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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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In the present study, we tested the hypothesis that neutrophil elastase (NE) might mediate remodeling of extracellular matrix by affecting fibroblast-mediated contraction of three-dimensional collagen
gels. Human lung fibroblasts were cast into type I collagen gels containing NE. After gelation, the
gels were released into medium and the area was measured by image analyzer. NE augmented gel
contraction (p < 0.001). This was not due to cell proliferation or to degradation to soluble collagen
fragments because the amounts of DNA and hydroxyproline were not altered. 
1-Protease inhibitor and the synthetic inhibitor of NE, L-680,833, when added in sufficient amount to inhibit free elastase
activity, blocked the contraction induced by NE. Furthermore, neutrophil granulocytes (PMN) in coculture, as well as conditioned media from PMN, resulted in an increased contractility (p < 0.001 for
both). Bronchoalveolar lavage fluid (BALF) from patients with increased PMN in their lower respiratory tract and free elastase activity had augmentive activity for gel contraction which could be partially blocked by the inhibitors. We conclude that NE augments fibroblast-mediated contraction of collagen gels. The findings support the notion that products secreted by PMN in inflammatory disorders may lead to rearrangement of extracellular matrix and could subsequently lead to tissue dysfunction.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Inflammatory diseases are characterized by the presence of various inflammatory cells and by an alteration of connective tissue. Fibroblasts are the main producers of extracellular matrix macromolecules. They are also known to generate traction force and therefore are thought to play an important role in tissue rearrangements (1). These alterations in tissue architecture may result in compromise of physiologic function. For example, in obstructive lung disorders such as chronic bronchitis, peribronchiolar fibrosis associated with airway narrowing is believed to be related to the development of airflow limitation (2, 3). Similar fibrotic responses, often with disruption of tissue function, occur both in other lung diseases and in inflammatory diseases in many diverse tissues. Tissue contraction is a regular feature of these conditions.
The inflammatory response is characterized by the recruitment of granulocytes and cells from the monocyte-macrophage lineage. These cells also are known to be able to modulate the fibrotic response by a number of mechanisms including the release of mediators capable of directing fibroblast recruitment, proliferation, and matrix production.
While neutrophilic inflammation is traditionally regarded as an acute response, the chronic presence of neutrophils is often associated with fibrotic responses including tissue contraction (4, 5). Thus, many fibrotic conditions such as idiopathic pulmonary fibrosis and cirrhosis of the liver are characterized by the persistent presence of polymorphonuclear leukocytes (PMN). Moreover, high levels of the proteolytic enzyme neutrophil elastase (NE) have been found in various inflammatory conditions including chronic bronchitis and cystic fibrosis (6, 7). This suggests the hypothesis that neutrophils may contribute to fibrosis by augmenting fibroblast-mediated tissue contraction and that elastase may play a role in this process.
To test this hypothesis, we used an in vitro model for tissue
remodeling, fibroblast-mediated contraction of collagen gels (8). In this culture system, human lung fibroblasts are cultured in a three-dimensional collagen gel. By culturing fibroblasts in
this manner, the cells are able to contract the gels. In the current study we demonstrate that human NE is capable of augmenting the fibroblast-mediated contraction of collagen gels.
Furthermore, we show that increased contractile activity can
be blocked both by 1-protease inhibitor and a synthetic inhibitor of neutrophil elastase, L-680,833. Finally, using both
whole neutrophils and samples from patients with neutrophilic inflammation, we provide evidence that elastase has the
potential to mediate this function in both cell culture systems
and in vivo. Our findings, therefore, support the notion that
NE may play a role in the rearrangement of extracellular
matrix, a process that could subsequently lead to tissue dysfunction.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Materials
Type I collagen (rat tail tendon collagen, RTTC) was extracted from rat tail tendons as previously described (9). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were removed carefully. After repeated washing with Tris-buffered saline (0.9% NaCl, 10 mM Tris, pH 7.5) and 95% ethanol, the collagen was extracted in 6 mM acetic acid. Protein concentration was determined by weighing a lyophilized aliquot from each batch of collagen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) routinely demonstrated no detectable proteins other than type I collagen. The RTTC was stored at 4° C until use.
