree of protection against airspace enlargement. It is also interesting that Shapiro has found that neutrophil elastase-deficient mice are partially (50–60%) protected from smoke-induced airspace enlargement (1). This protection level is essentially identical to that obtained in the present experiments with ZD0892.

Because of the protease–antiprotease hypothesis, it has long been believed that antiprotease might provide a treatment for emphysema. The potential problems with this approach, including the idea that emphysema is not mediated just by proteolytic attack, have been discussed by Snider and coworkers (45). As well, a report showing that mice lacking neutrophil elastase are susceptible to gram-negative infections (46) emphasizes the potential dangers of antiproteolytic therapy. On the other hand, our current results, using an extracellular elastase inhibitor, suggest that some specific types of antiinflammatory therapy might be beneficial in modifying or preventing the development of emphysema.

In summary, in this animal model, we have shown for the first time that administration of a serine protease inhibitor (ZD0892) ameliorates lung inflammation and parenchymal destruction induced by cigarette smoke. This is associated with evidence of decreased indices of matrix degradation, decreased TNF-{alpha} levels, and a decreased number of acute inflammatory cells in the bronchoalveolar lavage fluid. The study indicates that alteration of the protease–antiprotease balance is of importance in the genesis of cigarette smoke-induced matrix destruction and emphysema and that such pathophysiological alterations might be redressed by administration of synthetic, small molecule inhibitors of neutrophil elastase.


   FOOTNOTES
 
Supported by AstraZeneca R&D and grant MOP 42339 from the Canadian Institutes of Health Research.

Received in original form February 10, 2002; accepted in final form June 25, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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