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Published ahead of print on May 8, 2003, doi:10.1165/rcmb.2003-0113OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 613-619, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2003-0113OC

Serine Protease Inhibitors Modulate Smoke-Induced Chemokine Release From Human Lung Fibroblasts

Hiroki Numanami, Sekiya Koyama, Dan K. Nelson, Jeffrey C. Hoyt, Jon L. Freels, Michael P. Habib, Jun Amano, Masayuki Haniuda, Etsuro Sato and Richard A. Robbins

Research Service, Southern Arizona Veterans Health Care System, and Arizona Respiratory Center, University of Arizona, Tucson, Arizona; First Department of Internal Medicine, and Second Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan

Address correspondence to: Richard A. Robbins, M.D., Chief, Research Service Line, Southern Arizona Veterans Health Care System, 3601 S. 6th Ave., Tucson, AZ 85723. E-mail: Richard.Robbins2{at}med.va.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Smoking is associated with lung inflammation and a protease–antiprotease imbalance. We previously reported that cigarette smoke extract (CSE) stimulates human lung fibroblasts to release chemotactic cytokines. We hypothesized that serine protease inhibitors might modulate lung fibroblast release of chemotactic cytokines in response to CSE. To test this hypothesis, serine protease inhibitors (FK706, {alpha}1-antitrypsin, methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone, or N{alpha}-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) from human fetal lung fibroblasts by the blind-well chemotactic chamber. Metalloproteinases and cysteine proteinases were not examined in this study. Similarly, the release and gene expression of chemokines and nuclear factor-{kappa}B (NF-{kappa}B) activation were measured by means of enzyme-linked immunosorbent assay and reverse transcriptase–polymerase chain reaction. Release of NCA, MCA, chemotactic chemokines including interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor, and the expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA were attenuated by FK706. Furthermore, FK706 suppressed NF-{kappa}B activation. These data suggest that serine protease inhibitors attenuate the CSE-induced release of NCA and MCA from human fetal lung fibroblasts and that the inhibitory action of antiproteases might depend on NF-{kappa}B signaling pathway.

Abbreviations: {alpha}1-antitrypsin, {alpha}1AT • chronic obstructive pulmonary disease, COPD • cigarette smoke extract, CSE • granulocyte colony-stimulating factor, G-CSF • granulocyte-macrophage colony-stimulating factor, GM-CSF • Hanks' balanced saline solution, HBSS • human fetal lung fibroblasts, HFL-1 • high power fields, HPF • interleukin, IL • monocyte chemotactic activity, MCA • monocyte chemoattractant protein, MCP-1 • neutrophil chemotactic activity, NCA • nuclear factor-{kappa}B, NF-{kappa}B • reverse transcriptase–polymerase chain reaction, RT-PCR • methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone, SPCK • N{alpha}-p-tosyl-L-lysine chloromethyl ketone, TLCK


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cigarette smoking is the major factor leading to the development of chronic bronchitis, emphysema, and chronic obstructive pulmonary disease (COPD) (1). Current concepts suggest that the pathogenesis of emphysema is an imbalance between proteases and antiproteases in the lung (2). An infiltration of inflammatory cells, including neutrophils, monocytes, and macrophages, play a critical role in tipping the protease–antiprotease balance toward protease excess. Increased numbers of neutrophils and monocytes activated by cigarette smoke produce large amounts of proteases and oxidants that inactivate antiproteases, resulting in lung destruction (3). Protease inhibitors may protect the lung from the detrimental destruction by proteases. However, antiproteases may also modulate neutrophil migration, further reducing lung inflammation and destruction (4).

The lung fibroblast plays an important role in maintenance of the extracellular matrix and in tissue repair after injury. Synthesis of the extracellular matrix serves an important structural function by providing a frame network for organ integrity. In addition to this traditionally accepted function, there is evidence suggesting that fibroblasts may modulate inflammation by producing chemokines such as interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF in response to cigarette smoke extract (CSE) (5). Moreover, fibroblasts secrete a variety of proteases, including plasmin (6), fibroblast-activation protein (7), tissue-type plasminogen activator (8), urokinase-type plasminogen activator (9), and a calcium-dependent serine protease (10). Therefore, the fibroblast, because of its anatomic location, production, and protease secretion, is at a pivotal position to participate in and to direct communications between interstitial and vascular events in pulmonary inflammation and fibrosis.

