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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1596-1603, (2002)
© 2002 American Thoracic Society

Original Article

{alpha}1-Antitrypsin Determines the Pattern of Emphysema and Function in Tobacco Smoke–exposed Mice

Parallels with Human Disease

Yasutaka Takubo*, Alexei Guerassimov*, Heberto Ghezzo, Alexandra Triantafillopoulos, Jason H. T. Bates, John R. Hoidal and Manuel G. Cosio

Respiratory Division, Royal Victoria Hospital, Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada; Department of Medicine and Molecular Physiology and Biophysics, Colchester Research Facility, University of Vermont, Colchester, Vermont; and Division of Respiratory, Clinical Care, and Occupational Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah

Correspondence and requests for reprints should be addressed to M. G. Cosio, M.D., McGill University, Royal Victoria Hospital, Respiratory Division, Room L4.11, 687 Pine Avenue West, Montreal, PQ, H3A 1A1 Canada. E-mail: manuel.cosio{at}muhc.mcgill.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cigarette smoking in humans is associated with various patterns of emphysema and functional consequences. We tested the hypothesis that variations in {alpha}1-antitrypsin expression modulate the pattern of emphysema and functional consequences in cigarette smoke–exposed mice. We compared the effects of up to 6 months of cigarette smoke exposure in C57BL/6J (C57) mice and in low-{alpha}1-antitrypsin, C57BL/6J pa+/pa+ (pallid) mice. At the end of the experiment, we determined lung mechanical properties, the extent (mean linear intercept) and type of emphysema, and the cellular inflammatory response. After 4 months of cigarette smoking, pallid smoking mice, but not C57 smoking mice, had a significant increase in mean linear intercept. After 6 months of smoke exposure, C57 smoking mice and pallid smoking mice had similar degrees of emphysema. The pattern of emphysema in pallid smoking mice was more diffuse than in C57 smoking mice, affecting all airspaces. Pallid mice, but not C57 mice, developed a T cell inflammation in the alveolar wall after 6 months of smoking (p < 0.01). Although lung compliance was not changed in C57 smoking mice after smoke exposure, it increased significantly in pallid smoking mice over the 6 months of exposure (p < 0.0082). In summary, cigarette smoking induces emphysema in C57 and pallid mice, but the emphysema, inflammatory infiltrate, and resulting physiologic abnormalities were substantially different in the two strains, with the C57 and pallid mice exhibiting features similar to centrilobular and panlobular emphysema, respectively.

Key Words: {alpha}1-antitrypsin • emphysema • lung mechanics • mice • smoking


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For hypothesis testing in relation to pathogenetic mechanisms of chronic obstructive pulmonary disease (COPD), animal models are needed that represent the various patterns of lung destruction and function observed in humans. Because the major environmental factor that predisposes patients to COPD is long-term cigarette smoking, a cigarette smoke exposure model would be advantageous. A variety of animals have been exposed to cigarette smoke, including dogs, rabbits, guinea pigs, and rodents (1). The results in terms of lung pathology have been variable. To take advantage of the defined genome and the well-established methods of modulating gene expression, murine models of cigarette smoke exposure have been evaluated. Although airspace enlargement has been observed in the murine models, little attention has been given to how well this simulates the varied pathologic features in smoking-associated emphysema of humans. Moreover, no studies have been reported to date in which the pathologic alterations have been correlated to pulmonary function abnormalities, an essential step in the assessment of the functional consequences of emphysema.

