INTRODUCTION

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Keywords: cigarette smoke; emphysema; ₁-proteinase inhibitor; antioxidant defenses; mouse strains

Chronic obstructive pulmonary disease (COPD) is a major health burden and its prevalence is increasing worldwide (1). Cigarette smoke is the most prominent factor determining the morbidity and mortality of COPD (2). Also, epidemiological studies have revealed that pulmonary emphysema, which is a major component of COPD, is associated with cigarette smoking habit (3). However, it is well recognized that in a population of heavy smokers only 15% to 20% of the subjects develop COPD (4). The reason for this is unknown, but it is likely that individual factors such as different levels of antiproteases defense and antioxidant status may play an important role (5, 6).

It was thus of interest to investigate the effect of chronic cigarette smoke in different strains of mice potentially susceptible to the effects of cigarette smoke either because of a mild deficiency in their antielastase screen (C57BL/6J) (7, 8) or because of a sensitivity to oxidants (DBA/2) (9). Both these strains do not develop spontaneous emphysema. A strain of mice with normal antielastase screen and not sensitive to oxidants (ICR) was used as a comparison (Study 1).

An additional study (Study 2) was carried out to investigate if chronic cigarette smoke exposure could accelerate the development of emphysema in a strain of mice that develops emphysema spontaneously in its adult life. This was done in pallid mice in which the appearance of spontaneous emphysema is associated with a marked deficiency in serum ₁-PI (8, 10, 11).

The present report presents the results obtained in the course of these studies.

	METHODS

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Animals

Three-mo-old mice of the strains C57BL/6J, DBA/2, and ICR (supplied by Charles River, Calco, Italy), and 5- to 6-mo-old C57BL/6J pa/pa (pallid) from our colony, were used in this study. The mice were housed in groups of two to four in macrolon cages. Room temperature was kept at 22° to 24° C; and relative humidity at 40 to 50%; food and water were supplied ad libitum. All animal experimentation was approved by the Local Ethical Committee of the University of Siena.

Assessment of Antiprotease Levels and Antioxidant Status

Elastase inhibitory capacity (EIC) and antioxidant status were assayed in bronchoalveolar lavage (BAL) fluids. The trachea was isolated under light ether anesthesia and then cannulated with a 20-gauge blunt needle and with the aid of a peristaltic pump (P-1 Pharmacia) the lungs were lavaged in situ three times with 0.6 ml saline solution. The average fluid recovery was greater than 95%. BAL fluids were assayed for elastase inhibitory capacity (11) against porcine pancreatic elastase (PPE) (Type III, Sigma E0127) on Suc-Ala-Ala-Ala-pNA (12). EIC was expressed as micrograms of PPE inhibited per milliliter of BAL as previously reported (8).

Total antioxidant capacity was measured in cell-free BAL fluids according to Miller and coworkers (13). The antioxidant capacity of BAL was then compared with Trolox as "Trolox equivalent antioxidant capacity" (TEAC) (13). The TEAC in BAL samples is defined as the concentration (µmol/ml) of Trolox having the equivalent antioxidant capacity to 1 ml BAL fluid. This was measured under basal conditions as well as after the acute exposure to five cigarettes. For details of the cigarette exposure methodology see below.

Chronic Exposure to Cigarette Smoke

Study 1. Mice of the strains C57BL/6J, DBA/2, and ICR were exposed to either the smoke of three cigarettes/d, 5 d/wk for 7 mo (commercial Virginia cigarettes: 12 mg of tar and 0.9 mg of nicotine), or to room air (control animals), in especially designed macrolon cages (Tecniplast, Buguggiate, Italy), essentially according to Escolar and coworkers (14). These cages (42.5×26.6×19 cm) equipped with a disposable filter cover having 15 10-mm holes that enabled the air to flow out of the cages and thus to be continuously renewed. The smoke was produced by the burning of a cigarette and was introduced into the chamber with the airflow generated by a mechanical ventilator (7025 Rodent Ventilator, Ugo Basile, Biological Research Instruments, Comerio, Italy), at a rate of 33 ml/min. The rate was increased to 250 ml/min in the acute study in which five cigarettes were smoked within 20 min. A second mechanical ventilator was used to provide room air for dilution (1:8) of the smoke stream. Thus, by using this methodology three cigarettes/cage (or five cigarettes/cage) were used in the chronic (or acute) study. In the chronic study the mice were exposed to the smoke originated by three cigarettes once a day for the duration of 90 min.