The effect of protease inhibitors on cigarette smoke inflammation has not been extensively studied. We hypothesized that antiproteases might reduce the cigarette smoke–induced release of chemotactic factors from lung fibroblasts. The purpose of this study was to test this hypothesis by studying the effects of protease inhibitors on neutrophil and monocyte chemotactic activity released a human lung fibroblast cell line (HFL-1) exposed to CSE. In addition, we determined whether FK706, a synthetic serine protease inhibitor, might attenuate the production of inflammatory chemokines and chemokine mRNA expression, and because current concepts suggest that these chemokines are regulated at least in part by nuclear factor (NF)-{kappa}B (11), we also evaluated for NF-{kappa}B activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures
Human fetal lung fibroblasts (HFL-1, lung, diploid, human, passage 14) were purchased from the American Type Tissue Culture Collection (Rockville, MD). The HFL-1 cells were cultured according to previously described methods in Ham's F-12 medium with 10% heat-inactivated fetal bovine serum (5). After 2–4 d in culture, the cells had reached confluence and were then used for experiments.

Preparation of CSE
CSE was prepared with a modification of the method of Laurent and coworkers (12). Briefly, two cigarettes (CAMEL; R. J. Reynolds Tobacco Co., Winston-Salem, NC) without filters were combusted with a modified syringe-driven apparatus. The smoke was bubbled through 50 ml of Hanks' balanced salt solution (HBSS; GIBCO BRL, Grand Island, NY). The resulting suspension was adjusted to pH 7.4 and filtered through a 0.2-µm filter (Nalge Nunc International, Rochester, NY) to remove bacteria and large particles. CSE was stored at -80°C until used. Smoke extract prepared by this method contained < 10 ng/ml of bacterial endotoxin in 10% smoke extract solution, as determined by the Limulus test using Endotoxin Test Kit (Seikagaku Kogyo, Tokyo, Japan) (13).

Protease Inhibitors and Stimulants
FK706 (C26H32F3N4NaO7; Fujisawa Pharmaceutical Co., Osaka, Japan) was used as a serine protease inhibitor (14). After the cells had reached confluence, the culture medium was removed, the cells washed twice with serum-free Ham's F-12 medium, and the cells incubated in the presence or absence of CSE (0, 0.5, 1, 5, and 10%) for 72 h at 37°C in a humidified 5% CO2 atmosphere. In some experiments, HFL-1 cells were pretreated with several concentrations of the serine protease inhibitors, FK706 (5, 10, and 50 µg/ml), {alpha}1-antitrypsin ({alpha}1AT) (20, 100, and 200 µg/ml; Sigma, St. Louis, MO), methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (SPCK) (1, 5, and 10 µM; Sigma) or N{alpha}-p-tosyl-L-lysine chloromethyl ketone (TLCK) (10, 50, and 100 µM; Sigma). After 30 min incubation at 37°C, the HFL-1 cells were stimulated with CSE. The concentrations of FK706 were based on results showing that neutrophil elastase–induced lung hemorrhage of mice was significantly inhibited by intratracheal treatment with FK706 at the dose from 1–100 µg (14). In addition, the concentrations of other protease inhibitors were decided in accordance with previous studies (4). CSE was not cytotoxic as assessed by cell shape, cell detachment, and trypan blue exclusion after 72 h at 10% concentration (no deformity of cell shape, no detachment from culture dish and > 98% viability). The supernatant fluids were harvested and stored at -80°C until assayed. At least six HFL-1 fibroblast culture supernatant fluids were harvested for each experimental condition.

Effects of Protease Inhibitors on NCA and MCA in HFL-1 Supernatant Fluids
Polymorphonuclear leukocytes were purified from heparinized normal human blood by the method of Böyum (15). The resulting cell pellet consisted of > 96% neutrophils and > 98% viable cells as determined by trypan blue and erythrosin exclusion. The cells were suspended in HBSS (GIBCO) containing 2% bovine serum albumin (Sigma) at pH 7.4 to give a final concentration of 3.0 x 106 cells/ml. This suspension was used for the neutrophil chemotaxis assay.

