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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 487-494, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4818

Phosphodiesterase 4 Inhibitor Cilomilast Inhibits Fibroblast-Mediated Collagen Gel Degradation Induced by Tumor Necrosis Factor-{alpha} and Neutrophil Elastase

Tadashi Kohyama, Xiangde Liu, Yun Kui Zhu, Fu-Qiang Wen, Hang Jun Wang, Qiuhong Fang, Tetsu Kobayashi and Stephen I. Rennard

Department of Respiratory Medicine, University of Tokyo, Tokyo, Japan; University of Nebraska Medical Center, Omaha, Nebraska; Department of Respiratory Diseases, Jincheng Hospital, Lanzhou, China; and Mount Sinai Hospital, Toronto, Ontario, Canada

Address correspondence to: Stephen I. Rennard, M.D., Pulmonary and Critical Care Medicine, University of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, NE 68198-5125. E-mail: srennard{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tissue destruction, resulting in emphysema, can be a consequence of several pathologic processes. The current study evaluated the effects of the phosphodiesterase (PDE)4 inhibitor, cilomilast, and other PDE inhibitors on the ability of fibroblasts to degrade extracellular matrix. Using the three-dimensional collagen gel culture system, fibroblasts (HFL-1) were cultured with tumor necrosis factor (TNF)-{alpha}, known to induce matrix metalloproteinase (MMP) release, and/or neutrophil elastase (NE), which can induce MMP activation. On Day 4, gels containing TNF-{alpha} and NE were significantly degraded (20.8 ± 2.9% of original collagen content). Cilomilast (10 µM) inhibited this degradation (84.4 ± 8.4%). Amrinone, a PDE3 inhibitor, and zaprinast, a PDE5 inhibitor, had no effect. Gelatin zymography and immunoblotting revealed that fibroblasts cultured with TNF-{alpha} released increased amounts of latent MMP-1 and -9. The addition of NE resulted in the conversion of MMP-1 and -9 to their active forms, indicative of collagen degradation. Cilomilast inhibited the release of MMP-1 and -9, as well as conversion of MMP-1 to its active form. Using real-time PCR analysis, cilomilast's effect on MMP-1 release was not associated with the proteinase's mRNA expression, suggesting that the inhibition of release is regulated at the post-transcriptional level. These results suggest that cilomilast may be a potentially effective therapeutic agent in diseases characterized by excessive tissue destruction, such as emphysema.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • extracellular matrix, ECM • enzyme-linked immunosorbent assay, ELISA • fetal calf serum, FCS • human fetal lung fibroblasts, HFL-1 • horseradish peroxidase, HRP • matrix metalloprotease, MMP • neutrophil elastase, NE • polyacrylamide gel electrophoresis, PAGE • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • phosphodiesterase, PDE • reverse transcription, RT • rat tail tendon collagen, RTTC • sodium dodecyl sulfate, SDS • tissue inhibitor of metalloprotease, TIMP • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tissue architecture is altered both during normal wound healing and in many pathologic processes. The mechanisms that regulate remodeling of tissue structure, therefore, are likely important targets for therapeutic interventions in diseases which are characterized by altered tissue structure. Pulmonary emphysema, for example, is characterized by destruction of alveolar walls (1, 2). The action of enzymes with elastolytic activity has been thought important in leading to tissue destruction (1). Some evidence, however, suggests that enzymes with collagenolytic activity may also play a role (3). It is likely that multiple enzymes are involved. In this regard, recent studies suggest that there may be collaborative interactions among serine proteases and the matrix metalloproteases (MMPs) leading to degradation of extracellular matrix (ECM) (4).

The phosphodiesterases (PDEs) are a large family of intracellular enzymes that degrade cyclic nucleotides. Because of their potential for altering a variety of cellular responses, PDEs are appealing targets for therapeutic development (5, 6). PDE4 has been targeted because of its role in regulating both airway smooth muscle and inflammatory cells (7–9). PDE4 inhibitors have been suggested to affect fibroblast chemotaxis and contraction of ECM (10), suggesting that these agents may also be able to affect tissue remodeling.

