Synergistic neutrophil elastase-cytokine interaction degrades collagen in three-dimensional culture

Y. K. Zhu1, X. D. Liu2, C. M. Sköld3, T. Umino2, H. J. Wang2, J. R. Spurzem2, T. Kohyama2, R. F. Ertl2, and S. I. Rennard2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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-alpha , interleukin-1beta , and interferon-gamma ), 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). alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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)-alpha , interleukin (IL)-1beta , and interferon (IFN)-gamma , 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.


    METHODS
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INTRODUCTION
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-alpha , human recombinant IL-1beta , and human recombinant IFN-gamma were purchased from R&D Systems (Minneapolis, MN). alpha 1-Proteinase inhibitor (alpha 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-alpha , 5 ng/ml of IL-1beta , and 10 ng/ml of IFN-gamma ) (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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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-1beta , TNF-alpha , and IFN-gamma , 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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Fig. 1.   Effect of neutrophil elastase and cytomix on fibroblast-mediated collagen gel contraction. Fibroblasts were cast into collagen gels and cultured in serum-free DMEM alone or supplemented with 15 nM neutrophil elastase (NE), cytomix [10 ng/ml of tumor necrosis factor (TNF)-alpha , 5 ng/ml of interleukin (IL)-1beta , and 10 ng/ml of interferon (IFN)-gamma ], or a combination of NE and cytomix. Gel area was measured daily. Values are means ± SE for triplicate gels in a single representative experiment. The effect of cytomix, NE, and the combination was consistently observed in many experiments despite the fact that the actual amount of contraction in any given experiment was somewhat variable. See text for details.

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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Fig. 2.   Effect of NE and cytomix on collagen degradation in contracting collagen gels. Fibroblasts were cast into collagen gels containing NE, cytomix, or a combination of both. After 5 days, the gels and the medium in which they were floated were harvested, and hydroxyproline was measured as an index of collagen content in the gels and of the amount of collagen degraded in the medium. Values are means ± SE for 3 replicate gels/condition.

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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Fig. 3.   Collagen degradation in 3-dimensional gel culture: concentration dependence of NE. Fibroblasts were cast into collagen gels floating in culture medium supplemented with indicated concentrations of NE alone or in combination with cytomix. +, Presence; -, absence. Hydroxyproline content in the contracted gels was measured on day 3. Values are means ± SE for experiments performed in triplicate. *P < 0.0018 by Student's t-test with Bonferroni's correction.



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Fig. 4.   Effect of various cytomix concentrations on degradation of collagen in 3-dimensional gel culture in the presence of NE. Fibroblasts were cultured in 3-dimensional collagen gels and floated in culture medium containing indicated strengths of cytomix in the presence and absence of 15 µM NE. Cytomix 1, full strength; cytomix 2, 10% strength (1 ng/ml of TNF-alpha , 0.5 ng/ml of IL-1beta , and 1 ng/ml of IFN-gamma ); cytomix 3, 1% strength (0.1 ng/ml of TNF-alpha , 0.05 ng/ml of IL-1beta , and 0.1 ng/ml of IFN-gamma ). Values are means ± SE for 3 gels. *P < 0.0018 by Student's t-test with Bonferroni's correction.

To determine if increasing concentrations of cytomix would augment contraction in the presence of a constant concentration of neutrophil elastase, three preparations of cytomix (full strength, 10% strength, and 1% strength) were added to the medium surrounding the fibroblasts in floating gel culture, with and without neutrophil elastase, at a concentration of 15 nM (Fig. 4). After 3 days of culture, cytomix alone resulted in a 10.1 ± 0.1% reduction in hydroxyproline content in the gels. Cytomix (full strength) added to 15 nM elastase resulted in complete degradation of the collagen gels. Reducing the concentration of cytomix reduced the degree of collagen degradation, although at the lowest concentration tested (1% cytomix), collagen degradation was still relatively complete (47.6 ± 0.5%; P < 0.01 compared with full-strength cytomix). These data, therefore, show the concentration dependence of cytomix in the presence of neutrophil elastase.

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 alpha 1-PI. alpha 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, alpha 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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Fig. 5.   Effect of alpha 1-proteinase inhibitor (alpha 1-PI) on collagen degradation, induced by NE and cytomix, in 3-dimensional collagen gels. Fibroblasts were cast into 3-dimensional collagen gels and floated in culture medium. alpha 1-PI (100 nM) was added to the culture medium together with NE, cytomix, or the combination of NE and cytomix. Hydroxyproline was quantified in the contracted gels after 5 days of culture. Values are means ± SE for a single representative experiment incorporating triplicate gels for each condition. *P < 0.0018 by Student's t-test with Bonferroni's correction.