The purpose of the present investigation was to test the hypothesis that variations in {alpha}1-antitrypsin ({alpha}1-AT) expression modulate the type of disease and functional consequences in cigarette smoke–exposed mice. To test this hypothesis, we developed models that simulate the varied pathologic and physiologic features of human emphysema, and identified determinants of the distinct pathologic features. We compared the type of emphysema and functional consequences that develop after cigarette smoke exposure of C57BL/6J mice with that developing after smoke exposure of its congenic strain, the pallid mouse (C57BL/6J, pa+/pa+), a strain with reduced {alpha}1-AT levels and serum elastase inhibitory capacity (2). We considered it important to assess precisely not only the degree and distribution of airspace enlargement, but also the functional consequences of the emphysema, by measuring changes in elastic recoil of the lungs. We also examined the cellular inflammatory responses of the lung after 6 months of smoking in order to investigate possible differences between mouse strains and possible similarities with human emphysema. We hypothesized that C57BL/6J (C57) mice would develop emphysema localized to the center of the acinus, with concomitant alterations in the mechanical properties akin to human centrilobular emphysema (CLE), whereas pallid mice would develop a more diffuse pattern of emphysema and mechanical characteristics similar to those of human panlobular emphysema (PLE).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male C57BL/6J mice and male pallid mice (C57BL/6J, pa+/pa+) were divided into sham-exposed controls and cigarette smoke–exposed groups from 3 months of age (C57NS [C57 nonsmokers, n = 24], PNS [pallid nonsmokers, n = 25], C57S [C57 smokers, n = 24], and PS [pallid smokers, n = 25]). All groups were exposed for 2, 4, and 6 months.

Cigarette Smoke Exposure
The smoking animals were exposed daily to the smoke of two standard research nonfiltered cigarettes (2R1), generated by a cigarette smoke exposure system (Cigarette Laboratory at the Tobacco and Health Research Institute, University of Kentucky, Lexington, KY). Carboxyhemoglobin levels in the blood were monitored. The body weights of all mice were measured monthly.

Measurement of Lung Mechanics
Each mouse was weighed and then deeply anesthetized by intraperitoneal injection of xylazine (8 mg/kg) and pentobarbital (70 mg/kg) and tracheostomized, and the trachea was cannulated. The cannula was connected to a computer-controlled small animal ventilator (flexiVent; Scireq, Montreal, PQ, Canada) and regular quasi-sinusoidal ventilation commenced at a frequency of 150 breaths/minute and tidal volume of 5 ml/kg. The mouse was paralyzed with pancuronium bromide (0.8 mg/kg, administered intraperitoneally) and the chest was opened. After stabilization, lung mechanics were measured at positive end-expiratory pressure (PEEP) levels of 1 to 9 cm H2O applied in random order, by delivering a broad-band volume perturbation to the lungs for a period of 16 seconds, during which time the combined system of the ventilator and lungs remained closed to the environment (3).

The signals of cylinder pressure (Pcyl) and piston volume displacement (Vcyl) obtained during the perturbations were low-pass filtered and stored on a computer for analysis. The fast Fourier transforms of the data windows were used to calculate the input impedance of the lung (Zin), as described previously (4–6). The parameter tissue elastance (Htis) is equal to lung elastance at a frequency of {omega} = 1 rad/second = 0.16 Hz (7, 8), and we used Htis versus PEEP to calculate an equivalent pressure–volume curve for the lungs between 1 and 9 cm H2O. In some mice, we measured static pressure–volume curves by step inflation of the lungs to a total volume of 1.4 ml, followed by a similar stepwise deflation.

Lung Histology and the Quantitation of Emphysema
After measuring mechanics, animals were killed by exsanguination. The left lung was inflated with optimal cutting temperature fluid to a transpulmonary pressure of 25 cm H2O for 30 minutes, and then flash-frozen in isopentane and liquid nitrogen. Five-micrometer sagittal sections were cut, fixed, and stained with hematoxylin–eosin for histologic examination. Two slides per mouse were coded and analyzed. Airspace enlargement was quantified by the mean linear intercept (Lm) in 20 randomly selected fields per slide (9). Proportions of alveolar sacs, alveoli, and alveolar ducts in the lungs were analyzed by point counting (10, 11) in 10 random fields throughout the lung, using NIH Image software (12). Points falling on each alveolus and on alveolar sacs and alveolar ducts were identified and counted (13).

