1 Jincheng Hospital, Lanzhou 730050, China; 2 University of Nebraska Medical Center, Omaha, Nebraska 68198-5125; and 3 Karolinska Institute, S-171 76 Stockholm, Sweden
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ABSTRACT |
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Proteolytic degradation of
extracellular matrix is thought to play an important role in many lung
disorders. In the current study, human lung fibroblasts were cast into
type I collagen gels and floated in medium containing elastase, cytomix
(combination of tumor necrosis factor-, interleukin-1
, and
interferon-
), or both. After 5 days, gel collagen content was
determined by measuring hydroxyproline. Elastase alone did not result
in collagen degradation, but in the presence of fibroblasts, elastase
reduced hydroxyproline content to 75.2% (P < 0.01),
whereas cytomix alone resulted in reduction of hydroxyproline content
to 93% (P < 0.05). The combination of elastase and
cytomix reduced hydroxyproline content to 5.2% (P < 0.01).
1-Proteinase inhibitor blocked this synergy.
Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and
activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were
cleaved by elastase. We conclude that a synergistic interaction between
cytomix and elastase, mediated through cytokine induction of MMP
production and elastase-induced activation of latent MMPs and
degradation of TIMPs, can result in a dramatic augmentation of collagen
degradation. These findings support the notion that interaction among
inflammatory mediators secreted by mononuclear cells and neutrophils
can induce tissue cells to degrade extracellular matrix. Such a
mechanism may contribute to the protease-anti-protease imbalance in emphysema.
matrix metalloproteinases; collagen; fibroblasts; interleukin-1; tumor necrosis factor
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INTRODUCTION |
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PROTEOLYTIC DEGRADATION of extracellular matrix (ECM) is thought to play an important role in tissue remodeling. Such processes are likely important not only in the development of diseases such as pulmonary emphysema but also in the structural alterations that characterize alveolar and airway fibrosis in interstitial lung diseases and conditions such as chronic bronchitis and asthma. The mechanisms that lead to degradation of ECM are incompletely understood. Both serine proteases such as neutrophil elastase and metalloproteinases such as the matrix metalloproteinases (MMPs) have been suggested to play roles in a variety of lung disorders (6, 12, 21, 25, 28, 33, 36).
Tissue remodeling is a complex process involving not only the production and degradation of ECM but also its rearrangement. In this regard, one characteristic of tissue repair is contraction of the ECM. This contraction characterizes scar formation and is also present in most fibrotic conditions. The culture of fibroblasts in native type I collagen gels has been used to model this contractile process and to explore this aspect of tissue remodeling (1, 14).
The present study was designed to evaluate potential interactions
between inflammatory cytokines and the inflammatory mediator neutrophil
elastase. In this context, neutrophil elastase has been noted to
augment fibroblast contraction of three-dimensional collagen gels
(30), whereas proinflammatory cytokines, including tumor
necrosis factor (TNF)-, interleukin (IL)-1
, and interferon (IFN)-
, can inhibit this process (7, 40, 42). The
present study demonstrates that neutrophil elastase and the cytokines do not simply antagonize each other's effects but rather interact in a
potentially synergistic manner. Specifically, the cytokines can induce
the production of MMPs by fibroblasts. Neutrophil elastase can lead to
activation of the MMPs. In combination, therefore, cytokines and
elastase result in augmented degradation of ECM. The present study,
therefore, not only provides evidence for synergy between neutrophil
elastase and cytokines but suggests that the tissue remodeling
characteristic of many lung diseases may depend in large part on
interactions between neutrophil elastase and MMPs.
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METHODS |
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Materials.
Type I collagen was extracted from rat tail tendons by a previously
published method (10, 19). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were
carefully removed. After repeated washes with Tris-buffered saline and
95% ethanol, type I collagen was extracted in 4 mM acetic acid at
4°C for 24 h. Protein concentration was determined by weighing a
lyophilized aliquot from each lot of collagen solution. SDS-PAGE
routinely demonstrated no detectable proteins other than type I
collagen. Human neutrophil elastase was purchased from ECP (Owensville,
MO). Human recombinant TNF-, human recombinant IL-1
, and human
recombinant IFN-
were purchased from R&D Systems (Minneapolis, MN).