The current study, therefore, was designed to determine if the PDE4 inhibitor cilomilast could function to modulate ECM degradation. Fibroblasts were cultured in three-dimensional collagen gels in the presence of tumor necrosis factor-{alpha} (TNF-{alpha}), to induce MMP release, and neutrophil elastase (NE), which leads to activation of the MMPs. The ability of cilomilast to inhibit the degradation of collagen was then directly assessed in this in vitro system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human fetal lung fibroblasts (HFL-1) were purchased from the American Type Culture Collection (Rockville, MD). Human NE was purchased from Elastin Products Co. (Owensville, MO). NE was dissolved in phosphate-buffered saline (PBS) to a stock solution of 1 mg/ml. Human recombinant TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN) and dissolved in PBS to a stock solution of 10 µg/ml. Cilomilast (a kind gift from GlaxoSmithKline, King of Prussia, PA), amrinone, and zaprinast (both Sigma, St. Louis, MO) were dissolved in dimethylsulfoxide (Sigma) to a stock solution of 10 mM. Preliminary MTT experiments demonstrated that concentrations of drugs and the solvents ethanol and dimethylsulfoxide used in this study did not show any significant cytotoxicity in fibroblasts (data not shown).

Perchloric acid, normal propanol, and methanol were purchased from Fisher Chemical (Springfield, NJ). Glycine, tris, and all other supplements for polyacrylamide gel electrophoresis (PAGE) were purchased from Bio-Rad Laboratories (Hercules, CA). Medium and other supplements for cell culture were purchased from Invitrogen (Life Technologies, Grand Island, NY), except for fetal calf serum (FCS), which was purchased from Biofluid (Rockville, MD).

Type I Collagen
Type I collagen from rat tail tendons (RTTC) was extracted according to previously published methods (11, 12). Briefly, tendons were excised, and the tendon sheath and other connective tissues were removed carefully. After repeated washing with tris-buffered saline (TBS; 0.9% NaCl, 10 mM Tris, pH 7.5) and 95% ethanol, type I collagen was extracted in 6 mM hydrochloric acid at 4°C for 24 h. Protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecyl sulfate (SDS)-PAGE routinely demonstrated no detectable proteins other than type I collagen.

Cell Cultures
Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.25 µg/ml fungizone, and incubated with 5% CO2 at 37°C. Subconfluent fibroblasts were trypsinized (trypsin-EDTA; 0.05% trypsin, 0.53 mM EDTA-4 Na) and washed twice with DMEM to rid cells of trypsin before use in collagen gel culture.

Preparation of Collagen Gels
Collagen gels were prepared as described previously (12). Briefly, the appropriate amount of RTTC was centrifuged with distilled water, 4x concentrated DMEM, and fibroblasts so that the final mixture resulted in 0.75 mg/ml of collagen, 1x DMEM, and 5 x 105 cells/ml. Fibroblasts were routinely added last to minimize damage during the preparation of collagen gels. One-half milliliter of the mixture was cast into each well of 24-well tissue culture plates (Falcon, Becton-Dickinson Labware, Lincoln Park, NJ) for ~ 20 min at room temperature. Gels were released and transferred to 60-mm tissue culture dishes containing 5 ml of serum-free DMEM and cultured with 5% CO2 at 37°C for 4–5 d. To stimulate fibroblast-mediated degradation of collagen gels, TNF-{alpha} (10 ng/ml), NE (15 nM), or the combination of both were added to culture media in which gels were floated. To investigate the effect of PDE inhibitors on gel contraction and collagen degradation, PDE inhibitors (10 µM) were added to media containing NE, TNF-{alpha}, or the combination of both. Gel area was measured daily using an image analysis system (Optomax, Burlington, MA).