The following agents were purchased: NE purified from human
sputum (Elastin Products Co. Inc., Owensville, MO); 1-protease inhibitor (Prolastin; Miles Inc., Elkhart, IN); transforming growth factor-beta1 (TGF-
1; R&D Systems, Minneapolis, MN); prostaglandin E2 (PGE2; Sigma, St. Louis, MO), cell culture media except fetal calf
serum (FCS) (GIBCO, Life Technologies, Grand Island, NY); and
FCS (Biofluid, Rockville, MD). The synthetic human NE inhibitor L-680,833 (10) was a gift from Merck Sharp and Dohme, Rahway, NJ.
Human NE was dissolved in phosphate-buffered saline (PBS) to a
stock solution of 1 mg/ml. Prolastin was (according to manufacturer's recommendations) dissolved in diluent to a concentration of 10 mg/
ml. L-680,833 was dissolved in ethanol to a stock solution of 10
2 M. TGF-1 was dissolved to a stock solution of 1 µg/ml in 4 mM HCl and
0.1% bovine serum albumin. PGE2 was dissolved in PBS to a stock solution of 50 µg/ml.
Cell Culture
Human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in 100-mm tissue culture dishes (FALCON; Becton-Dickinson Labware, Lincoln Park, NJ) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 50 U/ml penicillin G sodium, 50 µg/ml streptomycin sulfate (penicillin-streptomycin, GIBCO), and 1 µg/ml amphotericin B (Parma-Tek, Huntington, NY). The fibroblasts were usually passaged weekly and cells between passage 10 and 20 were used. Confluent fibroblasts were trypsinized (trypsin-ethylenediaminetetraacetic acid [EDTA]; 0.05% trypsin, 0.53 mM EDTA-4 Na) and resuspended in DMEM without serum.
Fresh human neutrophil granulocytes (PMN) were obtained from healthy donors. All subjects were studied under University of Nebraska institutional review board approved guidelines after informed consent had been obtained. Venous blood was drawn into heparinized syringes. After dextran sedimentation (Dextran; Pharmacia, Uppsala, Sweden), the leukocytes were separated from the plasma layer by centrifugation (1,800 rpm, 10 min). Remaining red blood cells were eliminated by hypotonic lysis. The purified neutrophils were washed and resuspended in DMEM without serum and the cells were counted in a hemocytometer. Cytocentrifugation slides (Cytospin; Shandon, Runcorn, UK) stained with Diff-Quik (American Scientific, St. Louis, MO) revealed that > 90% of the cells were PMN, based on morphological features. The cells were kept on ice until use.
Collagen Gel Contraction Assay
Collagen gels were prepared as described previously (11). Briefly, RTTC, distilled water, 4× DMEM and fibroblast suspensions were mixed so that the final mixture resulted in 0.75 mg/ml of collagen, 3× 105 fibroblasts/ml gel, and a physiologic ionic strength of 1× DMEM. Fibroblasts were added after all the other ingredients had been mixed. The different mixtures were kept on ice during the preparation. A 550-µl portion of the gel solution was then cast into each well of a 24-well tissue culture plate with a 2 cm2 growth area (FALCON). Gelation occurred within 15 min at room temperature.
The ability of NE to affect fibroblast-mediated gel contraction was
measured by two different contraction assays: In the slow contraction
assay of Bell and coworkers (12), NE, with or without 1-protease inhibitor, was added to the gels just before they were cast. After gelation, which occurred within 15 min at room temperature, the gels were
released from the surface of the culture well using a sterile spatula and
transferred into 60-mm tissue culture dishes (FALCON) containing
5 ml of serum-free DMEM. In experiments including neutrophil granulocytes, these cells were added to the collagen gel solution just before the gels were cast. The synthetic inhibitor of NE, L-680,833, was
added to the media in which the released gels were floated. In experiments evaluating the specificity of the inhibitors, PGE2 was added in
the media while TGF-1 was mixed in the collagen gels and also
added in the media. The floating gels were cultured for up to 5 d, and the area of the gels was measured each day.
In the rapid contraction assay, which is a modification of the
method described by Tomasek and coworkers (13), fibroblasts cast
into type I collagen gels which remained attached to a 24-well tissue
culture plate (FALCON) were cultured for 4 d. An aliquot of 500 µl
of serum-free DMEM was added to the top of each gel. This media
was changed after 48 h. After 4 d of culture, the gels were released
into 5 ml of serum-free DMEM containing various concentrations of
NE (1010 M to 107 M). The area of the gels was then measured at
different time points.