Measurement of Lung Inflammatory Response
Two 5-µm sagittal sections per mouse were cut, fixed, and stained with specific immunostains for the assessment of alveolar wall inflammation: polymorphonuclear cells (PMNs), rat anti-mouse neutrophil monoclonal, diluted 1:33 (Cederlane Laboratories, Hornby, ON, Canada); lymphocytes, rat anti-mouse CD4+ monoclonal and rat anti-mouse CD8+ monoclonal (BD Biosciences Pharmingen, San Diego, CA), both diluted 1:250; alveolar macrophages (AMs), rat anti-mouse macrophages/monocytes, diluted 1:100 (Serotec, Raleigh, NC) and rat anti-mouse Mac-3, diluted 1:4,000 (BD Biosciences Pharmingen). Twenty sections per slide at x600 magnification were captured by a digital camera and analyzed with computer assistance. The length of all the alveolar wall in the image was measured, and all the cells in the field were counted to obtain the total number of cells in the lung. Results were expressed as the percentage of positive staining cells relative to total cells. Lungs were also homogenized and consequently digested with elastase, collagenase, and DNase to obtain cytospins (Thermo Shandon, Pittsburgh, PA) of lung cells. These cytospins were stained for AMs as described above, and AMs were expressed as a percentage of total cells.

Statistics
Data are expressed as means ± SEM. A Student t test and analysis of variance of a linear mixed-effects model were performed to compare differences between groups. Statistical significance was taken as p < 0.05. Because pairwise comparisons were of primary interest, a t test was performed followed by the Bonferroni correction within each set of comparisons. Because a description of the pattern of emphysema was an essential aim of this study, we did not take advantage of the large n value that would have been obtained by comparing the final point count among groups (the n value would have been approximately 2,000). Rather, we used the percentages for each animal to obtain group means and standard deviations for group comparisons (n is 12 in this case).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Both C57 and pallid mouse strains tolerated cigarette smoking well for up to 6 months of exposure. Carbon monoxide concentrations of 10–12% were reached immediately after smoking. Body weight of smokers tended to be lower than that of nonsmokers at 2, 4, and 6 months of smoke exposure in both C57 and pallid mice (Table 1) . Compared with C57S mice, PS animals had significantly lower body weights after 4 months (p < 0.001) and 6 months (p < 0.05) of smoke exposure.


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  		TABLE 1. Body weights of mice in grams* and number of animals in each group

 
Lm values in the nonsmoking groups increased slightly from the beginning to the end of the study, possibly as a reflection of lung growth, but these differences were not significant (Figure 1) . After 4 months of smoking, Lm values in C57NS and C57S mice were the same, but the PS group had a 16% increase in Lm compared with the PNS group (p < 0.005) and with the C57S group (p < 0.005). After 6 months, Lm was increased in both smoking groups: Lm in the C57S group was 17.9% larger than in the C57NS group (p < 0.001), and Lm in the PS group was also 17.9% larger than in the PNS group (p < 0.001).



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  		Figure 1. Mean linear intercept (Lm) for smoking and control mice at 2, 4, and 6 months of exposure. Lm was measured at a magnification of x100 and expressed as mean and SD for the whole lung. (n = 6 animals per group and time period.)

 
Figure 2 shows representative histologic sections from the C57S and PS groups and controls after 6 months of exposure. Emphysema is apparent in both strains of smoke-exposed mice, as evidenced by the destruction of alveoli, and the enlargement of alveolar ducts and alveolar sacs. Compared with the C57S group, changes are more extensive in the PS group.



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  		Figure 2. Black-and-white computer conversion (for Lm calculations) of lung photomicrographs of smoking and control mice after 6 months of exposure.

 
The proportions of airspaces (alveoli, alveolar sacs, and ducts) in the different groups are shown in Figure 3 . We determined the effects of aging and strain on the proportion and size of airspaces in response to smoking. With aging alone, mice had a progressive loss of alveoli and alveolar sacs and a progressive increase in alveolar duct size. The differences between strains were significant, with pallid mice showing more marked change than C57 mice (p < 0.001 after Bonferroni correction). The effect of smoking was less pronounced in C57 mice than in pallid mice. When compared with C57S mice, the loss of alveoli and alveolar sacs was greater in PS mice (t = 11.9 at 4 months and t = 8.66 at 6 months of smoking) as was the enlargement of alveolar ducts (t = 15.89 at 4 months and t = 12.7 at 6 months of smoking) (p < 0.001, Bonferroni corrected, for all comparisons).