In a pilot study, the efficiency of the smoke delivery system was tested in 12 mice by measuring blood HbCO by cooxymetry.

Study 2. Pallid mice were exposed to either the smoke of three cigarettes/day, 5 d/wk for 4 mo, or to room air (control mice) under the same experimental conditions as described above.

Morphology and Morphometry

In both studies (1 and 2), 24 h after the end of the chronic exposure to cigarette smoke, the animals were anesthetized with ether and then exanguinated by severing the abdominal aorta. The lungs were excised and fixed intratracheally with buffered formalin (5%) at a constant pressure of 20 cm H₂O for at least 24 h. All lungs were then dehydrated, cleared in toluene, and embedded under vacuum in paraffin. Two 7-µm transversal sections were made and stained with hematoxylin-eosin. Two pathologists blinded to the exposure protocol carried out morphological and morphometrical evaluation. Morphometric assessment included determination of the average interalveolar distance (mean linear intercept: Lm) (15) and of the internal surface area of the lungs (ISA) as estimated by the Lm method at postfixation lung volume (16). The alveolar destructive index (DI) was also determined, as described by Eidelmann and coworkers (17). DI represents for each pair of lungs the percentage of air spaces in which two or more breaks in the alveolar walls were detected.

For the determination of the Lm for each pair of lungs, 40 histological fields were evaluated both vertically and horizontally. Examination of these numbers of fields meant that practically the entire lung area was evaluated. For the assessment of the DI for each pair of lungs 20 histological fields were evaluated using a standardized plane with 20 points for a total of 400 points per section.

Biochemical Analysis of Lung Elastin

In both studies (1 and 2) at the end of chronic exposure to cigarette smoke, lung elastin was determined in each animal strain. The mice were anesthetized with sodium pentobarbital, sacrificed by severing the abdominal aorta and the lungs were immediately removed. The lungs were weighed, immediately homogenized in ice-cold water (1:4, wt:vol) and then used for the determination of insoluble elastin. Elastin was extracted by successive extraction with 1 M NaCl, chloroform-methanol, and by hot alkali treatment at 98° C for 50 min in 0.1 N NaOH (18). The insoluble, defatted residue remaining after NaOH extraction (19) was assayed for elastin with pancreatic elastase (0.1 mg in 2 ml 0.02 M borate buffer, pH 8.8, for 3 h at 25° C) to hydrolyze the alkali insoluble residue (operationally defined as elastin) into peptide fragments (20). After centrifugation, the supernatant peptides were then determined by the method of Lowry and coworkers (21) and taken as an estimate of elastin. Elastin peptides from bovine neck ligament, obtained by elastase digestion, were used as standard.

Statistical Analysis

For each parameter either measured or calculated, the values of the individual animals were averaged and the standard deviation and standard error mean were calculated. The significance of the differences was calculated using one-way ANOVA (F test). A p value of less than 0.05 was considered significant.

	RESULTS

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Assessment of Antiprotease Levels and Antioxidant Status

EIC in BAL fluids. The results of EIC in BAL fluids from the different strains of mice are shown in Figure 1. The EIC was determined in 10 animals of each strain. BAL fluids of ICR and DBA/2 mice had similar EIC values. In these strains 1 ml of BAL fluids inhibited approximately 42 µg of PPE. The BAL fluids of C57BL/6J mice exhibited significant lower values (about 40% in respect to DBA/2 mice) corresponding to 25.1 ± 4.5 µg of PPE inhibited by 1 ml of BAL fluids (p < 0.01).

View larger version (35K): [in this window] [in a new window]   Figure 1.   Elastase inhibitory capacity determined in BAL samples in various strains of mice. C57BL/6J mice show significant lower values (40%) with respect to DBA/2 and ICR mice. The values represent the mean (± SD) of the results obtained from 10 mice in each group. *p < 0.01.

Antioxidant status in BAL fluids. No difference in the mean baseline value of TEAC in BAL samples was found among the various strains of mice. The values ranged from 50 ± 7 to 63 ± 12 nmol Trolox/ml BAL. Acute cigarette smoke exposure increased the antioxidant defenses in ICR mice. On the other hand, BAL fluids of C57BL/6J and DBA/2 mice showed a decrease of their antioxidant defenses following cigarette smoke (Figure 2).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Antioxidant capacity in BAL samples of the various strains of mice determined after acute smoke exposure. Cigarette smoke decreases the antioxidant defenses in C57BL/6J and DBA/2 mice, but increases them in ICR mice. The results, expressed as percent of unexposed controls (± SD), represent the mean data obtained from six mice in each group. *p < 0.01.