To prevent attachment of released gels to the bottom of the 60-mm culture dishes, the floating gels were, during the first 24 h of culture, kept on a rocker platform (Bellco Biotechnology, Vineland, NJ) with continuous rocking (15 cycles/min) inside the culture incubator. The ability of the fibroblasts to contract the gels was determined by quantifying the area of the gels using an Optomax V image analyzer (Optomax, Burlington, MA).
Determination of Hydroxyproline Contents in Gels
The amount of hydroxyproline, which is directly proportional to the collagen content, was determined by a spectrophotometric assay (14). Briefly, after gel weight had been determined, the gels were added to 2 ml of 6 N HCl. The gels were then dissolved and hydrolyzed at 110° C for 12 h. After drying, the amount of hydroxyproline was assayed spectrophotometrically at 550 nm.
Measurement of Soluble Elastase Activity
Functional elastase activity was measured using a synthetic substrate, methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide (Calbiochem- Novabiochem Co., La Jolla, CA) (15). Serial dilutions of supernatants were incubated with 200 µl of 0.2 M substrate in 0.1 M HEPES, 0.5 M NaCl, and 10% dimethylsulfoxide at pH 7.5. After incubation for 24 h, absorbance of the product, p-nitroanilide, was measured at 414 nm. Purified human NE was used as a standard.
To determine the elastase activity in gels, the collagen gels were
dissolved by adding 0.5 ml of collagenase (Sigma), at a concentration
of 0.25 mg/ml, to each gel. The gels were then incubated for 24 h at
37° C and the dissolved gels were centrifuged (1,500 rpm, 10 min) and
stored at 
80° C until analyzed. The collagenase alone did not have
any elastase activity.
To estimate the number of cells in the dissolved gels, DNA was assayed fluorometrically with Hoechst dye 33258 (Sigma) by a previously published method (11, 16).
Conditioned Media from Cultured PMN
PMN-conditioned media was obtained by culturing the cells in 6-well
culture plates (FALCON). An aliquot of 3 ml of serum-free DMEM
containing 1 × 106 neutrophils/ml was added per well. To obtain degranulation of the PMN, the synthetic peptide n-formyl-met-leu-phe
(FMLP) was added at a concentration of 5 × 106 M. After incubating
the samples for 30 min under culture conditions, the PMN-conditioned media were collected, cells and debris were removed by centrifugation (3,000 rpm, 5 min), and the media were stored at 80° C
until use.
Bronchoalveolar Lavage (BAL)
In order to determine if neutrophils could release elastase in vivo
which could augment fibroblast-mediated collagen gel contraction, bronchoalveolar lavage fluid (BALF) was analyzed from seven patients with neutrophilia in the lower respiratory tract (Table 1). At the
time of BAL, all patients had pulmonary infiltration on their chest radiograph and were clinically suspected of having pneumonia. BAL
was performed as previously described (17, 18). Briefly, 20-ml aliquots
of sterile saline were infused through a flexible fiberoptic bronchoscope into each of three lobes and immediately aspirated. The first aliquots, which represent a sample enriched for bronchial material,
were processed separately. The final 4 × 20 ml aliquots, which were enriched for alveolar material, were pooled and used in the gel contraction assays as described subsequently. The BAL cells were counted in
a hemocytometer and the mucus was removed by filtration through a
nylon mesh. Cell differential counts were determined by counting 200 cells from slides prepared by cytocentrifugation (Cytospin; Shandon
Corp., Sewickley, PA) and stained with Diff-Quik. After the cells had
been removed by centrifugation, the BALF was stored at 80° C until
further analysis.
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The ability of BALF to contract collagen gels was measured by releasing fresh gels, made as previously described, into 5 ml of BALF diluted 1:4 in serum-free DMEM. Gels released in saline diluted 1:4 in serum-free DMEM were used as controls. The floating gels were cultured for 2 d and the area was determined as previously described.
Statistical Analysis
Results are expressed as mean ± SEM of three replica gels for each condition unless otherwise stated. Each experimental condition in Figures 1 and 5A was compared with control (fibroblasts alone) using repeated measures analysis of variance (ANOVA) with the statistical significance of each comparison adjusted for the multiple comparisons using a Bonferroni correction. Statistical comparisons of data displayed in Figures 3, 6, and 7 were made using two independent sample t tests, again assessing statistical significance after applying a Bonferroni correction. Statistical significance of differences between data between groups displayed in Figure 8 was assessed using paired t tests, after applying a Bonferroni correction. All significant p values reported have been "inflated" to reflect the multiple comparisons.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The Effect of Human NE on Fibroblast-Mediated Contraction of Collagen Gels
Two methods were used to assess fibroblast-mediated contraction of collagen gels in vitro. Human NE augmented the contraction in both.