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  		Figure 3. The proportion of alveoli, alveolar sacs, and alveolar ducts in (A) C57 smoking and control mice and (B) pallid smoking and control mice. There is a strain difference among the nonsmoking mice in the proportion of airspaces (p < 0.001). As the animals aged, smoking effects were more pronounced in pallid mice than in C57 mice (p < 0.001).

 
Lung inflammatory response is shown in Table 2 . The total number of cells per millimeter of alveolar wall was similar in all mice studied (range, 26.34 ± 3.16 to 31.05 ± 3.93). No differences in the percentage of inflammatory cells between smoking and nonsmoking C57BL/6J mice were found, except for an increase in AMs that was significant in lung cytospins (p = 0.00). A significant increase in T cells, mainly CD4+ cells (p < 0.01) and AMs (by lung cytospins, p < 0.01), was seen in pallid mice after smoking exposure. Neutrophils also tended to increase in pallid mice after smoking.


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  		TABLE 2. Mean and standard deviation of percentage of inflammatory cells in alveolar wall of C57bl/6J and pallid mice, exposed to smoke and controls

 
The lung elastic behavior and Htis at different PEEP levels in C57 and pallid mice at 2 to 6 months of cigarette smoke exposure are shown in Figure 4 . At all time points the minimum value of Htis occurs at a PEEP of about 3 cm H2O in both strains. Htis in the PNS and PS groups is not different after 2 months of exposure and does not differ from Htis in the C57NS or C57S groups. However, Htis in the PS group decreases significantly over time (p = 0.0082 by analysis of variance), and the differences between PNS and PS groups are significant at both 4 months (p = 0.0146) and 6 months (p = 0.0137) of smoke exposure. Although Htis seems to decrease slightly with smoking duration in the C57S group, the changes are not significant. At a PEEP of 9 cm H2O, no changes in elastance were observed in C57S mice compared with C57NS mice (Figure 4), but progressive decreases (p = 0.003) were observed in PS mice from 2 to 6 months of smoking (Figure 4). This decrease in elastance at 9 cm H2O indicates that the compliance at higher lung volumes increases in the PS group. Thus, in PS mice the normal plateau of the pressure–volume curve of the lung seen at high lung volumes is lost with increasing exposure to smoking.



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  		Figure 4. Tissue elastance (Htis) at different PEEP levels in C57 and pallid mice after 2 to 6 months of smoking exposure and in control mice. There is a significant decrease in Htis over time in PS mice (analysis of variance, p = 0.008), but not in C57S mice. The differences between PNS and PS mice are significant at both 4 months (p = 0.014) and 6 months (p = 0.013) of smoke exposure. (n = 6 animals per group and time period.)

 
To illustrate the extremes in response to cigarette smoke exposure, we present representative static pressure–volume curves from C57NS and PS animals at 6 months of exposure (Figure 5) . Compared with C57NS animals, PS animals had increased total lung capacities (lung volume at an inflation pressure of 25 cm H2O) and the slopes of the pressure–volume curves were steeper (indicating increased compliance). Figure 6 shows the dynamic pressure–volume curves between 1 and 9 cm H2O PEEP calculated from Htis in both C57 and pallid mice. The relationship between these slopes and the slopes obtained by the traditional pressure–volume curve in Figure 5A is shown in Figure 5B. The slopes were the same by both approaches over the ranges of PEEP tested. There were no changes in the slopes for the C57NS and C57S groups over time. However, changes in the slopes for the PS group increased significantly over time (p = 0.0037 by analysis of variance), and the differences between the slopes for the PNS and PS groups at 4 months (p = 0.027) and at 6 months (p = 0.00036) of smoke exposure were significant.



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  		Figure 5. (A) Static pressure–volume curves for two nonsmoking C57 mice and five pallid mice after 6 months of smoke exposure. The PS mice have larger lung volumes and are more compliant than the C57NS mice. (B and C) Comparison of quasi-static pressure–volume curves and pressure–volume curves between 0 and 9 cm H2O pressure derived from Htis for one C57NS mouse (B) and one PS mouse (C).

