Published ahead of print on April 10, 2003, doi:10.1164/rccm.200302-203OC
© 2003 American Thoracic Society
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-1-Antitrypsin Ameliorates Cigarette Smoke–induced Emphysema in the Mouse
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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Serine elastase inhibitors have been proposed as a treatment for cigarette smoke–induced emphysema, but little is known about whether such agents actually are effective. We recently reported that a synthetic serine elastase inhibitor, ZD0892, provided some protection against emphysema in a guinea pig model. For these experiments, we used transgenic mice that expressed extremely low levels of human
-1-antitrypsin (A1AT) but were tolerant of exogenous human A1AT. Mice were exposed to daily cigarette smoke for up to 6 months; some animals received 20 mg of human A1AT (Prolastin) every 48 hours. Treatment with A1AT produced an approximate twofold increase in serum A1AT levels and elastase inhibitory capacity and abolished smoke-induced elevations in lavage neutrophils and matrix breakdown products (desmosine and hydroxyproline) measured from 2 to 30 days of smoke exposure. A1AT oxidized to remove antiproteolytic activity did not increase serum elastase inhibitory capacity but did prevent neutrophil influx. Treatment with A1AT for 6 months provided 63% protection against increased airspace size (emphysema) and abolished smoke-mediated increases in plasma tumor necrosis factor-. We conclude that A1AT therapy ameliorates smoke-induced inflammation and matrix breakdown, possibly via an antiinflammatory mechanism related to tumor necrosis factor- suppression, and provides partial protection against emphysema.
Key Words: neutrophil • -1-antitrypsin • cigarette smoke • emphysema
Cigarette smoke–induced emphysema is generally believed to occur as a result of an imbalance between proteases derived from smoke-evoked inflammatory cells and the antiproteolytic defenses of the lung (reviewed in 1–3). Historically, this protease–antiprotease hypothesis arose from the finding that purified elastases, including pancreatic porcine elastase and human neutrophil elastase, would produce emphysema when instilled into the lungs of experimental animals and also from the observation that individuals deficient in -1-antitrypsin (A1AT), the major antiprotease in the lower respiratory tract, developed early emphysema, particularly if they smoked (1–3). These findings lead to the belief that neutrophils are the important inflammatory cells and neutrophil elastase the crucial protease in the development of emphysema, since neutrophil elastase is a potent elastolytic protease.
More recently, it has been shown that a variety of metalloproteases also can degrade elastin (1–3) and that mice lacking macrophage metalloelastase (MMP)-12 do not develop smoke-induced emphysema (4). These findings lead to an alternate formulation of the protease–antiprotease hypothesis in which macrophages and macrophage-derived metalloproteases were the agents of emphysema and a belief that neutrophils played a small role or no role in emphysema.
However, new studies have reemphasized the importance of neutrophil-derived proteases and A1AT. We showed that in fact both MMP-12 and neutrophils were required for acute smoke-induced matrix breakdown, the precursor of emphysema (5, 6), and that this process appears to run through MMP-12–mediated release of tumor necrosis factor- (TNF-) from alveolar macrophages, with subsequent endothelial activation, neutrophil influx, and matrix attack by neutrophil-derived proteases (7). Cavarra and colleagues (8) found that smoke-induced emphysema developed more rapidly in A1AT-deficient pallid mice compared with C57BL/6 mice or DBA mice, which have higher A1AT levels. The same group (9) previously showed that if a neutrophil influx was evoked with instilled formyl-methionylleucylphenylalanine (FMLP), the degree of resulting emphysema and amount of elastin loss were inversely proportional to serum A1AT levels in different strains of mice. Takubo and colleagues (10) found that serum A1AT levels influenced the type of emphysema in mice exposed to smoke. A1AT-deficient pallid mice developed a form of panlobular emphysema and a T lymphocyte infiltrate, whereas C57 mice developed centrilobular emphysema but no T-cell infiltrate.
Concomitant with the formulation of the original (neutrophil elastase–mediated) protease–antiprotease hypothesis, interest was expressed in the development of antiprotease therapy as a way of stopping, or at least slowing, the development of emphysema (11). Considerable effort has been devoted to developing neutrophil elastase inhibitors (reviewed in 12, 13), but most of the available in vivo experimental data on such agents have come from acute models such as elastase-induced pulmonary hemorrhage, and remarkably little information has been published on the use of antiproteolytic agents in emphysema. Surprisingly, as pointed out by Snider and colleagues (11), some reversible small molecular weight inhibitors of neutrophil elastase actually worsen experimental neutrophil elastase-induced emphysema, possibly because they increase the transport of neutrophil elastase into the interstitium. However, three synthetic neutrophil elastase inhibitors, FR901277 (14), ICI 200,800 (12, 15), and ONO-6618 (16), as well as A1AT (14), have been shown to provide partial-to-complete protection against morphologic or functional measures of emphysema after intratracheal instillation of neutrophil or porcine pancreatic elastase in laboratory animals.