In the slow contraction assay system, NE, added to the gels
just before they were cast, augmented contraction in a time-
and concentration-dependent manner (Figure 1). Specifically,
NE concentrations of 108 to 107 M increased the contraction
(p < 0.001 for both) whereas 109 M did not differ from control gels. After 3 d of culture, the fibroblasts did not further
contract the gels, suggesting that, under the culture conditions
used, the gels have reached their final size. Gels containing
NE (107 M) but no fibroblasts did not contract.
To assess whether the decreased size of the gels induced by NE was not due to a decreased amount of collagen in the gels, the amount of hydroxyproline was determined. Hydroxyproline content in the gels did not differ between the different conditions (Figure 2B), ruling out the possibility that the type I collagen had been degraded to soluble fragments during the culture period. Because the gels containing fibroblasts and NE had the highest contractility, the collagen concentration was increased in these gels as reflected by the amount of hydroxyproline in relation to the weight of the gels (Figure 2C).
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In the rapid contraction assay system, NE was added in the
media in which the gels were floated. In this "rapid" assay system, there was a time-dependent augmentation of fibroblast-mediated collagen gel contraction (Figure 3). Six hours after
release, the gels incubated in medium containing 107 M NE
had significantly contracted (p < 0.01) compared with control gels. After 24 h of incubation, NE, in concentrations of 108 to
107 M, further augmented contraction (p < 0.01 and p < 0.05, respectively) whereas 109 and 1010 M of NE did not
statistically differ from the control.
To determine whether the increased contractility induced by NE was the result of cell proliferation in the collagen gels, the amount of DNA was assayed fluorometrically. DNA content in the dissolved gels which is proportional to fibroblast cell number did not differ between the gels incubated with NE and the controls (data not shown).
Effect of 1-Protease Inhibitor on the Elastase-induced
Contraction of Collagen Gels
1-Protease inhibitor added alone did not affect gel contraction; however, the augmented contraction induced by NE was
completely abolished (Figure 4). Specifically, 58 nM (3 µg/ml)
or more of 1-protease inhibitor inhibited the elastase- (100 nM) induced contraction, whereas 38 nM (2 µg/ml) or less of
the inhibitor did not block the contraction induced by NE.
Functional elastase activity was detected in the dissolved gels
in which contraction had been augmented by NE. However,
the elastase activity decreased as a function of concentration
when 1-protease inhibitor was added to the gels. Importantly,
active elastase was undetectable when inhibition of the NE-
induced contraction had occurred, e.g., when 58 nM (3 µg/ml)
or more of 1-protease inhibitor had been added. The addition
of varying concentrations of 1-protease inhibitor demonstrated that inhibition of NE activity coincided with inhibition of gel contractile augmentation (Figure 4). Interestingly, the molar ratio when inhibition became complete was not at a 1:1
ratio of 1-protease inhibitor to elastase but at a ratio of 0.6:1, perhaps because some of the elastase in the commercially obtained material was inactive. Consistent with this, similar titration results were obtained quantifying functional elastase activity mixing NE and 1-protease inhibitor in test tubes without
collagen gels (data not shown). This result rules out the possibility that the collagen gels per se had inactivated the NE.
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Specificity of Inhibitors of NE on Fibroblast-mediated Contraction of Collagen Gels
PGE2, a well-known inhibitor of fibroblast-mediated gel contraction (19), inhibited both contraction of gels by control fibroblasts as well as contraction augmented by either NE or
TGF-1. In contrast, 1-protease inhibitor and a synthetic inhibitor of NE, L-680,833 (10), were able to inhibit NE-induced
contraction but did not have any effect on contraction induced
by TGF-1 (Table 2). The elastase activity in the dissolved gels
containing NE and NE + PGE2 was readily detectable. However, elastase activity was not detectable in gels treated with
1-protease inhibitor or L-680,833. The addition of these inhibitors alone did not affect contraction of control gels.
