Role of neutrophils and alpha 1-antitrypsin in coal- and silica-induced connective tissue breakdown

K. Zay, S. Loo, C. Xie, D. V. Devine, J. Wright, and A. Churg

Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Mineral dusts produce emphysema, and administration of dust to rats results in the rapid appearance of desmosine and hydroxyproline in lavage fluid, confirming that dusts directly induce connective tissue breakdown. To examine the role of neutrophils and alpha 1-antitrypsin (alpha 1-AT) in this process, we instilled silica or coal into normal rats or rats that had been pretreated with antiserum against neutrophils. One day after dust exposure, lavage fluid neutrophils and desmosine and hydroxyproline levels were all elevated; treatment with antiserum against neutrophils reduced neutrophils by 75%, desmosine by 40-50%, and hydroxyproline by 25%. By 7 days, lavage fluid neutrophils and desmosine level had decreased, whereas macrophages and hydroxyproline level had increased. By ELISA analysis, lavage fluid alpha 1-AT levels were increased four- to eightfold at both times. On Western blot, some of the alpha 1-AT appeared as degraded fragments, and by HPLC analysis, 5-10% of the methionine residues were oxidized. At both times, lavage fluid exhibited considerably elevated serine elastase inhibitory capacity and also showed elevations in metalloelastase activity. We conclude that, in this model, connective tissue breakdown is initially driven largely by neutrophil-derived proteases and that markedly elevated levels of functional alpha 1-AT do not prevent breakdown, thus providing in vivo support for the concept of quantum proteolysis proposed by Liou and Campbell (T. G. Liou and E. J. Campbell. Biochemistry 34: 16171-16177, 1995). Macrophage-derived proteases may be of increasing importance over time, especially in coal-treated animals.

emphysema; metalloproteases; serine proteases


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

RECENT EPIDEMIOLOGIC SURVEYS have come to the conclusion that exposure to mineral dusts is associated with chronic airflow obstruction (reviewed in Refs. 4, 5, 8, 25). The exact pathological changes that result in chronic airflow obstruction in this setting are unclear, but carefully performed radiographic and morphological studies on human autopsy lungs have shown that coal and silica exposure are both associated with emphysema, even in nonsmokers (6, 7, 19). Experimentally, emphysema as well as silicosis can be produced by administration of silica to rats (36).

Little is known about the mechanisms behind the appearance of emphysema in the lungs of dust-exposed workers, but the working assumption of most authors has been that dust should operate by increasing the proteolytic attack through recruitment of inflammatory cells. Brown et al. (9) observed persistently increased lavage proteolytic activity (measured by fibronectin degradation) along with increased numbers of polymorphonuclear neutrophils (PMNs) after quartz administration to rats. Hannothiaux et al. (16) administered silica to monkeys and found marked increases in macrophages; increased elastase activity against a synthetic substrate in some animals, along with increased elastase inhibitory capacity; and, in a few animals, an increase in alpha 1-antitrypsin (alpha 1-AT). These measures changed considerably over time and showed marked differences in absolute levels and patterns from animal to animal. More recently, Ishihara et al. (18), using silica administration to rats, found persisting increases in PMNs but not in macrophages and an increase in lavage fluid elastase activity but no increase in protease inhibitory capacity (measured as trypsin inhibitory capacity). The elastase activity was largely metalloprotease. Despite the lack of increase in macrophage number, they concluded that macrophages were the major source of elastase activity and that alpha 1-AT did not play a significant role.

Although these results favor the idea that there is increased protease activity in the air spaces after dust exposure, there is, quite evidently, no consensus about the role of PMNs vs. macrophages, the nature of the proteolytic activity, and the role of alpha 1-AT in this process.

Li et al. (20) have previously observed that intratracheal instillation of mineral dusts to rats results in the rapid appearance of desmosine (Des), a marker of elastin breakdown, and hydroxyproline (HP), a marker of collagen breakdown, in bronchoalveolar lavage (BAL) fluid, but it was not clear from that study whether PMNs, macrophages, or both cell types were most important in this process. In this study, we used an antiserum against rat neutrophils (APA) to decrease PMN levels after dust exposure and investigated how this manipulation affects connective tissue breakdown, elastase activity, and alpha 1-AT level, integrity, and activity to further examine the mechanisms of dust-induced connective tissue breakdown.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Sources of dusts. The silica used was alpha -quartz (Minusil-5) obtained from US Silica (Clarkstown, WV). The geometric mean particle size determined in our laboratory was 0.96 µm (geometric standard deviation = 2.2). The coal sample used was Penn State Coal Bank sample 1520, a subbituminous coal. The sample was obtained in a form ground to respirable size; the geometric mean particle diameter was 1.8 µm (geometric standard deviation = 3.5).