A1AT therapy has been tried in humans with A1AT deficiency. Recent data indicate that such treatment reduces levels of sputum leukotriene B4, a neutrophil chemoattractant, and reduces sputum elastase activity (17), but there is as yet no controlled trial in humans that has shown a protective effect of such therapy on the development of emphysema (17); indeed, a study of urinary desmosine (a marker of elastin breakdown) failed to show any decrease in urinary excretion with A1AT therapy during an 8-week trial (18).
Using a cigarette smoke model, we found that ZD0892, a synthetic antiserine protease with particular efficacy against neutrophil elastase, provided partial protection against smoke-induced emphysema in guinea pigs (19) (see DISCUSSION). We have also developed transgenic mice with the human A1AT gene under the control of the SpC promoter (described in detail by Dhami and colleagues [20]). These animals produce minute amounts of human A1AT in the lung parenchyma (mean 2.9 ± 1.4 µg/g of lung protein); the mean level of protein produced is far too low to be of therapeutic benefit. However, the animals are tolerant of repeated administration of exogenous human A1AT and thus can be used to test the potential value of supplemental A1AT as a treatment for smoke-induced emphysema. In this article, we report our experience using commercially available human A1AT (Prolastin, Bayer, West Haven, CT) on the development of emphysema in these mice. A portion of these results has been published in abstract form (21).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Study Design
We used CD-1 mice transgenic for human A1AT. The animals were exposed to whole smoke from two Kentucky 2R1 cigarettes 5 days per week; control mice were sham smoked (22) (see the online supplement).
Prolastin Purification and Administration
Exogenous human A1AT (Prolastin) was purchased from Bayer. Prolastin was purified as previously described (5); 20 mg of purified Prolastin were administered intraperitoneally in 1.0 ml of saline 24 hours before initial smoke exposure and then every 48 hours. Test groups were control, control + Prolastin, smoke, and smoke + Prolastin. For some experiments, the control + Prolastin group was omitted (see the online supplement).
Effects of Albumin
To ascertain whether the outcomes with Prolastin were due to the A1AT rather than a nonspecific protein effect, we examined the results of cigarette smoke exposure using similar groups given human albumin (see the online supplement).
Effects of Oxidized A1AT and Measurement of Elastase Inhibitory Capacity
To determine whether the antiproteolytic actions of A1AT were crucial to its effects, we oxidized Prolastin so that its antiproteolytic effects were lost, as described by Churg and colleagues (23) (see the online supplement). Elastase inhibitory capacity was measured as described by Churg and colleagues (23).
Collection of Lung Tissue and Lavage Fluid
Animals were sacrificed and the lungs removed from the chest cavity and lavaged with saline for cell counts or water for HPLC analysis of matrix breakdown products (24) (see the online supplement). At the 6-month time period, the lungs of the saline-lavaged animals were then inflated with formalin at a pressure of 25 cm of water for 24 hours.
Desmosine and Hydroxyproline Analysis
Analysis for levels of desmosine, a marker of elastin breakdown, and hydroxyproline, a marker of collagen breakdown, was performed by HPLC using our published protocol (24) (see the online supplement).
Analysis of Serum A1AT Levels
At the time of sacrifice, blood was obtained, and serum protein electrophoresis was run using a clinical protein electrophoresis apparatus. Amounts of A1AT in each band were determined by densitometry. To confirm that the bands seen in the -1 region represented A1AT, we performed a Western blot on serum samples (see the online supplement).
Evaluation of Airspace Size
For morphometric analysis, the formalin-fixed lungs were serially sectioned in a sagittal plane, and a random slice was submitted for paraffin embedding, sectioning, and staining by hematoxylin and eosin. To measure airspace size, we used 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 determined from the summed values of all sites (25).
Plasma TNF- Levels
Plasma TNF- levels were determined using the L929 cell assay as described by Wright and colleagues (19).