Mononuclear cells for the chemotaxis assay were obtained from normal human volunteers by Ficoll-Hypaque density centrifugation to separate red blood cells and neutrophils from mononuclear cells. The preparation routinely consisted of 30% large monocytes and 70% small lymphocytes determined by morphology and {alpha}-naphthyl acetate esterase staining (Sigma) with > 98% viability as assessed by trypan blue and erythrosin exclusion. The cells were suspended in HBSS containing 2% bovine serum albumin at pH 7.4 to give a final concentration of 5.0 x 106 cells/ml. The suspension was then used for the monocyte chemotaxis assay.

The chemotaxis assay was performed by a 48-well microchemotaxis chamber (NeuroProbe Inc., Cabin John, MD), as has been described previously (4, 5). Briefly, 25 µl of the harvested supernatant fluids were placed into the lower wells, and a 10-µm-thick polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA), with a pore size of 3-µm for neutrophil chemotaxis and 5-µm for monocyte chemotaxis, was placed over the bottom wells. The silicon gasket and upper pieces of the chamber were applied, and 50 µl of the cell suspension was placed into the upper wells above the filter. The chambers were incubated in humidified air in 5% CO2 at 37°C for 30 min for neutrophil chemotaxis and 90 min for monocyte chemotaxis. After incubation, the chamber was disassembled and nonmigrated cells were wiped away from the filter. The filter was then immersed in methanol for 5 min stained with Diff-Quik (American Scientific Product, McGraw Park, IL) and mounted on a glass slide. cells that completely migrated through the filter were counted by using light microscopy in ten random high power fields (HPF) per well.

Measurement of Chemokines in the Supernatant Fluids
The concentrations of IL-8, MCP-1, G-CSF, and GM-CSF were measured in the cell supernatant fluids using commercially available enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions in duplicate. The minimum concentrations detected by these methods were 10 pg/ml for IL-8, 5.0 pg/ml for MCP-1, 20 pg/ml for G-CSF, and 3.0 pg/ml for GM-CSF. The intra- and inter-assay variability of these kits were 4.6 ± 0.15% and 6.7 ± 1.45% for IL-8, 5.0 ± 0.85% and 5.1 ± 0.74% for MCP-1, 2.1 ± 0.35% and 8.6 ± 3.40% for G-CSF, and 2.6 ± 0.17% and 5.0 ± 0.58% for GM-CSF.

Evaluation of mRNA Expression
Chemokine mRNA was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR). HFL-1 cells were incubated with FK706 and CSE for 12 h and total cellular RNA was extracted from adherent cells using a modification of the methods of Chomczynski and Sacchi (16). The RNA was reverse transcribed using a commercially available kit (Promega, Madison, WI). One microgram of the reverse-transcribed DNA was then mixed with Ready to Go PCR Beads (Pharmacia, Piscataway, NJ) and the front and back primers of IL-8 and MCP-1, using a commercially available primer pairs (R&D Systems), added at 0.3 µM final concentration. PCR was performed in a Perkin Elmer model 480 thermal cycler (Perkin Elmer, Shelton, CT) using 94°C for 2 min and 20 cycles consisting of 94°C for 45 s, primer annealing at 55°C for 45 s, primer extension at 72°C for 45 s, followed by 72°C for an additional 7 min. ß-actin was used as a "housekeeping gene" with PCR. The DNA was subjected to agarose gel and the intensity of the bands quantified by densitometry. The results were expressed as the ratio of intensity to the ß-actin.

Measurement of NF-{kappa}B Activation
The activation of NF-{kappa}B was analyzed by commercially available NF-{kappa}B p65 transcription factor assay kits (Active Motif, Carlsbad, CA) according to the manufacturer's instructions in duplicate. HFL-1 cells were pretreated with FK706 and CSE for 1 h, and whole-cell extract was prepared as previously described (17). The protein concentration of the cell extract was determined by using a modified Bradford-based assay (18), and the amount of protein of cell extract was adjusted 10 µg per well. NF-{kappa}B activation was evaluated with reading optical density at 450 nm. The detection limit by this method was 0.5 µg cell extract per well.