Administration of mineral dusts and APA. Mineral dusts were administered to 200-g male Sprague-Dawley rats by intratracheal instillation under very light halothane anesthesia at a dose of 30 mg in 0.5 ml of saline. Li et al. and Wright et al. previously showed that this dose of silica produces rapid connective tissue breakdown (20) and also leads to emphysema over the course of 30 days (36). Control animals received only 0.5 ml of saline.

Polyclonal APA was obtained from Accurate Scientific (Westbury, NY). Initial tests showed that APA sharply decreased the BAL fluid PMN count 24 h after administration by intraperitoneal injection, and thus APA was given 24 h before dust exposure. Animals not receiving APA were given an equal volume of saline intraperitoneally. The maximum decrease in BAL fluid PMNs was ~75% after a dose of 1.25 ml of APA. Initial studies also showed that with this dose of APA, BAL fluid PMNs remained significantly depleted for 4 days after dust administration and then increased to control levels or greater by 7 days.

To carry out the actual experiments, groups of 10 rats were given saline (control), silica only, silica preceded by APA, coal only, or coal preceded by APA. At 1 day (24 h) or 7 days after dust administration, five animals from each treatment group were killed by a halothane overdose, and the lungs were rapidly removed and lavaged with 5 × 5 ml of ice-cold saline or cold water (see below).

BAL fluid Des analysis. BAL fluid Des was analyzed by HPLC as previously described (20). Because high salt concentrations interfere with Des analysis, one lung was lavaged with distilled water. The BAL fluid was lyophilized, then hydrolyzed in 2 ml of 6 M hydrochloric acid for 48 h at 110°C. A portion of this hydrolysate was used for Des analysis and a separate portion for HP analysis.

For Des evaluation, 0.5 ml of the hydrolysate was loaded onto a 50-mm cellulose column (Whatman cellulose CF11, Fisher Scientific); the column was then washed with 15 ml of a 4:1:1 n-butanol-acetic acid-water solution, and desmosines were eluted with 5 ml of water. The eluate was lyophilized, and the residue was dissolved in 1 ml of distilled water. Analysis was performed on a Waters HPLC system with a model 486 ultraviolet detector at 275 nm. Separation was obtained with a Nova-Pak C18 4-µm, 4.6 × 150-mm column and an eluant of 0.1 M phosphate buffer-acetonitrile (2.8:1 vol/vol). Twenty micromolar SDS was added as an ion-pairing agent. This procedure separates Des from isodesmosine. The latter is actually the major component in BAL fluid, but both are reported together here as "desmosine."

BAL fluid HP analysis. Fifty microliters of the hydrochloric acid hydrolysate were dried with a Waters Pico-Tag Vacuum Station and redried with 50 µl of a mixture of 2:1:1 ethanol-water-triethylamine, and the dried sample was derivatized with 50 µl of a mixture of 7:1:1:1 ethanol-water-triethylamine-phenylisothiocyanate for 20 min at room temperature. The sample was then dried again and finally dissolved in 700 µl of phosphate buffer for analysis. Analysis was performed with a Waters model 486 detector at 254 nm. To achieve separation, a Whatman Partisil ODS-2 C18 10-µm, 4.6 × 250-mm column was used. The mobile phase was programmed at a flow rate of 1.6 ml/min, starting with 100% solvent A (60 ml of acetonitrile mixed with 940 ml of a 138 mM acetate buffer, pH 6.4, containing 0.05% triethylamine), followed by a linear gradient to 50% solvent B (60% acetonitrile in water) for 15 min (see Ref. 20 for further details).

Purification of rat alpha 1-AT and production of antibody against rat alpha 1-AT. Rat alpha 1-AT was isolated from whole rat serum by a modification of the method of Travis and Johnson (33). After ammonium sulfate precipitation, the supernatant was dialyzed against 30 mM phosphate buffer, pH 7.4, and applied to a DEAE-Sephacel column (Pharmacia). Proteins were eluted from the column with a salt and pH gradient, and fractions containing alpha 1-AT were determined by inhibition of porcine pancreatic elastase (PPE). These fractions were concentrated and applied to a fast-flow Cibacron blue 3GA (Sigma) column to remove albumin. Purity of the eluted product was determined by SDS-PAGE and silver staining, and the product was tested for elastase inhibitory capacity with PPE. The purified product showed two bands with molecular masses of 62 and 54 kDa. Rabbits were immunized with biweekly injections of 400 µg of purified alpha 1-AT mixed with Freund's adjuvant.