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Effect of Cocultured PMN
Coculture of fibroblasts and PMN significantly augmented fibroblast-mediated contraction of collagen gels in a time-dependent manner (Figure 5A). Gels containing both cell types contracted more, at all time points, than gels that only contained fibroblasts. Gels containing only PMN did not contract and did not differ from gels without cells.
To determine whether the decreased gel size in the coculture system was partly due to dissolution of the type I collagen, the amount of hydroxyproline was measured in the gels. Five days of coculture of fibroblasts and PMN in the three- dimensional gel significantly reduced the amount of hydroxyproline in the gels (Figure 5B). Interestingly, the reduction of hydroxyproline required coculture because gels containing only PMN or only fibroblasts had the same amount of hydroxyproline content as gels alone. Despite the decreased amount of hydroxyproline in the gels, the concentrations of hydroxyproline measured as amount in relation to the weight of the gel were higher in gels from the coculture system (Figure 5C).
Both 1-protease inhibitor and L-680,833 were able to
block the augmented contraction induced by PMN (Figure 6).
Gels with both cell types in coculture had detectable elastase
activity, whereas the activity was undetectable when the
elastase inhibitors were added to the culture system (data not shown).
Effect of PMN-conditioned Media
To assess the possibility that increased contractility in the coculture system was due to a soluble factor, conditioned media from cultured PMN were assayed for their ability to augment
fibroblast-mediated contraction of collagen gels. The conditioned media had the ability to augment gel contraction mediated by fibroblasts. Moreover, conditioned media from PMN
stimulated with the peptide FMLP augmented contraction
more than conditioned media from unstimulated PMN (Figure 7). FMLP alone did not affect the gel contraction. The addition of 1-protease inhibitor and L-680,833 partially inhibited this augmented contraction. Elastase activity was detected in the neutrophil-conditioned media but was undetectable when 1-protease inhibitor and L-680,833 were added
(data not shown) suggesting that neutrophil-conditioned medium contained an augmentory activity in addition to NE.
Effect of BALF on Fibroblast-mediated Gel Contraction
To determine if the in vitro effect was observable with a biological sample, BALF samples from seven patients with neutrophilia in the lower respiratory tract (Table 1) were assayed
for their ability to affect fibroblast-mediated gel contraction.
BALF from all seven patients augmented fibroblast-mediated
contraction of the collagen gels (Figure 8). When 1-protease
inhibitor was added to the system, the contraction was significantly (p < 0.05) less (in BALF from six of seven patients, one
did not differ) compared with gels without the inhibitor. The
addition of the synthetic inhibitor L-680,833 to the media also
resulted in a trend toward inhibition of contraction (BALF
from five of seven patients had less contraction), which did not
reach statistical significance (p = 0.154). These data thus suggest the presence of multiple factors in the BALF that can
augment contraction and further suggest that elastase may
contribute a small part of this activity. Functional elastase activity was detectable in three of the patients' BALF (Patients
1, 4, and 5); it was undetectable in the remaining four patients.
The patients were therefore divided into two groups, with or
without detectable elastase activity. As shown in Figure 9, the
addition of 1-protease inhibitor to gels incubated in BALF
with detectable functional elastase activity resulted in an inhibition of contraction that was significantly more than the inhibition induced by adding 1-protease inhibitor to BALF containing no detectable elastase activity (21.7% ± 6.0, mean ± SEM, versus 7.0% ± 2.7; p < 0.05).
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The same experiment was done with the synthetic inhibitor of NE, L-680,833. The addition of L-680,833 to gels incubated in BALF with detectable functional elastase activity resulted in a more pronounced inhibition compared with gels in which the synthetic inhibitor was added to BALF with no detectable elastase activity. However, the difference did not reach statistical significance (18.5% ± 3.3 versus 4.6% ± 6.7; p = 0.154).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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In the current study, we demonstrate that human NE can stimulate fibroblast-mediated contraction of released collagen gels. The increased contractility could be blocked by 1-protease
inhibitor and a synthetic inhibitor of human NE, L-680,833.
We were also able to show that coculture of fibroblasts and
neutrophils as well as conditioned media from cultured neutrophils can augment fibroblast-mediated collagen gel contraction which could be blocked by the inhibitors. Finally, we
showed that a biological fluid (BALF) contains augmentive
activity for fibroblast-mediated gel contraction and that this
activity can be partly blocked by elastase inhibitors.