Statistical Analysis
Data were analyzed by one-way ANOVA with Fisher's protected least significant difference (Fisher's PLSD). In all cases, a P value of < 0.05 was considered significant. The data are expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Release of NCA and MCA from HFL-1 cells in Response to CSE
In response to smoke extract, HFL-1 released NCA (0% CSE 38.4 ± 6.7 cell/HPF; 0.5% CSE 50.4 ± 3.9 cell/HPF; 1% CSE 63.8 ± 4.2 cell/HPF; 5% CSE 76.9 ± 5.1 cell/HPF; 10% CSE 78.2 ± 5.1 cell/HPF; Figure 1, n = 6) and MCA (0% CSE 34.0 ± 3.0 cell/HPF; 0.5% CSE 36.2 ± 3.0 cell/HPF; 1% CSE 45.6 ± 3.4 cell/HPF; 5% CSE 53.4 ± 3.4 cell/HPF; 10% CSE 57.2 ± 2.1 cell/HPF; Figure 1, n = 6) in a dose-dependent manner after 72 h incubation. The lowest doses of smoke extract to stimulate HFL-1 were 0.5% for neutrophils and 1% for monocytes. Increasing concentrations of smoke extract progressively increased the release of chemotactic activity up to 10% (Figure 1, n = 6). CSE itself was not chemotactic for neutrophils and monocytes (data not shown).



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  		Figure 1. Neutrophil chemotactic activity (NCA, closed circles) and monocyte chemotactic activity (MCA, open circles) in response to CSE from HLF-1 monolayers after 72 h incubation (n = 6). Chemotactic activities are on the ordinate, and the concentration of CSE is on the abscissa. Values are expressed as mean ± SD. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured without stimuli.

 
Effects of Protease Inhibitors on Neutrophil and Monocyte Chemotactic Activity
FK706 inhibited, in a dose-dependent manner, NCA (5% CSE 94.6 ± 12.7 cell/HPF; CSE with 5 µg/ml FK706 78.9 ± 7.4 cell/HPF; CSE with 10 µg/ml FK706 52.2 ± 6.8 cell/HPF; CSE with 50 µg/ml FK706 47.4 ± 1.3 ccell/HPF; Figure 2A, n = 6) and MCA (5% CSE 67.5 ± 18.9 cell/HPF; CSE with 5 µg/ml FK706 58.0 ± 8.7 cell/HPF; CSE with 10 µg/ml FK706 53.5 ± 8.0 cell/HPF; CSE with 50 µg/ml FK706 45.7 ± 4.0 cell/HPF; Figure 2A, n = 6) from HFL-1 stimulated with CSE. FK706 alone had no effect on baseline release of NCA and MCA under control conditions (P > 0.05). The other serine protease inhibitors also reduced the CSE-induced NCA and MCA ({alpha}1AT NCA and MCA, 5% CSE 92.6 ± 17.6 cell/HPF and 64.7 ± 8.2 cell/HPF; CSE with 20 µg/ml {alpha}1AT 60.0 ± 10.4 cell/HPF and 53.3 ± 7.0 cell/HPF; CSE with 100 µg/ml {alpha}1AT 60.8 ± 25.1 cell/HPF and 50.4 ± 5.9 cell/HPF; CSE with 200 µg/ml {alpha}1AT 51.2 ± 4.0 cell/HPF and 43.0 ± 7.1 cell/HPF; Figure 2B, n = 6); (TLCK NCA and MCA, 5% CSE 94.7 ± 16.0 cell/HPF and 59.0 ± 6.1 cell/HPF; CSE with 10 µM TLCK 75.7 ± 17.8 cell/HPF and 48.9 ± 3.1 cell/HPF; CSE with 50 µM TLCK 68.8 ± 6.8 cell/HPF and 40.3 ± 2.6 cell/HPF; CSE with 100 µM TLCK 63.3 ± 3.8 cell/HPF and 36.2 ± 8.5 cell/HPF; Figure 3A, n = 6); (SPCK NCA and MCA, 5% CSE 74.5 ± 17.9 cell/HPF and 60.0 ± 2.1 cell/HPF; CSE with 1 µM SPCK 49.8 ± 10.9 cell/HPF and 43.6 ± 8.9 cell/HPF; CSE with 5 µM SPCK 43.0 ± 10.9 cell/HPF and 39.6 ± 6.2 cell/HPF; CSE with 10 µM SPCK 34.1 ± 7.7 cell/HPF and 35.4 ± 6.3 cell/HPF; Figure 3B, n = 6). None of these inhibitors altered baseline NCA and MCA release (P > 0.05).