Hydroxyproline Assay
Spectrophotometry was used to measure hydroxyproline, which is directly proportional to the collagen content of the gels (13). Briefly, the media surrounding gels were completely removed, and the gels were transferred to Eppendorf tubes. Gels were dissolved with collagenase (0.25 mg/ml, 50 µl/gel) for 2–4 h, centrifuged at 1,000 x g for 5 min, and the supernatant was harvested for hydroxyproline assay. Aliquots of 20 µl were mixed with 30 µl of 3.3 N NaOH so that the final concentration of NaOH was 2 N, and samples were hydrolyzed by autoclaving at 120°C for 20 min. Afterward, 450 µl of 0.056 M Chloramine-T (1.27 g Chloramine T in 20 ml 50% normal propanol and brought to 100 ml with acetate/citric acid buffer) was added and oxidation was allowed to proceed for 25 min at room temperature. Then 500 µl of Ehrlich's aldehyde reagent (p-Dimenthylamino-benzaldehyde dissolved in normal propanol/perchloric acid 2:1 vol/vol) was added to each sample and the chromophore was developed by incubating the samples at 65°C for 20 min. Absorbance of each sample was measured at 550 nm (Ultrospec 2000; Biochrom, Cambridge, UK).

Zymography
After fibroblasts were cultured in gels for 4 or 5 d, conditioned media (0.5–5 ml) were precipitated with ethanol and resuspended with 30 µl of ddH2O and subjected to SDS-PAGE under nonreducing conditions in 10% acrylamide gels containing 0.1% gelatin. After electrophoresis with Minigel 3 apparatus (Bio-Rad), gels were washed by gentle shaking for 2 h at room temperature in 2.5% (vol/vol) Triton-X 100. The gels were then incubated at 37°C with shaking in the metalloproteinase buffer (0.06 M Tri-HCL, pH 7.5, containing 5 mM CaCl2 and 1 µM ZnCL2) for 18 h at 37°C, and subsequently stained with Coomassie blue. Zones of proteolysis appeared as clear bands against a blue background. Supernatants from HT1080 cells were used as positive controls in the zymograms.

Western Immunoblotting Analysis
Samples of conditioned media (2 ml) from three-dimensional cultures were precipitated with ethanol, which were then resuspended in a 30 µl dH2O and equal volumes of 2x sample buffer (0.5 M Tris-HCL, pH 6.8, 10% SDS, 0.1% bromphenol blue, 20% glycerol). After heating for 4 min at 95°C, 30 µl of each sample was loaded into each well of 10% SDS polyacrylamide gels and electrophoresed. The protein in the gels was electrically transferred to a polyvinylidene difluoride membrane (Bio-Rad) at 20 V for 40 min with a semi-dry electrophoretic transfer cell (Bio-Rad) using electroblotting buffer (20 mM tris, pH 8.0, 150 mM glycine, 20% MeOH). After blocking nonspecific binding sites with 5% nonfat milk in PBS Tween at room temperature for 1 h, blots were incubated overnight at 4°C with primary antibody (1 µg/ml mouse antihuman MMP-1; Calbiochem, Cambridge, MA). After extensive washing, blots were incubated with rabbit anti-mouse IgG horseradish peroxidase (HRP) as second antibody (Rockland, Gilbertsville, PA) in conjunction with an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). HT1080 supernatants were used as positive controls.

Tissue Inhibitor of Metalloprotease-1 Assay
The concentration of tissue inhibitor of metalloprotease (TIMP)-1 in culture media was determined using an enzyme-linked immunosorbent assay (ELISA). Ninety-six-well ELISA plates were coated overnight at 4°C with 100 µl antihuman TIMP-1 antibodies (R&D Systems), diluted in Voler's buffer (pH 9.6). Plates were then washed three times in PBS with 0.05% Tween 20 (pH 7.2–7.4) and 100 µl of recombinant human TIMP-1 standards (31.25–4,000 pg/ml) were added in duplicate. Samples (diluted 1/100 with PBS) were added in triplicate to individual wells and incubated at room temperature for 2 h. After three washes, 100 µl of biotinylated anti-human TIMP-1 antibody (R&D Systems) diluted in PBS-Tween was added for 1 h. After another three washes, 100 µl of HRP–avidin conjugate (Zymed, South San Francisco, CA), diluted 1/20,000 in PBS-Tween, was added for 1 h. After the final three washes, 200 µl of TMB-substrate was added and color developed for 30 min at room temperature. The reaction was stopped by adding 50 µl of stop solution (1 M of H2SO4) and the degree of color generated was determined by measuring the optical density at 450 nm in a microplate reader (Bio-Rad).