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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Fig. 6.   Effect of fibroblast number on degradation of collagen in 3-dimensional gel culture induced by NE and NE together with cytomix. Collagen gels were prepared with indicated concentrations of fibroblasts and floated in culture medium in the presence of NE or NE together with cytomix. -, Absence; +, presence; HFL, human fetal lung fibroblasts given as initial concentration of fibroblasts in the gel. After 4 days, gels were harvested and hydroxyproline was quantified in the contracted gels. Values are means ± SE for experiments performed in triplicate. *P < 0.0014 by Student's t-test with Bonferroni's correction.

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-alpha was the most potent cytomix component at augmenting degradation of collagen. The combination of TNF-alpha plus neutrophil elastase resulted in 71.7 ± 6.4% degradation (P < 0.01 compared with TNF-alpha alone). IL-1beta together with neutrophil elastase augmented degradation much less (37.8 ± 6.6%; P < 0.05 compared with IL-1beta alone). IFN-gamma 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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Fig. 7.   Effect of individual components of cytomix on NE-induced degradation of collagen in 3-dimensional collagen gel culture. Fibroblasts were cast into collagen gels and floated in medium containing the individual components of cytomix or cytomix either alone or in combination with NE. After 5 days, hydroxyproline was quantified in the contracted gels. Values are means ± SE for triplicate gels within a representative experiment.


                              
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Table 1.   Effects of cytokines and elastase on collagen degradation in contracted gels

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-alpha and IL-1beta both induced the production of MMP-9, although less potently than cytomix. IFN-gamma 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-alpha and IL-1beta . The effect of neutrophil elastase added in the presence of IFN-gamma was not apparently different from that of neutrophil elastase alone.


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Fig. 8.   Gelatin zymography. Gelatin zymography was performed as described in METHODS to identify matrix metalloproteinase (MMP)-2 and MMP-9 in the medium in which fibroblasts in floating collagen gel culture were incubated. Media were harvested after 5 days of culture in the presence of cytomix or the components of cytomix added either alone or in the presence of NE. Positive control was HT-1080 cell-cultured conditioned medium, which was similarly processed. CM, cytomix. Nos. at right, molecular mass.

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-alpha added alone and IL-1beta added alone also resulted in a marked increase in the 52-kDa band, although less prominently than with cytomix. IFN-gamma 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-alpha and cytomix but not in the presence of IL-1beta 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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Fig. 9.   Western blot identifying MMP-1 in medium from fibroblasts in 3-dimensional collagen gel culture. To confirm the presence of MMP-1, fibroblasts were cast into collagen gels and floated in culture medium with various supplements. Media were then subjected to SDS-PAGE followed by Western blotting with antibodies for MMP-1 (see METHODS for details). Nos. at right, molecular mass.

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-alpha and IL-1beta were both able to induce production of MMP-3, although less potently than cytomix did. IFN-gamma did not induce detectable levels of MMP-3. Neutrophil elastase added together with TNF-alpha or IL-1beta resulted in the conversion of detectable amounts to lower molecular mass forms.


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Fig. 10.   Western blot for MMP-3. Fibroblasts were cast into collagen gels and floated in culture medium containing CM or its individual components either alone or with the addition of NE. Media were then subjected to SDS-PAGE followed by Western blotting with antibodies specific for MMP-3 (see MATERIALS AND METHODS for details). Nos. at right, molecular mass.

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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Fig. 11.   Western blot for tissue inhibitor of metalloproteinase (TIMP)-1 (A) and TIMP-2 (B). Fibroblasts were cast into collagen gels and floated in culture medium containing NE, CM, and the two in combination. Media were then subjected to SDS-PAGE followed by Western blotting with antibodies specific for either TIMP-1 or TIMP-2. Nos. at right, molecular mass.

No TIMP-2 was detected in control conditions or in cells incubated in the presence of neutrophil elastase (Fig. 11B). Cytomix, however, resulted in the presence of a prominent band at ~72 kDa. Addition of neutrophil elastase in the presence of cytomix completely eliminated this band.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta , TNF-alpha , and IFN-gamma , 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-1beta and TNF-alpha were more effective at inducing MMP production and at augmenting collagen degradation in the presence of neutrophil elastase than IFN-gamma , 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 alpha 2beta 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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Table 2.   Effect of alpha 1-proteinase inhibitor on collagen degradation induced by cytomix with elastase

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-1beta and TNF-alpha 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-1beta and TNF-alpha 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 alpha 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-1beta , TNF-alpha , and IFN-gamma added together and IL-1beta and TNF-alpha 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.


    ACKNOWLEDGEMENTS

We acknowledge the assistance of Lillian Richards and Mary Tourek with preparation of the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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