1-Proteinase inhibitor (
1-PI) was
purchased from Sigma (St. Louis, MO). Tissue culture supplements and
medium were purchased from GIBCO BRL (Life Technologies, Grand Island, NY). Fetal calf serum (FCS) was purchased from BioFluids (Rockville, MD).
Fibroblasts. Human fetal lung fibroblasts (HFL 1) were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in 100-mm tissue culture dishes (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 50 µg/ml of penicillin, 50 µg/ml of streptomycin, and 0.25 µg/ml of Fungizone. The fibroblasts were passaged every 3-5 days. Subconfluent fibroblasts were trypsinized (trypsin-EDTA; 0.05% trypsin, and 0.53 mM EDTA-4 Na) and used for collagen gel culture. Fibroblasts used in these experiments were between cell passages 16 and 19.
Preparation of collagen gels.
Collagen gels were prepared by mixing the appropriate amounts of rat
tail tendon collagen, distilled water, 4× concentrated DMEM, and cell
suspension so that the final mixture resulted in 0.75 mg/ml of
collagen, 4.5 × 105 cells/ml, and a physiological
ionic strength (19). Fibroblasts were always added
last to minimize damage during the preparation of the collagen gels.
The mixture (0.5-ml aliquots) was cast into each well of 24-well tissue
culture plates (Falcon, Franklin Lakes, NJ). Gelation occurred in ~20
min at room temperature, after which the gels were released and
transferred to 60-mm tissue culture dishes containing 5 ml of
serum-free DMEM and cultured at 37°C in 5% CO2 for
4-5 days. To demonstrate the effects of cytokines and elastase on
collagen gel contraction and collagen degradation, cytomix (10 ng/ml of
TNF-, 5 ng/ml of IL-1
, and 10 ng/ml of IFN-
)
(16), 15 nM elastase, or a combination of both was added to the culture medium. Gel area was measured daily with an image analysis system (Optimax V, Burlington, MA).
Hydroxyproline assay. Hydroxyproline, which is directly proportional to type I collagen content, was measured by spectrophotometric determination (2, 8). Briefly, the medium surrounding the gels was completely removed, and the gels were transferred to a glass tube (KIMAX, Fisher Scientific, St. Louis, MO) with 2 ml of 6 N HCl. O2 was removed by ventilation with N2 for 30 s. The gels were hydrolyzed at 110°C for 12 h. The samples were dried with a vacuum centrifuge and redissolved in distilled H2O before measurement. Hydroxyproline in the samples was reacted with oxidant (1.4% chloramine T in acetate-citric acid buffer; Sigma) and Ehrlich's reagent (0.4% p-dimethylaminobenzaldehyde; Sigma) in 60% perchloric acid (Fisher Chemical, Fair Lawn, NJ) at 65°C for 25 min, and hydroxyproline content was determined by spectrophotometer at 550 nm (DU 640, Beckmann).
Gelatinase activity assay. To investigate the activity of gelatinase, gelatin zymography was performed. The supernatant-conditioned media were concentrated 10-fold by lyophilization and dissolved in distilled water. Gelatin zymography was performed with a modification of a previously published procedure (17, 41). Samples were dissolved in 2× electrophoresis sample buffer (0.5 M Tris · HCl, pH 6.8, 10% SDS, 0.1% bromphenol blue, and 20% glycerol) and heated for 5 min at 95°C. Forty microliters of each sample were then loaded into each lane, and electrophoresis was performed at 45 mA/gel. After electrophoresis, the gels were soaked with 2.5% (vol/vol) Triton X-100 and gently shaken at 20°C for 30 min. After this, the gels were incubated in the metalloproteinase buffer (0.06 M Tris · HCL, pH 7.5, containing 5 mM CaCl2 and 1 µM ZnCl2) for 18 h at 37°C. The gels were then stained with 0.4% (wt/vol) Coomassie blue and rapidly destained with 30% (vol/vol) methanol, and 10% (vol/vol) acetic acid.