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  		Figure 2. Effects of FK706 (A) or {alpha}1AT (B) on NCA (black columns) and MCA (white columns) from HFL-1 stimulated with CSE (n = 6). NCA and MCA are on the ordinate and the experimental groups are on the abscissa. Values are expressed as mean ± SD. *P < 0.05 compared with supernatant fluids from HFL-1 cells cultured with CSE. **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with CSE. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with CSE.

 


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  		Figure 3. Effects TLCK (A) or SPCK (B) on NCA (black columns) and MCA (white columns) in response to CSE (n = 6). NCA and MCA are on the ordinate and the experimental groups are on the abscissa. Values are expressed as mean ± SD. *P < 0.05 compared with supernatant fluids from HFL-1 cells cultured with CSE. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with CSE.

 
The percentage of inhibition of NCA and MCA by FK706 (NCA: 16.7 ± 7.9% to 49.9 ± 1.4%, and MCA: 14.1 ± 12.9% to 32.3 ± 5.9%) was compared with that by {alpha}1AT (NCA: 35.2 ± 11.3% to 44.7 ± 4.4%, and MCA: 17.6 ± 10.9% to 33.6 ± 11.0%), TLCK (NCA: 20.1 ± 18.7% to 33.2 ± 4.0%, and MCA: 19.0 ± 3.9% to 38.8 ± 14.5%), or SPCK (NCA: 33.2 ± 14.6% to 54.2 ± 10.4%, and MCA: 27.3 ± 14.9% to 41.1 ± 10.5%). It was not significantly different between FK706 and {alpha}1AT (NCA: P = 0.99, and MCA: P = 0.73), TLCK (NCA: P = 0.53 and MCA: P = 0.26), or SPCK (NCA: P = 0.84 and MCA: P = 0.11).

Effects of FK706 on Chemokine Production from HFL-1
HFL-1 spontaneously released IL-8, MCP-1, G-CSF, and GM-CSF, but CSE stimulated the release of these chemokines from HFL-1 (Figures 4 and 5, n = 4). The releases of IL-8 (5% CSE 2,348.8 ± 147.2 pg/ml; CSE with 10 µg/ml FK706 854.3 ± 254.8 pg/ml; CSE with 50 µg/ml FK706 1,222.3 ± 283.2 pg/ml; Figure 4A, n = 4) and MCP-1 (5% CSE 860.3 ± 65.4 pg/ml; CSE with 10 µg/ml FK706 606.1 ± 68.2 pg/ml; CSE with 50 µg/ml FK706 667.5 ± 17.2 pg/ml; Figure 4B, n = 4) were significantly inhibited by FK706 from CSE-stimulated HFL-1. The concentrations of G-CSF (5% CSE 26.6 ± 2.4 pg/ml; CSE with 10 µg/ml FK706 16.9 ± 1.4 pg/ml; CSE with 50 µg/ml FK706 18.0 ± 1.8 pg/ml; Figure 5A, n = 4) and GM-CSF (5% CSE 56.7 ± 11.5 pg/ml; CSE with 10 µg/ml FK706 17.3 ± 4.8 pg/ml; CSE with 50 µg/ml FK706 31.8 ± 5.6 pg/ml; Figure 5B, n = 4) were also significantly attenuated by FK706 (Figure 5, n = 4). FK706 had no stimulating effect on release of these chemokines from HFL-1 (Figures 4 and 5, P > 0.05).



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  		Figure 4. Effects of FK706 on IL-8 (A) and MCP-1 (B) stimulated with CSE from HLF-1 monolayers after 72 h incubation (n = 4). The concentration of IL-8 and MCP-1 is on the ordinate and experimental groups are on the abscissa. Values are expressed as mean ± SD. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with CSE.

 


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  		Figure 5. Effects of FK706 on G-CSF (A) and GM-CSF (B) stimulated with CSE from HLF-1 monolayers after 72 h incubation (n = 4). The concentration of IL-8 and MCP-1 is on the ordinate and experimental groups are on the abscissa. Values are expressed as mean ± SD. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with CSE.