Pro-MMP-1 Assay
The amount of Pro-MMP-1 in the supernatants surrounding the gels was measured by ELISA kits, following manufacturer's instructions (R&D Systems). Briefly, Pro-MMP-1 in the sample was captured by the antibody on the microtiter plate. Subsequently, enzyme-linked monoclonal antibody specific for Pro-MMP-1 was added, followed by the addition of substrate solution. Color development was terminated by the addition of a stop solution, and absorbance was measured in a microtiter plate reader at 450 nm with correction wavelength set at 540 nm.

MMP-1 mRNA Detection by Real-Time Polymerase Chain Reaction Assay
To elucidate the effect of cilomilast on MMP-1 mRNA levels in HFL-1 cells in floating gels, real-time quantitative polymerase chain reaction (PCR) assay, utilizing reverse transcription (RT) and real-time PCR, was performed. Total RNA was isolated from HFL-1 cells cultured in collagen gels by the guanidinium thiocyanate-phenol-chloroform extraction method, as described by Chomczynski and Sacchi (14). Briefly, HFL-1 cells within the gels (7.5 x 105 cells) were lysed in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH7; 0.5% sarcosyl, 0.1 M 2-mercaptoethanol) and RNA was extracted from the solution by chloroform extraction. After isopropanol-precipitation, RNA was washed twice with 70% ethanol, dried, and resuspended in diethylpyrocarbonate-treated water. After DNase I treatment. 400 ng of total RNA was reverse transcribed with 20 µl of total volume of RT reaction solution containing 1X PCR buffer, 5.5 mM MgCl2, 500 µM of each dNTP, 2.5 µM random hexadeoxynucleotides, 0.4 U/µl RNase inhibitor and 2.5 U/µl MuLV Reverse Transcriptase. The RT reaction was performed at 25°C for 10 min, 48°C for 30 min, and 95°C for 10 min. After the RT reaction the PCR was performed. Briefly, the human MMP-1 specific primer pairs 5'-CGGTTTTT CAAAGGGAATAAGTACT-3' (5' primer), 5'-TCAGAAA GAGCAGCATCGATATG-3' (3' primer) or glycerol dehyde-3-phospate dehydrogenase (GAPDH) primers 5'-CCAGGAAAT GAGCTTGAGAAAGT-3' (5' primer), 5'-CCCACTCCTCCAC CTTTGAC-3'(3' primer) and Taqman probes were used for PCR amplification. The Taqman probe sequence for MMP-1 was 6FAM-AATGTGCTACACGGATACCCCAAG GACA-TAMRA and for GAPDH was 6FAM-CGTTGAGGG CAATGCCAGCCC- TAMRA. The Taqman real-time PCR was performed with 5 µl of RT product (100 ng total RNA), 1x Taqman Master Mix (Applied Biosystems, Foster City, CA), 300 nM primers, and 100 nM Taqman probe, in a total volume of 50 µl. PCR was performed at 50°C for 2 min, 95°C for 10 min, and 40 run at 95°C for 15 s, 60°C for 1 min on ABI PRISM 7700 detection system (Applied Biosystems). Each sample was run in duplicate data were analyzed using a sequence Detector V1.6 program (Applied Biosystems).

NE Activity Assay
To investigate the effect of cilomilast on functional NE, NE functional activity was quantified (15). NE (15 nM), cilomilast (10 µM), or TNF-{alpha} separately or in various combinations were added to the media, incubated, and harvested at 2, 8, 24, and 48 h. Elastase activity was measured using a synthetic substrate, methoxy-succinyl-alanyl-prolyl-valyl-p-nitroanilide (Calbiochem-Novabiochem Co., La Jolla, CA). The supernatants (100 µl) 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 2 h at 37°C, absorbance of the product, p-nitroanilide, was measured at 414 nm. Purified human NE was used as a standard.