Rearrangement of extracellular matrix may lead to altered tissue structure and tissue dysfunction (20, 21). Fibroblasts can generate a traction force thought to contribute to tissue remodeling (22). When fibroblasts are cultured in a three- dimensional collagen gel, they acquire a bipolar spindle-shaped form, more resembling in vivo conditions than conventional dish culture (9, 11, 25). When a collagen gel containing fibroblasts is detached immediately after gelation, the fibroblasts contract the gels over several days in serum-free medium. This model originally described by Bell and coworkers (12) has been designated "slow contraction" and is thought to mimic traction remodeling. In contrast, when fibroblasts are cultured without detachment for several days and then released, the fibroblasts contract the gels within hours (stress relaxation or "rapid" contraction) (8, 26). The rate of fibroblast contraction of collagen gels may vary with different fibroblast strains, collagen concentrations, cell number, and the presence of soluble mediators.
NE is the major protease released from neutrophils. It is a
29 kD serine protease stored in the azurophilic primary granules. The concentration of NE within the PMN has been estimated to be greater than 105 M (27). Thus the concentration
of NE in the immediate vicinity of the PMN is likely to be
high. NE has far-reaching effects on the environment. For instance NE is capable of degrading a wide range of extracellular matrix proteins, coagulation and complement factors as
well as immunoglobulins. Because neutrophils have an extremely high turnover and are believed to survive only 1 to 2 d
in the tissues, an effective protective screen is necessary. Thus,
the inhibition of NE activity in vivo occurs predominantly by
1-protease inhibitor with which it forms a 1:1 complex (28). In the current study, the contractile activity induced by 100 nM NE was totally inhibited by the addition of 58 nM 1-protease inhibitor, e.g., a ratio of approximately 0.6:1. The most
reasonable explanation for this finding is that all of the added
elastase was not active. We could confirm this finding in control experiments where 1-protease inhibitor and NE were
mixed in test tubes. This suggests that inactivation of NE in
the three-dimensional collagen gel was not occurring.
Both fibroblasts and neutrophils are capable of secreting a variety of proteases that potentially may interact with the type I collagen matrix. In the three-dimensional gel contraction assay, this may result in a smaller gel, i.e., the fibroblast-mediated "contraction" may in part be a result of a dissolution of the type I collagen gels. In the current study, gels without cells containing only NE maintained their original size during the culture period. Furthermore, the amounts of hydroxyproline, which corresponds to the collagen content (14) were, despite the significant contraction, unaltered in the gels containing NE, indicating a similar amount of collagen despite a smaller gel. However, due to a decreased gel size, the concentration of collagen was considerably higher in the gels contracted by NE. The results, however, do not completely rule out the possibility that some degradation has occurred because partially degraded collagen molecules, if trapped in the gel, would be in the hydroxyproline analysis and not measured as degradation.
On the other hand, in the coculture system with fibroblasts and neutrophils, the amount of hydroxyproline was lower in the contracted gels, indicating that part of the decreased gel size might be due to gel dissolution by various enzymes. Because the gels that only contained one cell type (fibroblasts or neutrophils) had an unaltered hydroxyproline content, we believe it is reasonable to assume that the enzymatic activity may originate from either cell type, but required coculture. The nature and impact of these enzymes on long-term culture requires further studies.
The inhibition of NE-induced contraction by 1-protease
inhibitor and L-680,833 was specific. Thus, 1-protease inhibitor and L-680,833 were only able to block contraction induced
by NE and did not have any effect on contraction induced by
TGF-1. In contrast, PGE2 was able to inhibit fibroblast-mediated contraction induced by both TGF-1 and NE as well as
contraction by control cells.
PMN, as well as NE, have the ability to stimulate fibroblasts and also have effects on extracellular matrix components. For example, when fibroblasts are exposed to PMN or PMN supernatants containing elastase, the ability of the fibroblasts to adhere to articular cartilage is enhanced (29). Also, it has previously been shown that fibroblasts embedded in collagen gels respond to elastase by increasing their migration through the matrix (29). These studies suggest that NE may alter fibroblast motility, e.g., when fibroblasts are exposed to NE they migrate more. However, the exact mechanisms by which NE augments fibroblast contraction remain to be defined. For example, it might be possible that it either acts directly on cells or on soluble mediators released by cells.