 


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  		Figure 6. Htis-derived pressure–volume curves between 0 and 9 cm H2O in nonsmoking and smoking animals (n = 6 for each group). There is a significant increase in compliance among PS mice over time (p < 0.0037 by analysis of variance) but not among C57S mice. The slope of the PS pressure–volume curve at 6 months was different than that for PNS (p = 0.0004), but no difference was found between C57S and C57NS at 6 months.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
When investigating the pathogenesis of emphysema in animal models, it is important to consider the known risk factors (cigarette smoking and {alpha}1-AT deficiency) and the varied morphology and physiology of the human disease. In the current investigation we demonstrate that cigarette smoke exposure results in morphologic emphysema in C57 mice and that the added risk of decreased {alpha}1-AT levels accelerates the development and modifies the pattern of emphysema, and produces alterations in lung mechanics not present in the smoke-exposed animals with normal {alpha}1-AT levels. Furthermore, the pattern of cellular inflammation in lungs after cigarette smoke exposure is qualitatively different between the two strains, with pallid mice developing a T cell infiltration in the lung parenchyma that was not present in the C57BL/6J mice. These patterns of abnormalities produced after smoke exposure of the different mouse strains closely parallel the different patterns of emphysema (14, 15) and inflammation (16) found in human smokers.

The pallid mouse was identified in 1931 (17) and, subsequently, by repeated backcrossing, placed on a C57BL/6J (C57) background (18), with the result that the only difference between C57 pa+/pa+ mice and normal C57 mice is a mutation in the pallid gene located on chromosome 2 (19). In the pallid mouse, the deficiency of {alpha}1-AT is due to an abnormality in the pallidin protein resulting from a nonsense mutation at codon 69 of the gene. Pallidin colocalizes and interacts with syntaxin-13 mediating vesicle docking and fusion to early and recycling endosomes (20, 21). The level of syntaxin-13 is reduced by half in pallid mice compared with C57 mice (20). The net result is an inability to secrete {alpha}1-AT normally into the circulation with consequent decrease in serum elastase inhibitory capacity (2). The {alpha}1-AT levels in pallid mice are 60% of those in congenic C57 mice and 45% of those in allogeneic BALB/c mice (2, 22), although the protein itself is normal. These levels of {alpha}1-AT correspond to the moderate deficiencies seen in human heterozygous MZ and SZ, and homozygous SS, carriers who have between 50 to 60% of the normal level (23, 24). Individuals with these moderate reductions in serum {alpha}1-AT are more susceptible to developing emphysema if they smoke (25–27).

Other alterations resulting from pallidin deficiency have been described, including defective secretion of neutrophil lysosomal enzymes (28), platelet storage pool deficiency, prolonged bleeding time, pigment dilution, kidney lysosomal enzyme elevation, and abnormal otolith formation (20). Whether these or other alterations resulting from pallidin deficiency have any role in the pathobiochemistry of emphysema in the model is not known. Thus, caution is needed in linking the lung abnormalities to the relative {alpha}1-AT deficiency in pallid mice. This issue is currently being addressed in the laboratory by experiments determining the effect of {alpha}1-AT replacement on the development of emphysema in pallid mice.

The effects of smoke exposure of both C57 and pallid mice were striking as demonstrated histologically (Figure 2) and by the increases in Lm (Figure 1). Both C57 and pallid mice developed emphysema after 6 months of cigarette smoke exposure. However, after only 4 months of exposure, the pallid mice developed microscopic emphysema as determined by Lm, but the C57 mice did not. Similar results were reported by Cavarra and coworkers (29). Collectively, these studies suggest that the deficiency of {alpha}1-AT in pallid mice accelerated the development of their emphysema. The small increase in Lm found in the nonsmoking controls over the time of the study is a well-established phenomenon thought to be due to lung growth and aging (13).