Chronic Exposure to Cigarette Smoke

Study 1. The lungs of the mice of the strains C57BL/6J, DBA/2, and ICR that had been exposed to room air showed a well-fixed normal parenchyma with normal airways (Figure 3A, 3C, and 3E). Seven months after exposure to cigarette smoke the lungs of ICR mice showed some areas of mild intraalveolar, peribronchial, and, in few cases, perivascular infiltration of mononuclear cells with the participation of some neutrophils, otherwise the parenchyma and airways appeared normal (Figure 3F). On the other hand, some areas of emphysema were prominent in the lungs of C57BL/6J and DBA/2 mice (Figure 3B and 3D). In a few cases foci of fibrosis were also seen intercalated with the emphysematous changes. Intraalveolar, peribronchial, and/or perivascular cellular infiltration was also seen in these lungs.

View larger version (87K): [in this window] [in a new window]   Figure 3.   Histologic sections from the lung of C57BL/6J (A), DBA/2 (C ), and ICR (E ) control mice showing a normal parenchymal structure. Lungs of a C57BL/6J mouse (B) and DBA/2 mouse (D) after chronic exposure to cigarette smoke show focal areas of emphysema. (E ) Lung of ICR mouse exposed to chronic cigarette smoke showing a normal appearance. H & E stain (original magnification: ×100).

Chronic exposure to cigarette smoke resulted in a significant increase of the Lm and a decrease of the ISA in the C57BL/ 6J mice (+12% and 8%, respectively) and in the DBA/2 mice (+21% and 8%, respectively) but not in the ICR mice (Table 1). Similarly, exposure to cigarette smoke induced a 3.1-fold increase of the DI in the C57BL/6J mice, a 4.2-fold increase in the DBA/2 mice (for both p < 0.05), but no significant changes in the ICR mice (Table 1).

The results of the biochemical analysis of lung elastin content, expressed as mg/lung, in the different groups for each strain are also entered in Table 1. Seven months after exposure to cigarette smoke, both DBA/2 and C57BL/6J mice had significantly lower values (18% and 17%, respectively) than air-exposed control animals. Air and cigarette smoke- exposed ICR mice had very similar values.

Study 2. The lungs of the pallid mice exposed to cigarette smoke showed clear areas of emphysema associated or not associated with areas of peribronchial, perivascular, intraseptal, and/or intraalveolar cellular infiltration (Figure 4B). Minor emphysematous changes were also seen in the lungs of the control pallid mice (Figure 4A); as previously reported by us, these changes were not accompanied by cellular infiltration (10). In the smoking pallid mice the value of the Lm was 10.2% greater and that of the ISA 12.2% smaller (for both p < 0.05) than those of the room air-exposed controls (Table 2). Also, in the smoking mice the DI was almost three times greater (p < 0.05) than in the control animals (Table 2). Some representative changes used in DI determination are shown in Figure 5A and 5B. The lungs of animals exposed to cigarette smoke had significantly lower elastin values than those of control animals (Table 2).

View larger version (105K): [in this window] [in a new window]   Figure 5.   (A) Histological section from the lung of a control pallid mouse showing an alveolus with an obvious break in one of the walls (arrowhead). (B) Histological section from the lung of a pallid mouse after chronic cigarette smoke showing representative changes in the alveolar walls. Three obvious breaks (arrowheads) are evident. H & E stain (original magnification: ×1,000).

	DISCUSSION

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The major findings of this study are (1) the different response to chronic cigarette smoke in mice with different ₁-PI levels and/or sensitivity to oxidants; (2) the acceleration induced by smoke exposure of the development of emphysema in a strain of mice with a marked deficiency of ₁-PI.

In the first study, mice of the strain ICR, with normal BAL fluids EIC, and that responded to the acute oxidant stress of cigarette smoke with an augmentation of the antioxidant capacity in their BAL fluids, were practically unaffected by the chronic cigarette smoke inhalation. On the other hand, mice of the strains C57BL/6J and DBA/2 that reacted to the acute stress of cigarette smoke with a fall of their BAL fluids antioxidant capacity developed pulmonary emphysema after long-term cigarette smoke inhalation. This suggests that the measurement of the BAL fluids TEAC after acute cigarette smoking could represent a rapid and simple screening test for identifying mouse strains that may be sensitive to the chronic effects of cigarette smoke. Also, in humans plasma TEAC was markedly reduced after smoking (22). However, in humans the predictive value of this result for the future development of cigarette smoke-induced emphysema is unknown.