Statistics
With the exception of the neutrophil counts, comparisons were made using analysis of variance. Because neutrophil counts were often zero, comparisons were performed with the nonparametric Kruskal-Wallis test. Values of p < 0.05 were considered significant. Regardless of statistical method, all graphs show means and SDs.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1A illustrates the serum protein electrophoresis strips from a 7-day experiment. These show the presence of a single band for mouse A1AT in all animals and a second band representing exogenous human A1AT in the animals receiving Prolastin treatment. The baseline mouse A1AT serum level was 5.0 ± 1.0 mg/ml, and after the Prolastin treatment, the mean ± SD ratio of human to mouse A1AT was 2.2 ± 0.3. Endogenous mouse A1AT levels were not altered by Prolastin administration. Similar results were seen at other time points. Figure 1B illustrates a Western blot of serum samples run against A1AT. Two bands are seen in the 51 kD range after administration of Prolastin, and only one band was seen in untreated mice, confirming that the bands observed in the electrophoresis in Figure 1A are A1AT.
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Figures 2–5 show the data for the 2-, 4-, 7-, and 30-day time periods, respectively, for lavage neutrophils, macrophages, hydroxyproline, and desmosine. Administration of Prolastin for 30 days to the sham-smoked control group had no effect on baseline inflammatory cell numbers. Cigarette smoke exposure induced a neutrophilia at all time points compared with control subjects (p < 0.03, p < 0.01, p < 0.01, and p < 0.01, respectively), which was reduced to control baseline by administration of exogenous A1AT. In contrast, smoke had no effect on lavage macrophages except for the 30-day period, at which time they were slightly but significantly elevated (p < 0.05).
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In the smoke-exposed animals, lavage hydroxyproline was significantly elevated at the 2-, 7-, and 30-day time periods (p < 0.05, p < 0.01, p < 0.001, respectively, compared with control subjects) and was elevated but did not achieve statistical significance at the 4-day period. In this same group, lavage desmosine was significantly elevated at all time periods (p < 0.03, p < 0.05, p < 0.04, and p < 0.05, respectively, compared with control subjects). Prolastin reduced the hydroxyproline and desmosine to control levels at all time points.
To ensure that the effects of Prolastin were not due to a nonspecific protein response, albumin, which has a molecular weight similar to that of A1AT, was administered for a 2-day smoking experiment using a protocol identical to that for Prolastin. Figure 6 shows that albumin provided no protection against smoke-induced lavage neutrophilia.
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To determine whether the antiproteolytic effects of A1AT are crucial to its ability to protect the lung, various acute (24-hour) experiments were performed with A1AT carefully oxidized to destroy its antiproteolytic activity as described by Churg and colleagues (23) (Figure 7) . Native A1AT almost doubled serum elastase inhibitory capacity, a result that corresponds to the increases in serum A1AT levels shown in Figure 1, whereas oxidized A1AT did not increase serum elastase inhibitory capacity at all (Figure 7A). Nonetheless, the oxidized form of the protein was just as effective as the native form in reducing lavage neutrophils to control levels after smoke exposure (Figure 7B). Both native and oxidized forms of A1AT also prevented smoke-induced increases in plasma TNF-
(Figure 7C).
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Figure 8 shows data from the 6-month time point. Smoke exposure again produced an increase in lavage neutrophils and also an increase in lavage macrophages. Administration of A1AT reduced neutrophil levels by approximately 75% and lavage macrophages by approximately 50% compared with the smoke-only groups. Airspace size (mean linear intercept [Lm]) was increased by 37% in the smoke-exposed animals compared with the sham-smoked control subjects. Administration of A1AT reduced airspace size by 63% compared with the smoke-exposed animals, but not to control levels. TNF-
levels were increased by cigarette smoke exposure, and A1AT abolished the increases.
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Figure 9 shows representative microscopic images of the lungs of the various treatment groups at 6 months. Increases in airspace, primarily the alveolar duct, size can be seen in the animals exposed to cigarette smoke (Figure 9C), and this increase is greatly reduced by administration of A1AT (Figure 9D).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In this article, we have demonstrated that administration of a naturally occurring serine protease inhibitor, A1AT, ameliorates smoke-induced emphysema. The observations support our previous findings that a synthetic serine protease inhibitor, ZD0892, also is protective. However, these conclusions are subject to a number of important limitations.
