Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5
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ABSTRACT |
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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
1-antitrypsin (
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
1-AT levels were increased
four- to eightfold at both times. On Western blot, some of the
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
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
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INTRODUCTION |
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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
1-antitrypsin
(
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
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
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 1-AT level, integrity, and
activity to further examine the mechanisms of dust-induced connective
tissue breakdown.
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MATERIALS AND METHODS |
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Sources of dusts. The silica used was
-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 1-AT
and production of antibody against rat
1-AT.
Rat
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
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
1-AT mixed with Freund's adjuvant.
Determination of BAL fluid
1-AT levels.
Rat BAL fluid
1-AT levels were
measured with a competitive ELISA test, with purified rat
1-AT as a standard. The plates were coated with 100 ng/well of rat
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
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
1-AT.
Proteins from BAL fluid were separated on a 12% polyacrylamide
resolving gel with purified rat
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
1-AT and goat anti-rabbit
horseradish peroxidase antibody and developed by enhanced
chemiluminescence (Amersham).
Isolation of 1-AT from
BAL fluid.
A rabbit anti-rat
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
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.
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RESULTS |
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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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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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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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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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Figure 6 shows BAL fluid
1-AT levels. At 1 day, dust
increased
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,
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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Figure 7 shows a Western blot for BAL fluid
1-AT run under reducing
conditions. No
1-AT-neutrophil
elastase complexes are seen, but dust treatment caused the formation of
many low-molecular-mass fragments.
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Figure 8 shows the ratio of methionine
sulfoxide to methionine in 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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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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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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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, 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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DISCUSSION |
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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
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 1-AT in vitro (21) and that
exposure of
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 1-AT in preventing
smoke-induced damage is also extremely controversial. Although there is
no doubt that severe
1-AT
deficiency in humans leads to early emphysema, reports on
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
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
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
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
1-AT, and thus true
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
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
1-AT at 1 day did roughly
correlate with PMN levels. There was also evidence of considerable
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
1-AT leads to fragmentation of
the complex (3), some of these breakdown products probably represent
the protective effect of
1-AT;
others may reflect proteolytic attack on the native protein, including
attack by free metalloproteases. Oxidation of methionine in
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
1-AT by
release of oxidants.
What these changes imply in terms of function is less certain. Rat
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
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
1-AT does not play a
major role in this system. The breakdown of
1-AT seen on the Western blots
also argues for care in interpreting data on association constants for
1-AT and neutrophil elastase. If the
1-AT is purified, as
here, with an antibody that retains both native
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
1-AT notwithstanding, it is
clear from the serine elastase inhibitory capacity analysis that large
quantities of functional
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
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
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
1-AT reflect high
tissue levels of
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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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