Also, the fall in BAL fluids antioxidant capacity observed in C57BL/6J and DBA/2 mice indicates that in these two strains of mice acute cigarette smoking impaired the antioxidant defenses at the level of the respiratory tract lining fluid, good evidence of local oxidative stress. Chronic oxidative stress after chronic cigarette smoke was associated with a significant decrease of lung elastin content and emphysema in these strains. This suggests a major role for elastolysis in the development of cigarette smoke-induced emphysema. Chronic oxidative stress (23, 24) may have resulted in emphysema either via a direct effect on lung matrix components such as elastin and collagen (25), and/or via an indirect effect by interfering with lung elastin synthesis and repair (26), inducing inactivation of ₁-PI (27), sequestration of neutrophils in the lung microvasculature (28), release of proinflammatory cytokines such as tumor necrosis factor (TNF)- and interleukin (IL)-8 (29), and expression of proinflammatory genes (30).

The present results show that a decrease in BAL TEAC and/or a moderate deficit in BAL EIC are associated with emphysema. These results, however, do not give any information about the pathogenic importance of these single factors. Also, the relevance of these factors is stressed by the results obtained in ICR mice. These mice have normal BAL EIC levels and when acutely exposed to cigarette smoke their BAL TEAC not only did not decrease, but was also significantly augmented. Using human plasma it has been shown that cigarette smoke depletes a number of factors such as protein sulfhydryls (31), ascorbic acid, vitamin E, -carotene, and selenium (24). If the situation in mouse BAL fluids is somehow similar, one can reasonably assume that in certain strains, such the ICR mice, this depletion does not occur. The mechanism for this is unknown. However, oxidative stress, including that produced by cigarette smoking, also causes up-regulation of antioxidant genes (reviewed in MacNee and Rahman [32]) and this process could be more or less effective in different strains.

In Study 2 we investigated whether chronic cigarette smoke exposure could accelerate the development of emphysema in pallid mice that spontaneously develop emphysema in their adult life (10). These mice have a marked deficiency in serum ₁-PI and EIC (8) as well as in BAL fluids EIC (Cavarra and coworkers, unpublished results). This is associated with a progressive increase of neutrophil elastase burden on lung elastin, assessed using an immunogold electron microscopic method, and with a progressive decrease of lung elastin content. The values of lung elastin content correlate inversely with the immunogold values of the elastase burden (11). Emphysema develops between 8 and 12 mo of life (10). These mice are particularly sensitive to the actions of neutrophil chemoattractants. After a large influx of neutrophils into the lungs (induced with FMLP) significant emphysema develops within 3 wk, whereas in non-₁-PI-deficient NMRI mice, the same influx of neutrophils has no effect (33). In pallid mice, chronic exposure to cigarette smoke significantly potentiated parenchymal destruction as assessed using three morphometric methods. It is suggested that this potentiation was related to the relative deficit of antiprotease screen in the lower respiratory tract of these mice in a situation of proteolytic burden as indicated by the significant decrease of the lung elastin content. However, which proteases are involved in the parenchymal destruction in cigarette smoking is a controversial matter. There is strong experimental evidence both for macrophage-derived proteases (34) as well for neutrophil proteases (37). The present study does not allow us to differentiate between these possibilities. But because the pallid mice are deficient in the serine protease inhibitor ₁-PI, our results indirectly suggest that neutrophil proteases play a role in connective tissue breakdown.

In humans a relationship between cigarette smoke-induced COPD and the ₁-PI phenotypes has been recently reported. Cigarette smoke COPD has been found to be associated with the MZ phenotype with an ₁-PI serum deficiency of approximately 50% as compared with the MM phenotype (38). This is also the ₁-PI deficiency level of the pallid mice (8) in which cigarette smoke was here found to induce potentation of the spontaneously occurring emphysema.

The results of the present studies (1 and 2) show that chronic cigarette smoke exposure has different effects in mice with different genetic backgrounds and that the results obtained in these strains of mice have a counterpart in the human situation.