An {alpha}1-AT deficiency in human smokers predisposes them to the development of PLE. Thus, ideally, smoke exposure of animals with relative {alpha}1-AT deficiency should also develop a more diffuse PLE-like emphysema. This was observed in the present investigation. As demonstrated in Figures 2 and 3, the emphysema patterns in the C57 and pallid mice are quite different. After 6 months, the C57S mice had an enlargement of alveolar ducts with preservation of alveoli that remained normal in size. In contrast, the PS group had a more generalized destruction of the parenchyma with loss of alveoli and alveolar sacs and enlargement of both remaining alveoli and alveolar ducts. We conclude that a deficiency of {alpha}1-AT not only accelerates the appearance of emphysema but also predisposes to a diffuse type of lung damage both in humans and mice.

An important consequence of the diffuse lung involvement in emphysema is the extent of elastic tissue loss. Humans with anatomically diagnosed PLE have significant loss of lung elastic tissue even when the emphysema is mild, whereas patients with CLE have normal levels of elastin until the emphysema is severe and most of the lung is destroyed (30, 31). Given the marked structural differences, it is not surprising that the lung function in the two forms of emphysema is different. Subjects with PLE have high compliance at low lung volumes and show early losses of elastic recoil, whereas those with CLE have low or normal compliance and decreases of recoil are not seen until the disease is advanced (14, 15, 32).

The present study provides a detailed description of the mechanical properties of the lung in mice and their changes with age and smoking exposure, an aspect not previously investigated in animal models. We believe that the measurement of lung elasticity and its relationship with emphysema is important to increase understanding of the disease in animal models, especially as it parallels the functional abnormalities of emphysema in human smokers. In the present investigation, the mechanical properties of the lung (Htis, pressure–volume curves, and compliance) in C57 and PS mice are different and resemble the mechanical abnormalities that accompany human CLE and PLE, respectively. Specifically, PS mice had an increase in lung compliance (Figure 6) (loss in elastance, Htis; Figure 4) with an increase in lung volume (Figure 5A) that progressed from 2 to 6 months of smoking, whereas for the same extent of emphysema as measured by Lm, the C57S mice did not show a change in compliance or a significant alteration in the static pressure–volume curve. These results emphasize the shortcomings of assessing emphysema by measuring only Lm. It could be argued from our results that the emphysema in the PS group is more severe than that in the C57S group, for the same Lm, because in the PS group it results in striking alterations in lung compliance.

The probable reason for the mechanical changes in pallid mice is the more diffuse lung abnormality that develops in these mice (Figures 2 and 3). This is likely related to loss of lung elastic tissue. Martorana and coworkers have found that the lung elastin content at 16 months of age in nonsmoking pallid mice is significantly lower than in congenic C57BL/6J mice (2), and a significant decrease in elastin content in the lung has also been reported by Cavarra and coworkers in pallid mice after only 4 months of smoking (29). The differences between the patterns of emphysema developed by the two congenic mouse strains are striking and emphasize the potential important role of {alpha}1-AT, not only in the rapidity of the production of emphysema, but also in the pattern of emphysema and the mechanical consequences that ensue.

Another important difference between the two mouse strains is the pattern of cellular inflammatory infiltrate that develops during smoke exposure. Pallid mice after 6 months of exposure had a significant increase in the number of T cells, mainly CD4+ cells, in the alveolar walls, an inflammatory pattern not seen in smoking C57BL/6J pa-/- mice. Other inflammatory cells, mainly PMNs, tended to increase in pallid mice after smoking exposure and AMs increased in both smoking strains. Of interest is that a T cell response in airways (33) and parenchyma (16) is also seen in human smokers who develop COPD, and the magnitude of cell infiltration is similar to that which mice develop (16). Furthermore, human smokers who do not develop emphysema have fewer CD4+ T cells per millimeter of alveolar wall than do nonsmokers (16), a finding also seen in C57BL/6J smokers when compared with nonsmokers. We have no clear explanation for this latter finding.