Immunoblot analysis of metalloproteinases. To confirm the identity of the MMPs that were produced and activated, immunoblots were performed. The supernatants from three-dimensional cultures were precipitated with 50% (vol/vol) ethanol, resuspended in equal volumes of distilled water and 2× sample buffer (0.5M Tris · HCl, pH 6.8, 10% SDS, 0.1% bromphenol blue, and 20% glycerol). After being heated for 3 min at 95°C, 30 µl of each sample were loaded into wells for electrophoresis. The proteins were transferred in electroblotting buffer (20 mM Tris, pH 8.0, 150 mM glycine, and 20% MeOH) at 20 V for 35 min. The blots were blocked in 5% nonfat milk in PBS-Tween at room temperature for 1 h and were then exposed to primary antibodies [MMP-1, MMP-3, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2; Calbiochem, Cambridge, MA] for 1 h and subsequently developed with the use of rabbit anti-mouse IgG horseradish peroxidase (Rockland, Gilbertsville, PA) in conjunction with an enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK).
Statistical evaluation. The results of the gel contraction assay, hydroxyproline measurements, and zymograms were confirmed by repeating the experiments on separate occasions at least three times. Data were generally taken from single representative experiments and are expressed as means ± SE of the three replicate determinations unless described otherwise. Group data were evaluated by analysis of variance (ANOVA). Differences between paired samples that appeared statistically significant were analyzed by Student's t-test with Bonferroni's correction.
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RESULTS |
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Effect of neutrophil elastase and cytomix on fibroblast-mediated
collagen gel contraction.
Under control culture conditions, fibroblasts contracted collagen gels.
In five separate experiments, each performed in triplicate, gel size
was reduced to 62.1 ± 2.5% after 1 day of culture and 41.9 ± 2.7% of the control level after 5 days in culture
(P < 0.01) compared with original size. Cytomix, the
combination of IL-1, TNF-
, and IFN-
, consistently inhibited
the contraction. Over the course of the five experiments, gel size was
reduced to 82.6 ± 2.0% of control size after 1 day and 75.5 ± 2.6% of control size after 5 days (P < 0.01 compared with fibroblasts alone). Neutrophil elastase consistently
augmented fibroblast-mediated collagen gel contraction. Over the course
of all five experiments in the presence of 15 nM neutrophil elastase,
fibroblasts contracted to 41.3 ± 4.5% of original size after 1 day and 11.7 ± 1.7% of original size after 5 days
(P < 0.01 compared with control fibroblasts). When
neutrophil elastase and cytomix were added concurrently, the degree of
contraction after 1 day (56.2 ± 1.6% of original size) was
intermediate between the augmented contraction observed with neutrophil
elastase alone and the inhibited contraction observed with cytomix
alone. Beyond 1 day, however, the rate of contraction in cells
incubated with cytomix and elastase concurrently accelerated such that
by day 5, the gels had contracted, on average for all experiments, to 2.9 ± 0.5% of original size. This was
significantly greater than the contraction seen with fibroblasts alone
(P < 0.01) and was also greater than that observed
with fibroblasts incubated in the presence of neutrophil elastase alone
(P < 0.01). An example of a representative experiment
is shown in Fig. 1.
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Effect of neutrophil elastase and cytomix on degradation of
collagen in three-dimensional collagen gel culture.
To estimate whether degradation of collagen was taking place during
incubations with neutrophil elastase and/or cytomix, hydroxyproline was
measured both in the contracted gels and in the surrounding medium.