Statistical Evaluation
Results are expressed as the mean ± SEM of three separate experiments, each performed in triplicate. ANOVA was performed and corrected with the Tukey test to make pairwise comparisons. Values of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PDE Inhibitors on Fibroblast-Mediated Collagen Gel Contraction
NE augmented and TNF-{alpha} inhibited fibroblast-mediated collagen gel contraction at all time points (Figure 1) . The effect of PDE inhibitors added to cultures varied according to the presence of other mediators. When added alone, the PDE4 inhibitor cilomilast (10 µM) inhibited collagen gel contraction (Figure 1). Inhibitors of other PDEs, amrinone (10 µM), and zaprinast (10 µM), had no effect (data not shown). Fibroblasts cultured with TNF-{alpha} and NE significantly reduced the size of collagen gels on Day 4 (Control: 50.9 ± 0.5%; NE: 21.9 ± 0.5%, TNF-{alpha} + NE: 9.5 ± 0.6%; Figure 1). When the three PDE inhibitors were each added to gels with TNF-{alpha} and NE together, only cilomilast significantly inhibited the synergistic effect of TNF-{alpha} and NE on Day 4 (17.3 ± 0.2%).



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  		Figure 1. Effect of cilomilast on collagen degradation induced by TNF-{alpha} and NE. Gels were released into tissue culture dishes containing 5 ml of serum-free DMEM with TNF-{alpha} (10 ng/ml) and/or NE (15 nM) in the presence or absence of cilomilast (10 µM) Vertical axis, gel size as % of initial area; horizontal axis, time after release (d). Data are presented are means ± SEM for triplicate gels in a single experiment. Similar results were obtained in three duplicate experiments. *P < 0.05. Unseen error bars are contained within plot symbols. Open squares, control; open circles, TNF-{alpha}; filled squares, cilomilast; open diamonds, NE; open triangles, TNF-{alpha} + NE; filled triangles, TNF + NE + cilomilast.

 
Effect of PDE Inhibitors on Collagen Degradation
The synergistic interaction between NE and TNF-{alpha} leading to smaller final size of collagen gels depends on degradation of the collagenous matrix (16). To elucidate the effect of PDE inhibitors on collagen degradation, hydroxyproline content was assessed in the gels after 4 d of culture. NE and TNF-{alpha} together resulted in significant degradation of the collagen gels (Figure 2A) . The PDE4 inhibitor cilomilast inhibited this degradation (control: 100%; TNF-{alpha} + NE: 20.8 ± 2.9%; TNF-{alpha} + NE + cilomilast: 84.4 ± 8.4%; P < 0.05, n = 3). Neither amrinone nor zaprinast had an effect on degradation induced by TNF-{alpha} and NE. The ability of cilomilast to inhibit TNF-{alpha}– and NE-mediated degradation was concentration-dependent, with significant inhibition observed at 10 µM (Figure 2B).




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  		Figure 2. Effect of cilomilast on collagen degradation induced by TNF-{alpha} and NE. Fibroblasts were cast into collagen gels containing NE (15 nM) and/or TNF-{alpha} (10 ng/ml) with or without cilomilast (10 µM; A) or NE and TNF-{alpha} with or without different concentrations of cilomilast (B). After 4 d, gels were harvested and hydroxyproline was measured. Vertical axis, hydroxyproline content expressed as percentage to control gels; horizontal axis, culture conditions. Data presented are the mean ± SEM for three separate experiments, each performed in triplicate. *P < 0.05

 
Effect of Cilomilast on MMP-2 and MMP-9 Activity
To elucidate whether collagen degradation is related to MMP-2 and MMP-9 release from the fibroblasts, gelatinases released by fibroblasts cultured in three-dimensional collagen gels were assessed by gelatin zymography (Figure 3A) . Under control conditions, fibroblasts released primarily MMP-2 (gelatinase A) into surrounding media, as identified by its characteristic molecular weights of 72 kD (latent form) and 66 kD (active form). TNF-{alpha}, NE, and cilomilast did not markedly alter the release of either the latent or active form of MMP-2 (Figure 3B).