Neutrophils are rapidly recruited at the inflammatory site and can release a variety of proteolytic enzymes and oxygen radicals to the extracellular environment. In vivo, extravasated neutrophils are believed to undergo apoptosis and subsequently be ingested by local macrophages (30) or even fibroblasts (31). When neutrophils are cultured in monolayer, more than 50% of them have morphological features of apoptosis after 24 h of culture (32). In the present study, the neutrophils were cultured in a three-dimensional collagen gel for up to 5 d. It seems therefore reasonable to assume that a majority of the neutrophils in our assay system had undergone apoptosis after this culture period. However, the impact of the three-dimensional matrix model on neutrophil apoptosis merits further studies. While our separation method yielded a rather pure PMN population, we cannot completely exclude some contamination of mononuclear cells. However, because blood monocytes are able to attenuate fibroblast combination in a three-dimensional collagen gel (33), we believe that the augmentative activity originates from PMN.
In the present study, we demonstrate the 1-protease inhibitor and a synthetic inhibitor of NE, L-680,833, were able to
block the increased contractility induced by NE released in a
coculture system or from elastase containing conditioned media from cultured neutrophils. Moreover, we found that conditioned media from FMLP-stimulated cells contracted the gels
more than control conditioned media suggesting that the augmentory factors are released by degranulation of neutrophils.
However, the contractile activity was only partially blocked by
the inhibitors, suggesting the presence of multiple augmentory
factors including other proteases released by neutrophils in
this in vitro system. In addition, BALF from patients with neutrophilia in the lower respiratory tract augmented the fibroblast-mediated gel contraction. The contractile activity could
partially be blocked by 1-protease inhibitor although L-680,833
was less effective. This suggests a role for elastase as a potential mediator fibroblast contractile activity in vivo and raises
the possibility that other serine proteases are also active in this regard.
Chronic inflammatory disorders are often characterized by the presence of activated neutrophils. Previous workers have suggested that these neutrophils can contribute to the tissue disruption which often characterizes these disorders. The current study suggests another potential role for neutrophil products, namely altering the repair process. In this regard, tissue contraction is also a regular feature of the fibrosis associated with chronic inflammatory disorders. By contributing to augmented tissue contraction, NE may help drive this process. Other mediators are also known to modulate various aspects of tissue repair. The role of NE in vivo in disease states, therefore, must be viewed in the context of these other mediators. The mechanisms by which elastase might interact with these other mediators, moreover, will be important lines for future investigation.
In conclusion, the present study demonstrates that human
NE can augment fibroblast-mediated contraction of collagen
gels and that it specifically can be blocked by 1-protease inhibitor and a synthetic inhibitor of NE, L-680,833. The findings support the notion that excess products secreted by neutrophils in inflammatory disorders may lead to rearrangement
of extracellular matrix and can contribute to fibrosis and tissue dysfunction.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Stephen I. Rennard, M.D., Pulmonary and Critical Care Medicine Section, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-5300. E-mail: srennard{at}mail.unmc.edu
(Received in original form May 11, 1998 and in revised form November 10, 1998).
The work was supported in part by the Larson Endowment, University of Nebraska Medical Center, Omaha, Nebraska. Dr. C. M. Sköld is supported by grants from the Swedish Council for Work Life Research, the Swedish Heart-Lung Foundation, King Oscar II Jubilee Foundation, the Swedish Society of Medicine, the Karolinska Institute (Erik och Edith Fernströms stiftelse), Astra-Draco AB, and Glaxo-Wellcome AB.Acknowledgments: The authors are delighted to acknowledge Dr. James Anderson for assistance with the statistical analyses and Ms. Lillian Richards for assistance in the preparation of the manuscript.
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References |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1. Weibel, E. R., and R. G. Crystal. 1997. Structural organization of the pulmonary interstitium. In R. G. Crystal, J. B. West, E. R. Weibel, and P. J. Barnes, editors. The Lung: Scientific Foundations, 2nd ed. Lippincott-Raven, Philadelphia.
2. Brewster, C. E., P. H. Horwarth, R. Djukanovic, J. Wilson, S. T. Holgate, and W. R. Roche. 1990. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 3: 507-511 .
3. Roche, W. R., R. Beasley, J. H. Williams, and S. T. Holgate. 1989. Subepithelial fibrosis in the bronchi of asthmatics. Lancet i: 520-524 .
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