Control fibroblasts cultured over 5 days resulted in 8.0 ± 0.4%
of the hydroxyproline being recovered in the surrounding medium. Over
the 5 days of culture, neutrophil elastase resulted in an increase in
solubilization of hydroxyproline, with 27.0 ± 1.9% of the
hydroxyproline recovered in the surrounding medium (P < 0.05 compared with control cultures). In contrast, neutrophil elastase added alone in the absence of fibroblasts did not result in
solubilization of collagen (see Concentration dependence of neutrophil elastase and cytomix in combination on collagen degradation in three-dimensional gel culture). Cytomix added alone to
fibroblasts in three-dimensional collagen gels also increased collagen
degradation slightly, with 18.1 ± 1.8% of the hydroxyproline
recovered in the surrounding medium (P < 0.05). In
contrast with the modest degradation observed with either neutrophil
elastase or cytomix alone, the combination of the two resulted in
75.5 ± 0.5% of the hydroxyproline being recovered in the
surrounding medium (P < 0.01 compared with both
neutrophil elastase alone and cytomix alone). The increase in
hydroxyproline recovered in the surrounding medium was exactly matched
by a decrease in the hydroxyproline recovered in the contracted gel
assayed after 5 days (Fig. 2). In seven
separate experiments, each performed in triplicate, the reduction in
hydroxyproline contained within the gel was consistently observed; in
contrast to control cultures, neutrophil elastase alone reduced the
hydroxyproline content to 75.2 ± 3.4%, cytomix reduced collagen
content to 92.9 ± 4.4%, and the two together reduced the
hydroxyproline content of the gels to 5.2 ± 3.4%
(P < 0.01 compared with either cytomix or neutrophil
elastase alone).
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Concentration dependence of neutrophil elastase and cytomix in
combination on collagen degradation in three-dimensional gel culture.
To determine if the interaction between neutrophil elastase and cytomix
with regard to collagen degradation in three-dimensional collagen gel
culture was concentration dependent, various concentrations of elastase
(Fig. 3) and cytomix (Fig.
4) were added to the medium in which the
fibroblasts cultured in floating collagen gels were incubated.
Neutrophil elastase added alone caused a concentration-dependent increase in collagen degradation over a 3-day culture period. In the
presence of cytomix, there was also a concentration-dependent increase
in collagen degradation, but the effect was markedly steeper (Fig. 3).
Over the concentration range tested, it was not possible to observe a
minimal effect concentration for the neutrophil elastase. However, 40 nM elastase resulted in complete degradation of the collagen gels in
the presence of cytomix, which constituted a maximal response. In
contrast, only 25.8 ± 0.6% degradation took place with 40 nM
elastase in the absence of cytomix. Thus, although these data do not
permit calculation of a 50% effective concentration, they clearly
demonstrate concentration dependence of neutrophil elastase for
degradation of collagen in three-dimensional gel culture both in the
absence and, more markedly, in the presence of cytomix.
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Role of active elastase.
To determine if neutrophil elastase activity was required for the
augmentation of collagen degradation in three-dimensional collagen gel
culture, experiments were done in the presence of 1-PI.
1-PI added alone did not affect collagen degradation
(Fig. 5). In contrast, the degradation
induced by elastase alone was completely blocked, and the augmentation
of collagen degradation induced by elastase in the presence of cytomix
was markedly attenuated. Interestingly,
1-PI, although
it blocked the partial degradation that occurred in the presence of
neutrophil elastase alone, had no effect on the slight degradation that
was consistently observed in the presence of cytomix alone.
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Effect of fibroblast number on neutrophil elastase- and
cytomix-induced degradation of collagen in three-dimensional collagen
gel culture.
To determine the role played by fibroblasts in the collagen degradation
induced by neutrophil elastase and cytomix, collagen gels were prepared
without fibroblasts and with varying initial numbers of fibroblasts.