Determination of BAL fluid alpha 1-AT levels. Rat BAL fluid alpha 1-AT levels were measured with a competitive ELISA test, with purified rat alpha 1-AT as a standard. The plates were coated with 100 ng/well of rat alpha 1-AT and blocked with PBS containing 3% BSA at pH 7.4. A 50-µl sample was added into the wells, followed by 50 µl of anti-rat alpha 1-AT antibody. After the solution was mixed, the plate was incubated for 3 h at room temperature. After incubation, the plate was washed five times with PBS. One hundred microliters of a secondary antibody (goat anti-rabbit IgG-horseradish peroxidase, 1:10,000) were measured into each well. After 1 h of incubation at room temperature, the plate was washed five times with PBS. The color reaction was developed with ELISA substrate (3,3',5,5'-tetramethylbenzidine; ICN Biochemicals). One hundred microliters of substrate were added into each well and incubated for 5 min. Color development was stopped with 100 µl of a 2 M HCl solution. Absorbance was measured at 450 nm.

Western blot analysis of BAL fluid alpha 1-AT. Proteins from BAL fluid were separated on a 12% polyacrylamide resolving gel with purified rat alpha 1-AT and Rainbow molecular-weight markers (Amersham) as controls. After electrophoresis, the protein bands were immobilized on a nitrocellulose membrane. The membrane was probed with rabbit anti-rat alpha 1-AT and goat anti-rabbit horseradish peroxidase antibody and developed by enhanced chemiluminescence (Amersham).

Isolation of alpha 1-AT from BAL fluid. A rabbit anti-rat alpha 1-AT affinity column was prepared with CNBr-activated Sepharose 4B (Pharmacia) as a matrix following the instructions of the manufacturer. After the CNBr-activated Sepharose 4B matrix was swollen in a 0.1 M HCl solution, the beads were washed with copious amounts of the same solution. The rabbit anti-rat alpha 1-AT ligand was coupled to the matrix in carbonate buffer at pH 8.3. The unbound fraction was washed out with five gel volumes of coupling buffer, and then the remaining active sites were blocked with 0.1 M Tris · HCl buffer at pH 8.0. The product was washed with three cycles of alternating washes with 0.1 M acetate buffer containing 0.5 M NaCl, pH 4.0, and 0.1 M Tris · HCl containing 0.5 M NaCl, pH 8.0. The column was equilibrated with 30 mM phosphate buffer, pH 6.7.

The BAL sample was loaded and washed into the column with the same buffer. The eluted fraction was discarded. The bound rat alpha 1-AT fraction was eluted from the column with a 1% triethylamine solution. The sample was lyophilized and processed for HPLC analysis.

HPLC analysis of BAL fluid for methionine and methionine sulfoxide. The purified alpha 1-AT solutions from the BAL fluid were lyophilized, and the residue was dissolved in 1 ml of a 4 M KOH solution. After being flushed with nitrogen, the tubes were sealed and the samples were hydrolyzed at 110°C for 16 h. The hydrolysates were neutralized with 6 M HCl.

Analysis was performed as previously described (21). Fifty microliters of the sample were dried with a vacuum drying system and redried in 50 µl of 2:2:1 ethanol-water-triethylamine. The dried samples were derivatized with 50 µl of a solution of 7:1:1:1 ethanol-water-triethylamine-phenylisothiocyanate for 20 min at room temperature. The derivatized samples were dried again and dissolved in 700 µl of phosphate buffer solution for analysis. Analysis was performed with a Waters HPLC system. Separation was achieved on a Whatman ODS-2 C18 10-µm, 4.6 × 250-mm column and with the same buffers as described in BAL fluid HP analysis for HP separation. Absorbance was read at 254 nm. All operations were performed at room temperature. To obtain a signal, all BAL fluids from each treatment group were pooled.

BAL fluid elastase activity. Elastase activity in BAL fluid was measured by colorimetric assay. N-succinyl-Ala-Ala-Ala p-nitroanilide (SLAPN; Sigma) was used as the substrate. The assay was carried out in 0.2 M Tris · HCl (pH 8.0) with and without the addition of 10 mM EDTA as a metalloelastase inhibitor or 10 M N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone as a serine elastase inhibitor.