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  		Figure 3. MMP-2 and MMP-9: gelatin zymography. Media were harvested after 4 d of culture in the presence of NE (15 nM) and TNF-{alpha} (10 ng/ml), added separately or together with or without cilomilast (10 µM). Gelatin zymography (A) was performed, and density of each band was measured by using NIH image 1.61 (B). Each band was compared with the band TNF-{alpha} + NE. The data shown are mean ± SEM for triplicate samples. *P < 0.05

 
MMP-9 (gelatinase B) was not present in control cultures, but in the presence of TNF-{alpha}, a band with the molecular weight of 92 kD was induced. Cilomilast decreased this induction (Figures 3A and 3B). In addition, NE in the presence of TNF-{alpha} resulted in conversion of latent MMP-9 to an 84-kD moiety corresponding to the active form. Although when cilomilast was added with NE and TNF-{alpha}, there was some decrease in the activated MMP-9 (100% versus 55.7 ± 18.6%), this reduction was not significant (P = 0.075; Figures 3A and 3B).

Effect of Cilomilast on Induction and Activation of MMP-1
Immunoblotting was used to determine the effect of cilomilast on MMP-1. Under control conditions, MMP-1 was detectable with an apparent molecular size of 52 kD, corresponding to the latent form of MMP-1 (Figures 4A and 4B) . NE alone appeared to reduce MMP-1 whereas TNF-{alpha} appeared to induce MMP-1 release. In the presence of both TNF-{alpha} and NE, not only was MMP-1 release induced, but clearly some of the MMP-1 was converted to lower molecular weight 42 kD and 20 kD forms, corresponding to active MMP-1. Cilomilast reduced the amount of MMP-1 detectable in both higher and lower molecular weight forms in all conditions (Figures 4A and 4B).




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  		Figure 4. MMP-1 immunoblot. Fibroblasts were cast in collagen gels and floated in cultured media in the presence of NE (15 nM) and TNF-{alpha} (10 ng/ml), separately or together, with or without cilomilast (10 µM). Media were then subjected to SDS-PAGE, followed by Western blotting with antibodies for MMP-1. (A) Western blot. Molecular weight markers and various additions added to media are as indicated. (B) Density of each band was measured by using NIH image 1.61. Each band was compared with the band of TNF-{alpha} + NE. The data shown are mean ± SEM for triplicate samples *P < 0.05.

 
Effect of Cilomilast on MMP-1 Release
Pro-MMP-1 was detectable by ELISA in the media in which collagen gels containing fibroblasts were cultured. NE and TNF-{alpha} each augmented the release of MMP-1. When NE and TNF-{alpha} were combined, this release was further increased. Cilomilast alone had no effect on MMP-1 release. When cilomilast was added together with TNF-{alpha}, NE, or both, however, the augmented release of MMP-1 was significantly diminished (Figure 5) .



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  		Figure 5. Pro-MMP-1 ELISA assay. Fibroblasts in three-dimensional gels were incubated with TNF-{alpha} (10 ng/ml) with or without NE (15 nM) and/or cilomilast (10 µM) for 4 d. Pro-MMP-1 accumulation in the surrounding media was measured by ELISA. Vertical axis, MMP-1 release expressed as percent to group of TNF-{alpha} + NE. Horizontal axis, condition. Data presented are means ± SEM from three independent experiments. *P < 0.05

 
To determine whether the cilomilast inhibition of MMP-1 release by HFL-1 was associated with changes in MMP-1 gene expression, real-time RT-PCR analysis was performed. After 6 h of incubation, TNF-{alpha} and NE slightly increased the mRNA level of MMP-1. However, cilomilast had no effect on MMP-1 mRNA expression (Figure 6) .



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  		Figure 6. MMP-1 mRNA content. HFL-1 fibroblasts cultured in three-dimensional gels were incubated with designated conditions. After 6 h, the MMP-1 mRNA content was quantified by Taqman RT-PCR. Vertical axis, MMP-1 mRNA which were normalized with GAPDH and expressed as % control. Horizontal axis, conditions. The data presented are the means ± SEM from three separate experiments, each duplicated.