These gels were then cultured in the presence of neutrophil elastase
alone, cytomix alone, and a combination of both. In the absence of
fibroblasts, neither neutrophil elastase alone nor neutrophil elastase
added together with cytomix induced any degradation of the collagen
gels (Fig. 6). In contrast, with the
addition of fibroblasts to the gels, neutrophil elastase resulted in
some degradation of collagen within the gels over the 3-day incubation
period. In the presence of neutrophil elastase, 1 × 105 fibroblasts/ml resulted in 10.9 ± 0.3%
degradation of collagen (P < 0.05). Fibroblasts
(6 × 105/ml) resulted in 24.9 ± 1.3%
degradation (P < 0.01 compared with no cells and
P < 0.05 compared with 105
fibroblasts/ml). In the presence of neutrophil elastase and cytomix, there was much more marked degradation of collagen in the gels, which
was strikingly dependent on fibroblast concentration. Fibroblasts (1 × 105/ml) resulted in 48.7 ± 1.6%
degradation (P < 0.01 compared with control gels), and
6 × 105 fibroblasts/ml resulted in complete
degradation of the collagen gels over a 3-day time period (Fig. 6).
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Effect of the individual cytokines contained within cytomix on the
degradation of collagen in three-dimensional collagen gels alone and in
the presence of neutrophil elastase.
To determine which components of cytomix were responsible for the
augmented degradation of collagen observed in the presence of
neutrophil elastase, fibroblasts were cast into collagen gels and
floated in culture medium containing either cytomix or the same
concentration of the individual cytokines contained in cytomix, either
alone or in the presence of neutrophil elastase. Over a 5-day time
course, neutrophil elastase alone resulted in a 24.8 ± 3.6%
degradation of collagen within the gels. Cytomix added together with
neutrophil elastase resulted in near-complete degradation of the
collagen. TNF- was the most potent cytomix component at augmenting
degradation of collagen. The combination of TNF-
plus neutrophil
elastase resulted in 71.7 ± 6.4% degradation (P < 0.01 compared with TNF-
alone). IL-1
together with neutrophil
elastase augmented degradation much less (37.8 ± 6.6%;
P < 0.05 compared with IL-1
alone). IFN-
added
together with neutrophil elastase did not result in an augmentation of
degradation compared with neutrophil elastase alone (27.0 ± 9.1%; P > 0.05). This effect of augmented degradation
in the presence of neutrophil elastase was paralleled by the effects of
the cytokines added alone in the absence of neutrophil elastase (Fig.
7; Table
1).
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Effect of cytomix and neutrophil elastase on MMP-2 and MMP-9
production by fibroblasts in three-dimensional collagen gel culture.
MMP-2 and MMP-9 released by fibroblasts in three-dimensional gel
culture in the presence of cytomix, the individual components contained
within cytomix, and neutrophil elastase were assessed by gelatin
zymography (Fig. 8). Under control
conditions, fibroblasts primarily released MMP-2 into the
surrounding medium. This was identified by gelatinase activity with the
characteristic molecular masses of 72 and 66 kDa and confirmed
by Western blot (data not shown). As noted in Fig. 8, the
majority of the MMP-2 appeared to be in the latent 72-kDa form.
Neutrophil elastase appeared to convert some of the latent MMP-2 to a
lower molecular mass form of 66 kDa, corresponding to the active
form of MMP-2. In the presence of cytomix, there was marked induction
in the release of MMP-9, identified by its gelatinase activity and its
characteristic 92-kDa size. MMP-9 was also confirmed by Western
blotting (data not shown). Neutrophil elastase added together with
cytomix converted the 92-kDa form to an 83-kDa form that corresponded
in size to active MMP-9. TNF- and IL-1
both induced the
production of MMP-9, although less potently than cytomix. IFN-
was
not obviously different from control cultures with regard to MMP-2 and
MMP-9 induction. In the presence of neutrophil elastase, lower
molecular mass forms corresponding to the activated forms of MMP-2 and
MMP-9 were observed in the presence of TNF-
and IL-1
. The effect
of neutrophil elastase added in the presence of IFN-
was not
apparently different from that of neutrophil elastase alone.
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Effect of cytomix and neutrophil elastase on MMP-1.