Pallid and C57 mice are congenic and the only difference between the two strains is the deficiency in pallidin that results in the partial deficiency of {alpha}1-AT (among other abnormalities) that pallid mice exhibit. Could {alpha}1-AT deficiency promote a T cell response to smoking? There is evidence in the literature that this might be the case. Breit and coworkers in 1982 presented evidence of in vivo T cell hyperresponsiveness in {alpha}1-AT-deficient individuals, on the basis of the observation that delayed-type hypersensitivity responses in subjects with {alpha}1-AT were accelerated (34). In vitro experiments suggest that proteases from leukocytes and {alpha}1-AT may modulate the function of immunologically competent cells (35–37). Furthermore, Breit and coworkers (38) and Guerassimov and Cosio (39) showed a dose–response effect of {alpha}1-AT in the proliferative response of human peripheral blood and mouse spleen lymphocytes stimulated with phytohemagglutinin or concanvalin A. These data suggest that serum proteases or their inhibitors ({alpha}1-AT) may contribute to modulation of the immune response. We speculate that the T cell response that pallid mice develop may be directly related to the low levels of {alpha}1-AT found in these mice. How this T cell response affects the development of emphysema will require additional studies, but it raises the possibility that T cells may be important mediators of the disorder.

Do the implications of this study extend beyond those pertaining to individuals with known deficiency of {alpha}1-AT? A significant number of white smokers with normal serum {alpha}1-AT levels develop predominantly PLE (14, 15). However, the Japanese, an ethnic group with no {alpha}1-AT deficiency, develop CLE only when they smoke (40). One possible explanation is the existence of {alpha}1-AT polymorphic variants that migrate with normal alleles but that are dysfunctional and thus predispose to the development of PLE. Variants of {alpha}1-AT with normal serum levels (and antielastase activity) have been associated with emphysema in smokers (41, 42). A restriction fragment length polymorphism of the 3' flanking sequence of the {alpha}1-AT gene has been described and found to be present in 17% of subjects with COPD and 5% of matched control subjects. It is believed that the region of the gene containing the polymorphism contains motifs that may prevent the increased expression of {alpha}1-AT during inflammation or other forms of acute stress. In addition, we have examined {alpha}1-AT phenotypes of 40 smokers with COPD and normal serum {alpha}1-AT levels. When the group was divided according to the type of emphysema, patients with PLE showed a prevalence of the PiM3 allele of 58% (normal expected, 9–10%) and of the PiMZ allele of 12% (normal expected, 2–3%). The prevalence of the different alleles in the CLE subjects was similar to that of the general population. The high prevalence of M3, which has been considered a normal variant of the M allele, suggests that studies of the regulation and response to acute stress of this variant could be important in determining the basis for the PLE that occurs in smokers with normal serum {alpha}1-AT levels (our unpublished observations).

Exposure to cigarette smoke is not the only way to produce emphysema in animals. Genetic manipulations, such as overexpression of interleukin-13 (43) or interferon-{gamma} (44), or induction of apoptosis of endothelial cells (45), has been described to produce an emphysema-like pattern in mice. The question remains as to the relevance of these models to the human disease that is induced by smoking. We therefore thought it important to retain as many of the pathogenic features of the human disease as possible in an animal model, because the understanding of the disease would necessitate first producing emphysema by the usual insult, smoking, and only then pursuing potential mechanistic pathways could become relevant to the pathogenesis of the disease.

In summary, we have produced emphysema in both C57BL/6J and pallid mice by chronically exposing them to cigarette smoke. We demonstrated that the rate of development of the emphysema as well as its pattern within the lungs, and especially the resulting lung mechanical abnormalities, were different between the two congenic strains, with the C57 and pallid animals developing a localized or more diffuse emphysema exhibiting similarities to human CLE and PLE, respectively. Furthermore, the pattern of inflammation in the two strains is also different, with pallid mice developing a prominent T cell inflammation not seen in C57BL/6J mice, a difference that could possibly be mediated by the partial deficiency of {alpha}1-AT in pallid mice. We believe that these results have implications not only for cigarette smokers who develop CLE and those individuals with known {alpha}1-AT deficiency, but also for the large number of COPD subjects who have normal basal serum {alpha}1-AT levels, but develop PLE.


   FOOTNOTES
 
Supported by the National Institutes of Health (NIH, Type 2RO1, GK37615-11), the Alpha One Foundation, and Astra Canada.

* Y.T. and A.G. contributed equally to the work presented. Back

Received in original form February 1, 2002; accepted in final form September 25, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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