Although both agents are serine protease inhibitors and in particular have the ability to effectively neutralize neutrophil elastase, it is unclear to what extent antiprotease activity is actually the important mechanism of action. Lavage neutrophils and plasma TNF- were elevated in our study of guinea pigs exposed to smoke for 6 months (19) and in the current study at 6 months, and in both species antiprotease treatment, whether with ZD0892 or A1AT, had the effect of decreasing neutrophil influx and lowering plasma TNF- levels at 6 months, although ZD0892 provided only a partial decrease in TNF- levels compared with the complete return to control values seen with A1AT.
As well, in this study, we observed that oxidized A1AT, a protein without antiprotease activity, still managed to suppress the acute smoke-induced neutrophil response, even though it did not increase serum elastase inhibitory capacity. We cannot absolutely exclude the possibility that mouse neutrophil elastase is still effectively inhibited by oxidized A1AT, but this seems unlikely, as in general, oxidized A1AT has a several order of magnitude lower association constant for elastases compared with the native protein. Moreover, as shown by Campbell and colleagues (26), rapid inhibition of neutrophil elastase at a microscopic level is absolutely crucial to preventing proteolytic injury to the lung. These observations suggest that, in this model, exogenous A1AT is not acting primarily through an antielastase mechanism.
We have previously observed that oxidized A1AT suppressed pulmonary inflammation and matrix breakdown caused by crystalline silica, a potent inducer of persisting pulmonary neutrophil influx, and also found that A1AT suppressed silica-induced whole-lung nuclear factor-
B upregulation (23). In this study, native and oxidized A1AT were both able to abolish acute smoke induced increases in plasma TNF-. These findings suggest that A1AT has considerable antiinflammatory effects as well as antiprotease effects and that antiinflammatory suppression of neutrophil influx may be the major protective mechanism in our smoke emphysema model as well. Other antiproteases have previously been shown to have antiinflammatory effects; for example, secretory leukocyte protease inhibitor downregulates nuclear factor-B activation and neutrophil influx after IgG instillation into the lung (27). Whether ZD0892 is also acting by an antiinflammatory mechanism remains to be determined.
The role of TNF- in mediating the various manifestations of chronic obstructive pulmonary disease is controversial. We have previously shown that in an acute animal model, mice lacking TNF- receptors do not develop either acute inflammation or matrix breakdown after smoke exposure (28). In many, but not all, human studies, TNF- levels in lavage, sputum, or serum are higher in smokers than nonsmokers or in smokers with clinical chronic obstructive pulmonary disease compared with those without (for a brief summary, see Thurlbeck [25]). High TNF- levels are thought to be the cause of cachexia and muscle wasting in some patients with chronic obstructive pulmonary disease (29), but high TNF- levels may in fact be continuously driving the inflammatory process in the lungs as well. Attempts to show that TNF- promoter polymorphisms, which are associated with differences in TNF- production, can be related to differences in loss of pulmonary function have produced contradictory results (28). However, as noted in the introduction, we found that release of TNF- from macrophages appears to be the mechanism by which MMP-12 causes acute cigarette smoke-induced inflammation (7). It is interesting in this regard that Joos and colleagues (30) recently reported that polymorphisms in the MMP-1 and MMP-12 promoters were associated with differences in the rate of functional decline in smokers. If the mechanism we proposed from an animal model (7) holds true in humans, these promoter variations might be translated into differences in TNF- production.
The correspondence between neutrophil influx and TNF- levels in the animals exposed to smoke long term is not exact. These variations may be related to species/treatment differences, as in the guinea pig plasma, TNF- was more than doubled by smoke exposure and only partially suppressed with ZD0892 (19), whereas in this study the increase in plasma TNF- was relatively small but could be completely abolished with A1AT even though neutrophil levels remained somewhat elevated. We cannot determine from these experiments whether antiprotease therapy is directly suppressing TNF- release or suppressing another inflammatory response that leads to TNF- release; nonetheless, our findings in this and our previous studies suggest that therapies directed against TNF- may be beneficial.
Another observation of interest is that neither ZD0892 nor A1AT is totally protective. We have found in both cigarette smoke-induced and crystalline silica-induced models of matrix breakdown that, acutely, levels of breakdown products consistently correlate with lavage neutrophil levels and not with lavage macrophage levels (5, 6, 23). After smoke exposure, both ZD0892 (19) and A1AT completely suppressed acute/short-term neutrophil influx and matrix breakdown at the same dose used for the long-term experiments, and one would presume, a priori, that similar complete protection would occur in the long term. However, it is clear that this protective ability disappears to a certain extent over time, as mice treated with Prolastin had a small but significant increase in lavage neutrophils at 6 months compared with control subjects. Similarly, guinea pigs treated with ZD0892 also had increased numbers of lavage neutrophils by 6 months, although the increase was not statistically significant compared with control subjects.