BAL samples were assayed in triplicate wells of 96-well flat-bottom plates (VWR Canlab, Toronto, ON). Each well contained 100 µl of assay buffer, 50 µl of substrate (0.5 mg/ml), and 50 µl of BAL sample. Negative control wells contained 150 µl of assay buffer and 50 µl of substrate. Background absorbance from each BAL sample was assessed by incubating 150 µl of assay buffer with 50 µl of BAL sample. The incubation was run for 1 day at room temperature. The absorbance of each well was determined at a 405-nm wavelength.

Serine elastase inhibitory capacity. BAL fluid serine elastase inhibitory capacity was determined by kinetic colorimetry with a defined amount of SLAPN as the substrate. Each well of a 96-well plate contained 10 µl of 1.5 M Tris buffer, pH 8.8, 150 µl of BAL fluid, and 50 µl of 0.1 mg/ml of PPE in 0.2 M Tris. The plate was incubated for 10 min at room temperature, 50 µl of SLAPN substrate were added, and the plate was read immediately at 415 nm. The results are expressed as percent inhibition of a test well containing PPE but no BAL fluid. In this system, normal rat serum produced ~70% inhibition.

Statistics. Differences among groups were determined by two-way analysis of variance with SYSTAT (34). In some instances, the data values were log transformed to produce normal distributions before analysis. Values of P <=  0.05 were considered significant.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Figure 1 shows BAL fluid PMN levels 1 and 7 days after dust instillation. At 1 day, there was a 60-fold increase in BAL fluid PMNs with silica exposure and a 170-fold increase with coal exposure compared with those in control rats. With both dusts, APA administration significantly reduced PMN numbers by ~75%. Histological examination (Fig. 2) confirmed that antibody treatment greatly reduced PMN numbers in the air spaces and tissues. By 7 days, PMN numbers had significantly decreased (~60% with silica and by >99% with coal administration). With both dusts, there was no significant difference between dust and dust-APA treatment on day 7.


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Fig. 1.   Bronchoalveolar lavage (BAL) fluid polymorphonuclear neutrophil (PMN) levels. Values are means ± SD. At 1 day, BAL fluid PMNs were increased after both coal (Co) and silica (Si) administration; anti-rat neutrophil antiserum (APA) reduced these values by ~75%. By 7 days, PMN levels had significantly declined from 24-h levels but were still elevated relative to control level (Ctrl; Ctrl at 7 days is too small to be visible). * Significantly greater than Ctrl (P <=  0.05). a Significantly less than dust without APA (P <=  0.05) b Significantly less than 1-day value (P <=  0.05).


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Fig. 2.   A and B: representative light micrographs from lungs of Si-treated animal at 1 day. There is an intense inflammatory infiltrate centered around alveolar ducts. Numerous PMNs are visible in air spaces and tissue. C and D: representative light micrographs from lungs of animal treated with anti-PMN antibody and Si at day 1. Compared with Si treatment alone, there is a marked decrease in inflammatory cells, but some PMNs are still present. Hematoxylin and eosin stain. Original magnifications: ×160 in A and C; ×400 in B and D.

Figure 3 shows similar data for BAL fluid macrophage numbers. At 1 day, macrophage numbers showed small (1.5- to 2-fold) increases with both dusts, but only coal-APA treatment produced a significant increase compared with that in control rats. At 7 days, significant increases were seen in silica-, silica-APA-, and coal-treated animals, with absolute values two to three times those of control animals; the silica- and coal-treated animals showed significant increases in macrophage numbers compared with those on day 1.


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Fig. 3.   BAL fluid macrophage levels. Values are means ± SD. At 1 day, macrophage numbers are elevated 1.5- to 2-fold, but only with Co-APA treatment are they significantly increased. By 7 days, macrophage numbers are 2-3 times greater than control numbers and are significantly greater than 24-h values for both Si and Co treatments. * Significantly greater than Ctrl (P <=  0.05). b Significantly greater than 1-day value (P <=  0.05).

Figure 4 shows BAL fluid levels of Des. At 1 day, Des levels were increased about four- to fivefold with both dusts compared with control levels; APA treatment produced significant reductions in Des levels (~40% after silica and 50% after coal administration). In silica-treated animals, Des levels decreased at 7 days to 50% of the 1-day values, and in coal-treated animals, 7-day Des values decreased to ~40% of the 1-day values.