 
Effects of FK706 on mRNA Expression from HFL-1
Semiquantitative RT-PCR was performed to evaluate the effect of FK706 on chemokine mRNA expression in HFL-1. FK706 suppressed CSE-induced IL-8 mRNA dose-dependently (5% CSE 0.80 ± 0.08; CSE with 10 µg/ml FK706 0.67 ± 0.07; CSE with 50 µg/ml FK706 0.42 ± 0.07; Figure 6, n = 3). CSE-induced MCP-1 mRNA were significantly attenuated by the high concentration of FK706 (5% CSE 0.95 ± 0.03; CSE with 10 µg/ml FK706 0.87 ± 0.06; CSE with 50 µg/ml FK706 0.71 ± 0.03; Figure 6, n = 3).



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  		Figure 6. Effects of FK706 on IL-8 and MCP-1 mRNA expression from CSE-stimulated HFL-1. Results with RT-PCR for IL-8, MCP-1, ß-actin (A), and densitometry data with IL-8 mRNA and MCP-1 mRNA are expressed as a ratio of IL-8 mRNA (black columns) or MCP-1 mRNA (white columns) to ß-actin mRNA (B, n = 3). A indicates negative control (NC, lines 1and 2), 5% CSE (lines 3 and 4), 5%CSE with 50 µg/ml of FK706 (lines 5 and 6), 5%CSE with 10 µg/ml of FK706 (lines 7 and 8), and 50 µg/ml of FK706 (lines 9 and 10). The ratio of density is on the ordinate and the experimental groups are on the abscissa. Values are expressed as mean ± SD. *P < 0.05 compared with supernatant fluids from HFL-1 cells cultured with CSE. **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with CSE. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with CSE.

 
Effects of FK706 on NF-{kappa}B Activation from HFL-1
NF-{kappa}B was activated by CSE. FK706 significantly inhibited NF-{kappa}B activation on HFL-1 in response to CSE (5% CSE 0.191 ± 0.046; CSE with 5 µg/ml FK706 0.100 ± 0.023; CSE with 10 µg/ml FK706 0.105 ± 0.042; CSE with 50 µg/ml FK706 0.085 ± 0.043; Figure 7, n = 4). FK706 did not enhance baseline NF-{kappa}B activation (P > 0.05).



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  		Figure 7. Effects of FK706 on NF-{kappa}B activation from CSE-stimulated HFL-1 (n = 4). NF-{kappa}B activation was evaluated with reading optical density at 450 nm. NF-{kappa}B activation (optical density at 450 nm) is on the ordinate and the experimental groups are on the abscissa. Values are expressed as mean ± SD. *P < 0.05 compared with supernatant fluids from HFL-1 cells cultured with CSE. **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with CSE. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with CSE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that HFL-1 release NCA, MCA, and chemokines including IL-8, MCP-1, G-CSF, and GM-CSF in response to CSE. Several structurally different serine protease inhibitors attenuated the release of NCA and MCA from CSE-stimulated HFL-1. FK706, a novel serine protease inhibitor, suppressed the expression of IL-8, MCP-1, G-CSF, and GM-CSF. These protease inhibitors had no effect on NCA or MCA released by unstimulated HFL-1. Consistent with these results, FK706 treatment of HFL-1 also showed the suppressive effect on expression of IL-8 and MCP-1 mRNA and activation of NF-{kappa}B significantly. These results suggest that serine protease inhibitors might have some direct actions to modulate the interaction between CSE and HFL-1 in the inflammatory disease.

Cigarette smoke is a complex aerosol of compounds, of which 4,800 have been identified (19). Although the mechanisms of the developing smoke-associated disease are not fully understood, protease and antiprotease imbalance is a largely accepted theory of lung destruction from smoking (20, 21). Increased numbers of neutrophils and monocytes activated by cigarette smoke produce large amounts of proteases and oxidants responsible for lung destruction (3), and cigarette smoke inactivates {alpha}1AT, the major antiprotease in the lung parenchyma (22). Antiproteases have been reported to modulate neutrophil migration in response to CSE from lung epithelial cells (4). Furthermore, cigarette-induced connective tissue breakdown can be prevented by administration of excess {alpha}1AT in mice (23). The current study revealed that serine protease inhibitors attenuated NCA, MCA, and chemokine production from HFL-1 in response to CSE. In this context, protease inhibitors may reduce lung injury not only directly by preventing destruction of connective tissue, but also indirectly by attenuating recruitment of neutrophils and monocytes to the site of cigarette smoke–induced lung inflammation.