 
Effect of Cilomilast on TIMP-1 Release
As MMP activity can be regulated by the presence of inhibitors, prominently TIMP-1, the effect of cilomilast on release of TIMP-1 was investigated by ELISA. Detectable TIMP-1 was released from fibroblasts cultured in three-dimensional collagen gels under control conditions (118 ± 50 ng/ml). TNF-{alpha} and NE individually demonstrated a nonsignificant trend toward reducing the detectable TIMP-1 released (Figure 7) . TNF-{alpha} combined with NE significantly decreased the detectable TIMP-1 (P < 0.05). Cilomilast had no effect on detectable TIMP-1.



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  		Figure 7. TIMP-1 ELISA assay. Fibroblasts in three-dimensional gels were incubated with TNF-{alpha} (10 ng/ml) and/or NE (15 nM) with or without cilomilast (10 µM) for 4 d. TIMP-1 accumulation in the surrounding media was measured by ELISA. Vertical axis: TIMP-1 release expressed as % control. Horizontal axis: condition. The data presented are the means ± SEM from three separate experiments. *P < 0.05

 
Effect of Cilomilast on NE Activity
To determine if cilomilast resulted in direct inhibition of NE activity, cultures were incubated with NE, TNF-{alpha}, or a combination of both in the presence and absence of cilomilast. NE activity was detectable only in cultures with added NE, and this activity fell with time, becoming undetectable after 24 h. Cilomilast had no effect on NE activity at any of the times tested (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study demonstrates that the PDE4 inhibitor cilomilast can modulate the ability of fibroblasts cultured in three-dimensional collagen gels to degrade their surrounding matrix. When incubated with TNF-{alpha}, cultured fibroblasts released increased amounts of MMP-1 and -9. The addition of NE resulted in the conversion of the latent MMPs to their active forms. Cilomilast inhibited the release of both MMP-1 and MMP-9, and also inhibited the conversion of these proteases to sizes corresponding to their active forms. The cilomilast inhibition of pro-MMP-1 release was not associated with alterations in MMP-1 mRNA expression, suggesting that inhibition of release was regulated at the post-transcriptional level. By inhibiting MMP release and activation, cilomilast inhibited degradation of collagen gels.

The PDEs are a family of enzymes that degrade cyclic nucleotides (17). By virtue of their ability to regulate intracellular cyclic nucleotide concentrations, PDEs can exert potent effects on regulation of a variety of cellular responses. PDE4 is able to degrade cyclic AMP to 5' AMP and is present in a variety of cell types, including lymphocytes, monocytes (9), macrophages (17), mast cells, eosinophils (7, 18), and airway epithelial cells (19). By virtue of inhibiting cyclic AMP degradation in airway smooth muscle cells, PDE4 inhibitors may function as bronchodilators and are being developed for this purpose clinically. A variety of PDE4 inhibitors including rolipram, denbufylline (20), and tolafentrine (21) have been shown to have a variety of effects on inflammatory cells, including inhibition of cytokine release. The current study suggests that PDE4 inhibitors may also affect tissue remodeling by inhibiting the release and activation of MMPs.

The MMPs are a large family of proteolytic enzymes. Various members are released by a variety of cell types (22, 23). The MMPs are generally released as latent forms and are activated by a variety of mechanisms, including proteolytic cleavage. Because some MMPs are able to activate others, it is likely that proteolytic cascades are responsible for MMP activation. The various MMPs have varying substrate specificities. Together, however, they are able to degrade all components of the ECM (22–24). As a result, MMPs are believed to play a major role in remodeling of tissue architecture both during normal healing processes and in disease states such as pulmonary emphysema (3, 25, 26).

Emphysema is characterized by destruction of alveolar walls (1, 2). The role of proteases in this tissue destruction was initially suggested by the classic studies of Laurell and Ericksson (27). Individuals deficient in the {alpha}1 serine protease inhibitor are at increased risk for the development of emphysema (2). The major substrate for {alpha}1 protease inhibitor is NE. Various elastolytic enzymes infused into the lower respiratory tract of animals are capable of inducing emphysema (28). Taken together, these studies strongly support a pathogenetic role for neutrophil elastase in the development of pulmonary emphysema.