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Fig. 4.   BAL fluid desmosine levels. Values are means ± SD. At 1 day, levels are increased 4-5 times with Si and Co treatments and significantly reduced (by 40-50%) with APA. At 7 days, desmosine levels remain elevated but are significantly less than 1-day values for Si and Co treatments. * Significantly greater than Ctrl (P <=  0.05). a Significantly less than dust without APA (P <=  0.05). b Significantly less than 1-day value (P <=  0.05).

Figure 5 shows BAL fluid HP levels. At 1 day, there were small but significant (1.5-2 times) increases in HP with silica and coal treatments compared with the control values. APA treatment produced a 20% decrease in HP levels with silica (P < 0.02 compared with silica alone) and a similarly small but not significant decrease with coal administration. By 7 days, HP levels with silica treatment had nearly doubled over the 1-day levels and with silica-APA treatment were four times the 1-day values. With coal treatment, 7-day HP values were approximately double those of control values and were slightly but not significantly elevated over day 1 values.


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Fig. 5.   BAL fluid hydroxyproline (HP) levels. Values are means ± SD. At 1 day, there is a 1.5- to 2-fold increase in HP levels with Si and Co treatments and a reduction with APA (only reduction for Si-APA is significant). At 7 days, there is a marked increase in HP levels for both Si treatment groups and a small increase for both Co treatment groups. * Significantly greater than Ctrl (P <=  0.05). a Significantly less than dust without APA (P <=  0.05). b Significantly greater than 1-day value (P <=  0.05).

Figure 6 shows BAL fluid alpha 1-AT levels. At 1 day, dust increased alpha 1-AT levels by five to eight times compared with the control levels and APA treatment decreased this elevation by ~40% with both silica and coal, but the differences were not significant. At 7 days, alpha 1-AT levels had decreased by ~50% in both silica- and coal-treated animals (only the difference for silica is significant) but were still four to six times the control levels.


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Fig. 6.   BAL fluid alpha 1-antitrypsin (alpha 1-AT) levels. Values are means ± SD. Levels are increased 4- to 8-fold with all treatments, and there is a nonsignificant reduction with APA at 1 day. At 7 days, alpha 1-AT levels with Si and Co treatments are ~60% of 1-day levels. * Significantly greater than Ctrl (P <=  0.05). b Significantly less than 1-day value (P <=  0.05).

Figure 7 shows a Western blot for BAL fluid alpha 1-AT run under reducing conditions. No alpha 1-AT-neutrophil elastase complexes are seen, but dust treatment caused the formation of many low-molecular-mass fragments.


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Fig. 7.   Western blot for rat alpha 1-AT run under reducing conditions. Lane 1, purified rat alpha 1-AT; lane 2, saline control, 1 day; lane 3, Si, 1 day; lane 4, Si-APA, 1 day; lane 5, Co, 1 day; lane 6, Co-APA, 1 day; lane 7, saline control, 7 days; lane 8 Si, 7 days; lane 9, Si-APA, 7 days; lane 10, Co, 7 days; lane 11, Co-APA, 7 days. Data are derived from combined BALs for each treatment group. Note increase in native alpha 1-AT with dust treatments as well as numerous smaller degradation fragments. Nos. at right, molecular mass.

Figure 8 shows the ratio of methionine sulfoxide to methionine in alpha 1-AT purified from BAL fluid. At 1 day, this ratio was increased with all treatments. At 7 days, the proportion of methionine sulfoxide was slightly higher with all treatments compared with the 1-day values. The ratio never exceeded ~10% in any group.


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Fig. 8.   Ratio of methionine to methionine sulfoxide in affinity-purified BAL fluid alpha 1-AT. Bars represent single values derived from combined BALs for each treatment group. There is an increase with dust treatment and a trend toward reduction with APA treatment at 1 day. Overall amount of methionine oxidized is fairly small (never more than ~10%).

Figure 9 shows BAL fluid serine elastase inhibitory capacity expressed as a percentage of pure PPE (without any inhibitor) activity. At 1 day, silica treatment resulted in an ~70% inhibition compared with only 10% for control BAL fluid. Coal also increased the inhibitory activity, albeit to a much lesser degree. APA treatment decreased BAL fluid inhibitory capacity with both dusts, but the differences were not significant. At 7 days, the elastase inhibitory capacity was significantly lower (75% for silica and 60% for coal compared with day 1 values). For both dusts, the inhibitory capacity at 7 days was about two to three times that of control BAL fluid.