To determine if MMP-1 was induced and/or activated by cytomix either
alone or together with neutrophil elastase, Western blotting was
performed (Fig. 9). Control conditions
produced small amounts of material that migrated with an apparent
molecular size of 52 kDa. Neutrophil elastase added alone resulted in
no marked change in this band. The addition of cytomix resulted in a
marked increase in release of this band. TNF- added alone and
IL-1
added alone also resulted in a marked increase in the 52-kDa
band, although less prominently than with cytomix. IFN-
caused a
marginal change from control values. In the presence of neutrophil
elastase, a small but readily detectable amount of material was
detected at 42 kDa in the presence of TNF-
and cytomix but not in
the presence of IL-1
alone. In the presence of cytomix, neutrophil
elastase also resulted in the presence of a 20-kDa band corresponding
completely in size to the active form of MMP-1.
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Effect of cytomix and neutrophil elastase on induction of MMP-3 in
fibroblasts in three-dimensional collagen gel culture.
To determine if cytomix or its components induced MMP-3 and to
determine if neutrophil elastase could potentially activate MMP-3,
Western blotting with antibodies to MMP-3 was performed (Fig.
10). In control cultures, no detectable
MMP-3 was observed. Neutrophil elastase had no effect when added alone.
Cytomix potently induced the production of MMP-3 as evidenced by a
prominent 57-kDa band. Smaller amounts of lower molecular mass forms
were also present with cytomix alone. Neutrophil elastase added
together with cytomix, however, increased the prominence of these lower molecular mass forms. TNF- and IL-1
were both able to induce production of MMP-3, although less potently than cytomix did. IFN-
did not induce detectable levels of MMP-3. Neutrophil elastase added
together with TNF-
or IL-1
resulted in the conversion of
detectable amounts to lower molecular mass forms.
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Effect of cytomix and neutrophil elastase on TIMP-1 and TIMP-2 in
fibroblasts in three-dimensional collagen gel culture.
To determine if TIMP-1 and TIMP-2 were induced by cytomix and cleaved
by elastase, Western blot analysis was performed (Fig. 11) and further confirmed by reverse
zymography (data not shown). Under control conditions, the medium from
fibroblasts in three-dimensional collagen gels produced a prominent
band at 30 kDa that corresponded to free TIMP-1 (Fig. 11A).
Less prominent bands were observed at ~58 kDa, corresponding to
TIMP-1-MMP-3 and -1 complexes. Neutrophil elastase added to fibroblasts
in three-dimensional collagen gel culture resulted in a decrease in
both free TIMP-1 and TIMP-1 at higher molecular masses. Cytomix added
to fibroblast cultures resulted in a slight but readily detectable
increase in observable TIMP-1 both free and complexed with MMPs. The
addition of neutrophil elastase in the presence of cytomix resulted in
a reduction in stainable TIMP-1 at all bands.
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DISCUSSION |
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The present study demonstrates synergistic interactions between
inflammatory mediators with regard to tissue remodeling. Specifically, fibroblasts cultured in three-dimensional collagen gels are able to
contract these gels, a system that has been used to model both normal
wound healing and fibrosis (1, 14). Neutrophil elastase can augment this contractile process (30), a result
confirmed in the current study. The cytokines IL-1, TNF-
, and
IFN-
, alone and combined as cytomix, can inhibit this process. When
added together, however, after an initial delay, neutrophil elastase added together with cytomix results in a marked contraction of collagen
gels. This contraction is associated with a marked degradation of the
collagen within the gel. The current study further demonstrates that
this augmented degradation is due to induction of production of MMPs by
the fibroblasts in culture as a result of their exposure to cytomix and
by conversion of these MMPs by neutrophil elastase to lower molecular
mass species that correspond to their active forms. Of the components
of cytomix, IL-1
and TNF-
were more effective at inducing MMP
production and at augmenting collagen degradation in the presence of
neutrophil elastase than IFN-
, but the combination was more
effective than any individual component.