These findings may imply that over the long term, different mediators of neutrophil influx (and possibly of TNF- release) appear, mediators that are not suppressed by antiserine protease therapy. These mediators might include matrix breakdown products created by other proteases, including macrophage-derived proteases, as matrix breakdown fragments are neutrophil chemoattractants (31). It is also possible that other elastolytic serine proteases produced by neutrophils, for example, proteinase 3 and cathepsin G, play a role and that this role become increasingly important over time. Some indirect support for this idea exists, as Shapiro (2) reported that mice lacking neutrophil elastase are only approximately 60% protected against smoke-induced emphysema; however, against this notion is the fact that A1AT should inhibit proteinase 3 and cathepsin G activity. Finally, it is possible that matrix breakdown might be caused by another process entirely, for example, apoptosis of pulmonary endothelial and epithelial cells, a phenomenon that has been reported in human emphysema (32).
Regardless of mechanism, the results we obtained with antiserine protease therapy and the data reported by Shapiro using neutrophil elastase knockout mice (2) suggest that interference with neutrophil proteolytic attack could be of value in treating/preventing emphysema in smokers. Even if this approach is not completely protective, a 50 to 70% reduction in the severity of emphysema may be of clinical benefit.
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FOOTNOTES |
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Supported by grant 42,539 from the Canadian Institute of Health Research and Bayer Canadian Blood Partnership Fund.
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 February 11, 2003; accepted in final form April 7, 2003
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 T. H. March, J. A. Wilder, D. C. Esparza, P. Y. Cossey, L. F. Blair, L. K. Herrera, J. D. McDonald, M. J. Campen, J. L. Mauderly, and J. Seagrave Modulators of Cigarette Smoke-Induced Pulmonary Emphysema in A/J Mice Toxicol. Sci., August 1, 2006; 92(2): 545 - 559. [Abstract] [Full Text] [PDF]
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 C. Persson, D. Subramaniyam, T. Stevens, and S. Janciauskiene Do Native and Polymeric {alpha}1-Antitrypsin Activate Human Neutrophils In Vitro? Chest, June 1, 2006; 129(6): 1683 - 1692. [Abstract] [Full Text] [PDF]
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 I. Petrache, I. Fijalkowska, L. Zhen, T. R. Medler, E. Brown, P. Cruz, K.-H. Choe, L. Taraseviciene-Stewart, R. Scerbavicius, L. Shapiro, et al. A Novel Antiapoptotic Role for {alpha}1-Antitrypsin in the Prevention of Pulmonary Emphysema Am. J. Respir. Crit. Care Med., June 1, 2006; 173(11): 1222 - 1228. [Abstract] [Full Text] [PDF]
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P. A. Martorana, R. Beume, M. Lucattelli, L. Wollin, and G. Lungarella Roflumilast Fully Prevents Emphysema in Mice Chronically Exposed to Cigarette Smoke Am. J. Respir. Crit. Care Med., October 1, 2005; 172(7): 848 - 853. [Abstract] [Full Text] [PDF]
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A. Churg, R. D. Wang, H. Tai, X. Wang, C. Xie, and J. L. Wright Tumor Necrosis Factor-{alpha} Drives 70% of Cigarette Smoke-induced Emphysema in the Mouse Am. J. Respir. Crit. Care Med., September 1, 2004; 170(5): 492 - 498. [Abstract] [Full Text] [PDF]
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 R. L. Keith, Y. E. Miller, T. M. Hudish, C. E. Girod, S. Sotto-Santiago, W. A. Franklin, R. A. Nemenoff, T. H. March, S. P. Nana-Sinkam, and M. W. Geraci Pulmonary Prostacyclin Synthase Overexpression Chemoprevents Tobacco Smoke Lung Carcinogenesis in Mice Cancer Res., August 15, 2004; 64(16): 5897 - 5904. [Abstract] [Full Text] [PDF]
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M. J. Tobin Chronic Obstructive Pulmonary Disease, Pollution, Pulmonary Vascular Disease, Transplantation, Pleural Disease, and Lung Cancer in AJRCCM 2003 Am. J. Respir. Crit. Care Med., January 15, 2004; 169(2): 301 - 313. [Full Text] [PDF]
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R. A. Stockley "Knock-out" Mouse: Down But Not Out Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 145 - 146. [Full Text] [PDF]
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