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Fig. 9.   BAL fluid elastase inhibitory capacity. Values are means ± SD. There is a significant increase for all treatments at 1 day and a nonsignificant reduction with APA treatment. Levels are significantly lower for Co and Si treatments at 7 days. * Significantly greater than Ctrl (P <=  0.05). b Significantly less than 1-day value (P <=  0.05).

Figures 10 and 11 show BAL fluid serine- and metalloelastase-like activities, respectively. At 1 day, there was no increase in serine elastase activity, but silica-APA treatment showed a significant increase in metalloelastase activity. At 7 days, increases in serine and, particularly, metalloelastase activity (>10-fold) were seen with silica and silica-APA treatment. Coal-treated groups showed roughly a doubling of metalloelastase activity and no increase in serine elastase activity.


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Fig. 10.   BAL fluid serine elastase-like activity. Values are means ± SD. A marked increase is seen with both Si treatments at 7 days. * Significantly greater than Ctrl (P <=  0.05).


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Fig. 11.   BAL fluid metalloelastase-like activity. Values are means ± SD. A 2-fold increase is seen with Si-APA treatment at 1 day. At 7 days, all groups show an increase compared with control levels and the 2 Si treatments show a marked increase compared with 1-day levels. * Significantly greater than Ctrl (P <=  0.05) b Significantly greater than 1-day value (P <=  0.05).

Because graphs of the type shown in Figs. 1, 3-6, and 8-11 hide interanimal variations and thus may hide correlations between variables, Table 1 was constructed to show statistical correlations between PMN and macrophage numbers and measures of connective tissue breakdown, alpha 1-AT levels, and elastase levels at 1 and 7 days. PMN numbers showed correlations with most of these measures after silica treatment, including metalloprotease activity, and fewer correlations after coal treatment. Macrophage levels after silica treatment showed no correlations. After coal treatment, macrophage levels did correlate with HP and metalloelastase levels at 7 days.

                              
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Table 1.   Correlations of inflammatory cells and various measures


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

As described in the introduction, there is little information and no consensus about the mechanism(s) of dust-induced emphysema. We hypothesized that this process might proceed through the same pathways that have traditionally been invoked for the development of emphysema in smokers, namely, a smoke-induced inflammatory infiltrate of PMNs that release neutrophil elastase and an oxidative inactivation of alpha 1-AT, the major antiproteolytic protein in the lower respiratory tract, leading to an imbalance of proteolytic activity and proteolytic defense, with resulting destruction of elastin and collagen (reviewed in Refs. 13, 29, 32).

Mineral dusts also evoke inflammatory infiltrates, and in support of this general theory, we have shown that mineral dusts that generate active oxygen species by surface catalysis in aqueous solution (for example, coal, silica, and asbestos) will oxidize methionine residues in alpha 1-AT in vitro (21) and that exposure of alpha 1-AT to silica in vitro is associated with a loss of inhibitory capacity against pancreatic elastase; this process is hydrogen peroxide mediated (37). On the face of it, these findings could support a proteolysis-antiproteolysis imbalance mechanism.

However, the whole question of whether the proteolysis-antiproteolysis theory of smoke-induced emphysema is correct and which proteolytic enzymes, matrix components, and antiproteolytic substances are important is greatly debated (13, 29, 32). It has become clear that, besides neutrophil elastase, a variety of other proteolytic enzymes including 72- and 92-kDa gelatinase, matrilysin, and the recently described macrophage metalloelastase all have significant elastase activity (15, 30, 31). There is increasing evidence that these other proteases play a role in smoke-related disease. For example, Hautamaki et al. (17) created mice in which the gene for macrophage metalloelastase has been knocked out. These mice fail to develop cigarette smoke-induced emphysema, suggesting a major role for macrophage-derived proteolytic enzymes in smoke-induced emphysema and, at least in that model, implying that neutrophil-derived proteases play a minor role at best. Selman et al. (28) have shown upregulation of interstitial collagenase expression in the lung in smoke-exposed guinea pigs, and Finlay et al. (14) reported increased expression of gelatinase B and interstitial collagenase in macrophage culture supernatants from patients with emphysema.