The culture of fibroblasts in three-dimensional collagen gels has been
utilized as an in vitro system to evaluate tissue repair and remodeling
(1, 14). When cultured in three-dimensional gels composed
of native type I collagen, fibroblasts orient themselves along the
collagen fibers. Both fibroblast proliferation and protein production
in three-dimensional collagen gel culture differ markedly from those in
routine tissue culture conditions (1). Through interactions that depend in part on
2
1-integrins, fibroblasts can exert a
tensile force on the collagen fibers. If the gels are unrestrained, for
example in floating gel culture, the fibroblasts cause the gels to
contract. This contraction can be modified by a variety of exogenous
agents, which can either stimulate or inhibit collagen gel contraction
(7, 30, 34, 40, 42).
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In vivo, tissue remodeling is a complex process. Fibrous connective tissue is produced in both normal wound healing and in fibrotic disease. In addition, both newly synthesized and preexisting ECM can be degraded. Although the mechanisms that regulate the degradation of extracellular collagen are incompletely understood, it is likely that MMPs play a prominent role.
The MMPs are a complex family of proteins (4, 39). More
than 20 members have been identified. All share the structural similarity of a metal ion at the active site. They differ, however, in
their mechanisms of activation and in their substrate specificity. Many
MMPs are capable of degrading components of the ECM, hence the class
name. Together they have been demonstrated to degrade all components of
the extracellular milieu (4, 27, 39). Other studies
(3, 18, 35) have demonstrated that cytokines are capable
of inducing MMPs by fibroblasts in routine tissue culture. The current
study has confirmed these results and demonstrated that fibroblasts in
three-dimensional collagen gel culture can produce MMP-1, -2, -3, and
-9. It is likely that other MMPs are also produced by fibroblasts in
this culture system, and these additional MMPs may also play a role in
connective tissue turnover. The current study also demonstrates that
the production of MMP-1, -3, and -9 is markedly induced in
three-dimensional culture by the addition of cytomix as well as by
IL-1 and TNF-
added alone.
The catalytic activity of the MMPs is regulated at multiple levels
including transcription, secretion, activation, and inhibition. A
critical step in the control of MMP activity is regulated by the
generation of active enzyme species with proteolytic activity from
latent precursors. Several proteases can serve to activate the
proteolytic cascade, which can lead to degradation of ECM. In this
regard, several MMPs including, membrane-type MMPs, are constitutively
active and are capable of activating MMP-2 (23). In
addition, MMP-3, when activated, can activate MMP-2 and -9 (24). Finally, serine proteases are also capable of
activating several of the MMPs. The MMPs produced in response to
cytokines in the present study were observed in sizes that corresponded to their latent forms. Neutrophil elastase had no effect on MMP production. Elastase did, however, have a clear effect in converting the MMPs to lower molecular mass forms that corresponded to active MMPs. Thus the current study supports the concept that cytokines such
as IL-1 and TNF-
can induce the production of MMPs but that
maximal collagen degradation is achieved only in the presence of an
activator such as neutrophil elastase.
The MMPs can also be regulated by the presence of inhibitors of MMPs. As a class, these are referred to as TIMPs (5). Four TIMPs have been described. The current study demonstrates production of TIMP-1 and TIMP-2 by fibroblasts in collagen gel culture. Interestingly, TIMP-2 was detected only after the addition of cytomix, and cytomix also augmented the production of TIMP-1. Neutrophil elastase, in addition to converting the MMPs to sizes corresponding to their active forms, also appeared to cleave TIMP-1 and TIMP-2. Thus neutrophil elastase may result in activation of MMPs both by converting latent MMPs to their active forms and by eliminating the naturally occurring inhibitors of MMPs.
Multiple mechanisms exist for activation of MMPs. In particular, they can be activated by a number of endopeptidases (20). In turn, active MMP-3 is capable of activating MMP-1, MMP-8, and MMP-9 (29, 32). It is likely, therefore, that proteolytically activated cascades play an important role in activation of MMP-induced degradation of ECM. The current study supports a role for neutrophil elastase as an activator of this cascade.