Recent studies, however, have suggested that proteases combined with NE can also play a role. Mice deficient in the macrophage elastase MMP-12, for example, are resistant to the development of cigarette smoke–induced emphysema (29). It is likely, therefore, that enzymes in addition to NE will also play a role in the development of emphysema. In the current study, cytokine induction of MMP production was insufficient to lead to tissue degradation in three-dimensional collage gels populated by lung fibroblasts. Elastase alone was also insufficient to degrade ECM. The concurrent addition of both TNF-{alpha}, which induced MMP production, and NE, which led to conversion of the MMPs to their active forms, however, was sufficient to induce degradation of the ECM. Such a collaborative interaction is likely to occur in tissues where a variety of concurrent stimuli will routinely be present. The ability of cilomilast, therefore, to inhibit the activation of MMPs raises the possibility that it could function effectively to inhibit tissue proteolysis in in vivo settings. Such an action may be important in modulating tissue remodeling in diseases such as emphysema.

The current study utilized the in vitro system of fibroblasts cultured in gels composed of native type I collagen. This three-dimensional culture system has been used as a model of tissue repair and scar contraction (30, 31). Fibroblasts cultured in such a system are also able to respond to cytokines and can, moreover, be induced to release enzymes that can degrade the ECM. It is likely that MMP-1, also known as fibroblast collagenase, is the enzyme most critical for the initial step in degrading native type I collagen fibers (3). The pathway that leads to activation of MMP-1 in the three-dimensional culture system remains undefined. It is likely that a proteolytic cascade leading to the activation of several MMPs, however, is responsible. In this regard, cilomilast inhibited the activation of both MMP-9 and MMP-1. There was also inhibition of release of these proteases. It is plausible that cilomilast is also inhibiting the release of other MMPs not evaluated in the current study and that these enzymes are responsible for activation of the protease cascade leading to collagen degradation.

The mechanism by which NE activates MMPs is also undefined. Although it is possible that direct cleavage of the MMPs could lead to activation, MMP activation requires several days, whereas neutrophil elastase activity decreases over the first 24 h of culture. It seems likely, therefore, that NE is activating fibroblasts, thus initiating the mechanisms which result in MMP activation. There are several mechanisms by which NE could activate fibroblasts. These include activation of the protease-activated receptors (PARs) or cleavage of other cell surface regulatory molecules. Activation of cell surface receptors by elastase is an appealing mechanism, as this raises a number of potential ways for cilomilast, by inhibiting PDE4, to attenuate the NE effect.

The MMP-1 release and activation is regulated by a variety of factors via different mechanisms (32–34). In this regard, PGE2 significantly inhibited cytokines and induced collagen degradation, presumably through the mechanism of increasing cAMP (35). In the current study, three kinds of PDE4 inhibitors (cilomilast, amrinoe, and zaprinast) were investigated. Although cilomilast significantly inhibited the MMP-1 release and activation, neither amrinone nor zaprinast had any effect on collagen degradation and MMP-1 release or activation. Whether the difference is due to their differences in structure or chemical activity in terms of regulating cAMP levels or modulating cytokine release by fibroblasts remains to be defined. However, cilomilast inhibited MMP-1 release and activation without altering MMP-1 mRNA expression, suggesting that inhibition of the MMP-1 release may be regulated at the level of post-transcription. In this regard, MMP-3 and -8 were released by chodrocytes with upregulation of mRNA expression (36, 37). However, MMP-1, -13, and -14 mRNA expression was not altered under basal conditions, even after stimulation by IL-1ß (36, 37).

Many chronic diseases are characterized by excessive destruction of normal tissue structures. The current study suggests that PDE4 inhibitors may be potential therapeutic agents in such diseases. By virtue of their ability to inhibit the release and activation of tissue-degrading MMPs, agents with activity similar to cilomilast could prove important therapeutic regulators of tissue remodeling.


   Acknowledgments
 
The authors acknowledge the excellent secretarial support of Ms. Lillian Richards and editorial assistance of Ms. Mary C. Tourek.

Received in original form January 15, 2002

Received in final form May 14, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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