Similarly, recent studies (11, 35) have shown that elastin is not the only matrix component that is abnormal in smoke-induced emphysema. In both animals and humans, there is increased collagen as well, and the development of increased collagen is preceded in guinea pigs by collagen breakdown. These observations again imply that, at least for cigarette smoke, neutrophil elastase is unlikely to be the only proteolytic enzyme of importance.

The role of alpha 1-AT in preventing smoke-induced damage is also extremely controversial. Although there is no doubt that severe alpha 1-AT deficiency in humans leads to early emphysema, reports on alpha 1-AT levels and/or activity against neutrophil elastase in smokers have been contradictory, with some studies finding decreased levels and/or activity in smokers and others finding no differences from nonsmokers (reviewed in Refs. 13, 29, 32). Although Carp et al. (12) reported that 50% of the methionine residues in alpha 1-AT from cigarette smokers was oxidized (thus decreasing antiproteolytic activity), other investigators have not been able to reproduce this finding (reviewed in Ref. 13). It has also been proposed that the differences between smokers and nonsmokers are actually to be found in the association constants for alpha 1-AT and neutrophil elastase, with a decreased constant in smokers (23).

In these experiments, we have used a model of mineral dust exposure to examine connective tissue breakdown mechanisms directly relevant to dusts and possibly applicable to cigarette smoke as well. The results are complex, appear to be somewhat different between coal and silica, and are probably subject to various interpretations. However, it is clear that with silica treatment, at 1 day both Des and HP levels in BAL fluid correlate with the number of BAL fluid PMNs and that reducing PMN levels reduces connective tissue breakdown. This is true of coal as well at 1 day, although the statistical correlations are not as good, probably because of greater variation in inflammatory cell numbers. Thus this model does support the idea that dust-evoked PMNs can produce connective tissue breakdown. The role of macrophages, which increase in number somewhat by 7 days, is less obvious. For silica treatments, BAL fluid HP levels are markedly greater and for coal slightly greater at 7 days compared with those at 1 day, despite major decreases in absolute PMN counts, thus suggesting a role for macrophages; with coal administration, macrophage levels do, in fact, correlate with HP levels at 7 days (this is not true for silica). Nonetheless, in a broad sense, these results support the growing consensus that inflammatory cell-mediated connective tissue breakdown in the lung reflects contributions from proteases derived from both PMNs and macrophages (13, 29, 32; see below).

We also found a considerable increase in BAL fluid alpha 1-AT levels at both 1 and 7 days, in sharp contrast to the results reported by Ishihara et al. (18) and Hannothiaux et al. (16). Ishihara et al. (18) used trypsin inhibitory capacity as a surrogate for alpha 1-AT, and thus true alpha 1-AT concentrations were unknown. Hannothiaux et al. (16) used inhalation rather than instillation exposures, a procedure that usually puts smaller amounts of dust into the lung and evokes less of a PMN infiltrate, and this may account for the small and inconstant increases in alpha 1-AT that these authors reported (in fact, all of the parameters they measured showed small and inconstant increases, perhaps suggesting that the total silica dose was really very low).

In our hands, the increase in alpha 1-AT at 1 day did roughly correlate with PMN levels. There was also evidence of considerable alpha 1-AT breakdown, and the amount of breakdown is probably underestimated on the Western blot because a polyclonal antibody such as we used will almost certainly detect cleavage fragments less efficiently than the whole protein. Because complexing of neutrophil elastase with alpha 1-AT leads to fragmentation of the complex (3), some of these breakdown products probably represent the protective effect of alpha 1-AT; others may reflect proteolytic attack on the native protein, including attack by free metalloproteases. Oxidation of methionine in alpha 1-AT clearly also does occur in this environment, and the level of oxidized methionines roughly parallels PMN numbers at 1 day, supporting the idea that inflammatory cells may damage alpha 1-AT by release of oxidants.

What these changes imply in terms of function is less certain. Rat alpha 1-AT contains 10 methionines (27), of which only 5-10% are oxidized. This level is far different from the oxidation of 50-60% of methionines seen with coal dust in vitro (21) and claimed by Carp et al. (12) in human smokers. Arguably, these oxidized methionines could all be active-site methionines, which in theory should reduce alpha 1-AT activity by 50-100%. However, the elastase inhibitory capacity data make it clear that this is not the case (see below). These results suggest that oxidative damage to alpha 1-AT does not play a major role in this system. The breakdown of alpha 1-AT seen on the Western blots also argues for care in interpreting data on association constants for alpha 1-AT and neutrophil elastase. If the alpha 1-AT is purified, as here, with an antibody that retains both native alpha 1-AT and breakdown products, then the measured association constant can be spuriously decreased by inclusion of nonfunctional small-molecular-mass fragments in the assay.