In this context, Okada and colleagues (22) could not
observe activation of MMP-9 by neutrophil elastase, whereas Ferry et al. (11) did observe activation. In the current study,
neutrophil elastase added to fibroblasts in three-dimensional collagen
gel culture in the presence of cytomix resulted in conversion of MMP-9 to a species corresponding to its active form. Although this result is
consistent with the results of Ferry et al. (11), we did not observe direct activation of MMP-9 by neutrophil elastase (data not
shown). It is possible that generation of active MMP-9 by neutrophil
elastase was indirect and depended on a sequence of proteolytic events.
That proteolytic cleavage was required, however, is supported by the
fact that the addition of 1-PI to the neutrophil
elastase blocked activation.
Aminophenylmercuric acid (APMA) is an activator of MMPs (13). Addition of APMA (1 mM) to control collagen gels after 2 days of culture resulted in a 12.5 ± 0.6% degradation of collagen. In contrast, addition of APMA to cytokine-stimulated gels resulted in 100% degradation that took place over 6 h (data not shown). Elastase, therefore, is not the only mechanism by which MMP activation can lead to collagen degradation. In addition, the relatively slow collagen degradation that results from the addition of elastase to cultures suggests that elastase may be activating cellular processes rather than directly activating the MMPs, the presumed mechanism of action of APMA.
Tissue remodeling is a complex process. Altered tissue remodeling can play an important role in many pathophysiological processes in which altered tissue structure leads to altered function. In the lung, tissue remodeling plays a prominent role in many disorders including pulmonary fibrosis, pulmonary emphysema, chronic bronchitis, and asthma. All these conditions are now recognized as chronic inflammatory disorders. Interestingly, a role for both MMPs and neutrophils has also been suggested in all these conditions (6, 12, 25, 28, 36-38).
Many studies demonstrate close interactions between inflammatory
mediators and the remodeling process (9, 31). The current study demonstrates synergy between the inflammatory protease neutrophil elastase and the cytokines IL-1, TNF-
, and IFN-
added together and IL-1
and TNF-
added individually. Because it is likely that both acute inflammatory processes and chronic inflammatory conditions are associated with the concurrent production of neutrophil elastase together with these cytokines, the synergistic interactions described in the current study may play an important role in determining the
structural consequences of inflammatory processes.
Several studies have evaluated the relationship between collagen content and contraction of collagen gels. Gels prepared with increasing concentrations of collagen are contracted less by fibroblasts (26, 43). The augmented contraction observed in the current study after an initial delay is consistent with these results. That is, at the time when collagen degradation is presumably reducing the content of collagen within the gels, collagen gel contraction is accelerating. Because contraction of collagen gels is associated with apoptosis of fibroblasts (15), the synergistic interaction leading to increased collagen degradation could be a crucial mechanism for removal of fibrotic tissues; increased degradation of collagen is associated with augmented contraction, which, in turn, is associated with apoptosis and, hence, removal of fibroblasts.
Whether tissue injury and inflammation are followed by effective repair with restoration of normal function or whether they are followed by abnormal repair with consequent remodeling and loss of function is crucially important in lung disorders. The current study demonstrates that synergistic interactions between proinflammatory cytokines and the inflammatory mediator neutrophil elastase can lead to degradation of extracellular collagen and can augment contraction in an in vitro model system. It is likely that such synergistic interactions contribute to tissue remodeling in inflammatory lung diseases as well.
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ACKNOWLEDGEMENTS |
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We acknowledge the assistance of Lillian Richards and Mary Tourek with preparation of the manuscript.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant R01-HL-64088-02.
Address for reprint requests and other correspondence: S. I. Rennard, Pulmonary and Critical Care Medicine, Univ. of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, NE 68198-5125 (E-mail: srennard{at}unmc.edu).
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. Section 1734 solely to indicate this fact.
Received 13 October 2000; accepted in final form 6 June 2001.
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