Oxidation of methionines and proteolytic breakdown of alpha 1-AT notwithstanding, it is clear from the serine elastase inhibitory capacity analysis that large quantities of functional alpha 1-AT are still present in the BAL fluid and, by implication, in the interstitium, the major site of proteolytic attack. The fact that inflammatory cell-mediated proteolysis can proceed in the presence of an inhibitor has been demonstrated in vitro in a variety of systems (22, 24, 26). Proteolysis may occur in this circumstance because proteolytic enzyme bound to the neutrophil surface is relatively insensitive to inhibition and because enzyme bound to substrate may be inaccessible to large-molecular-mass inhibitors such as alpha 1-AT (24). Probably more important is the phenomenon of "quantum proteolysis" described by Liou and Campbell (22). They showed that because proteases are released from neutrophils in small bursts of highly concentrated enzyme, proteolysis will proceed until enzyme diffuses sufficiently that an enzyme-to-inhibitor ratio of 1:1 or less is achieved. Because of the shape of the curve relating neutrophil elastase concentration and neutrophil elastase diffusion distance, they propose that inhibitors such as alpha 1-AT may limit proteolysis by compartmentalizing it to localized regions but cannot totally prevent it in the face of an inflammatory influx, even when the inhibitor is present, as in our experiments, at extremely high concentration. Assuming, as appears reasonable, that high BAL fluid levels of alpha 1-AT reflect high tissue levels of alpha 1-AT, our present observations appear to provide in vivo support for the concept of quantum proteolysis.

The free elastase data in our model are also of interest but must be interpreted with considerable caution. The SLAPN substrate is not specific for neutrophil elastase, and the detection of both serine and metalloprotease activities with this substrate has been previously reported (for example, Ref. 10); the cautious and probably correct approach is, as other authors (10) have indicated, to regard this activity as "elastase-like," and we are using the term "elastase" in this sense. It is also possible that the SLAPN assay, which was designed to detect serine elastase activity, underestimates metalloelastase-like activity. Moreover, assay of metalloprotease activity itself can be problematic because of binding of the enzymes to the tissue inhibitor of metalloproteinase when they are extracted from tissues (30).

What is apparent from the free elastase data is the presence of active metalloproteases, particularly at 7 days. Given the good correlation between PMN counts at 7 days and metalloprotease activity with silica exposure, as well as the correlation between macrophage numbers and metalloprotease activity with coal exposure, these observations suggest that both neutrophil- and macrophage-derived metalloproteases are playing a role and that the important proteases change over time and are different for the different dusts; in particular, elevated HP levels after coal exposure appear to be a reflection of macrophage-derived metalloproteases, whereas silica effects appear to be primarily PMN mediated. The nature of the serine protease-like activity seen in the face of high functional alpha 1-AT levels (7-day silica treatment) is unclear.

As noted, proteolysis is fundamentally an interstitial process and thus one could argue that BAL fluid elastolytic activity is of little relevance. However, it is clear from examining tissue sections and also from correlating tissue and BAL fluid data that air space and tissue inflammatory cell counts, particularly neutrophil counts, do correlate, suggesting that the proteolytic activity present in the air spaces is also present (at least for a time) in the interstitium. In addition, we propose that BAL fluid proteolytic activity can be directly important in matrix breakdown. Mineral dusts such as silica produce considerable epithelial damage including ulceration (1); although the intact epithelium may be able to prevent access of proteases to the matrix, sufficient epithelial damage may allow a direct attack by air space proteases on the underlying tissue. Indirect support for this idea comes from the recent observations by Adamson and Prieditis (2), who found that initial treatment with bleomycin followed by silica administration increased both silica particle transport into the interstitium and levels of fibrosis compared with silica alone. If relatively enormous silica particles can gain access to the matrix because of epithelial injury, it is highly likely that air space macromolecules can also do so.


    ACKNOWLEDGEMENTS

This study was funded by Medical Research Council of Canada Grant MT6907. The high-performance liquid chromatography equipment used was purchased with a grant from the British Columbia Health Research Foundation.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: A. Churg, Dept. of Pathology, Univ. of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5.

Received 4 September 1998; accepted in final form 30 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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