We investigated the effect of protease inhibitors on HFL-1 because lung fibroblasts constitute 35–40% of the cells in the interstitium of the lung and are activated to proliferate and synthesize various chemokines during inflammation (5, 24). Moreover, fibroblasts have been reported to produce large amounts of the chemokines, IL-8, MCP-1, G-CSF, eotaxin, RANTES, and GM-CSF in response to various stimuli, including CSE (5, 25), suggesting the contribution to certain disease states. In the present study, CSE stimulated the release of chemokines and an increase in NCA and MCA. These observations are consistent with the concept that fibroblasts may be an important source of neutrophil and monocyte chemoattractants in lung inflammation. The present study confirmed that fibroblasts had the potential for contributing to airway inflammation by releasing NCA and MCA, and suggested that an imbalance between protease and antiproteases in the lower respiratory tract might augment lung inflammation by modulating the responsiveness of fibroblasts. However, a limitation of these studies was that they were done in vitro with a human fibroblast cell line. The effects of protease inhibitors on primary cultures of human airway fibroblasts and demonstrating this phenomenon in vivo are important issues for future research.

FK706 is a water-soluble, chrolmethyl ketone derivative that inhibits serine proteases (14). The inhibition constant (Ki) value for human neutrophil elastase is 4.2 nM. This compound inhibits human neutrophil elastase activity and porcine pancreatic elastase activity with respective IC50 values of 83 nM and 100 nM. FK706 acts against the elastase-induced lung hemorrhage and elastase-induced skin edema in animal models (14). FK706 consists of a trifluoromethyl ketone motif, as an active site, with a molecular mass of 0.59 kD. In this study, we demonstrated that FK706 blocked the release of inflammatory chemokines, suppressed the expression of IL-8 and MCP-1 mRNA, and suppressed NF-{kappa}B activation. It seems possible that FK706 may directly block human lung fibroblasts activation of NF-{kappa}B, preventing expression of inflammatory chemokines during cigarette smoke–induced lung inflammation.

We showed that several structures different protease inhibitors attenuated the release of chemotactic activity for inflammatory cells from lung fibroblasts. FK706 was compared with other protease inhibitors, and there were no significant differences in the inhibition of NCA and MCA. These results suggest that the FK706 might have anti-inflammatory effect similar to other antiproteases in this experimental condition. However, a lower concentration of FK706 on a weight basis was needed to inhibit enzyme activity because it has a lower molecular mass than {alpha}1AT (FK706: molecular mass = 0.59 kD, IC50 = 0.049 µg/ml, {alpha}1AT: molecular mass = 54 kD, IC50 = 1 µg/ml). Low molecular inhibitors might have advantage of better penetration into the lung if given systemically. Moreover, FK706 is specific for elastase-type endopeptidase and it has no inhibitory effect against other serine proteases such as human pancreatic trypsin, human pancreatic chymotrypsin, and leukocyte cathepsin G (14).

Lower respiratory tract inflammation is a common feature in smoking-associated diseases. Cigarette smoke is known to increase oxidative stress, and the latter is known to activate NF-{kappa}B, a major nuclear factor inducing the release of several inflammatory chemokines. Expression of several genes, such as cyclooxygenase-2, matrix metalloproteinase-9, inducible nitric oxide synthase, tumor necrosis factor, IL-8, and cell surface adhesion molecules is regulated by NF-{kappa}B (11). Although it is not fully understood which signaling pathways were stimulated and/or inhibited in response to protease inhibitors, several antiproteases including {alpha}1AT, TLCK, N{alpha}-p-tosyl-L-phenylalanine chloromethyl ketone, and secretory leukocyte protease inhibitor reduced activation of NF-{kappa}B (26–28). Metalloproteinases have been suggested to regulate chemokine activity (28), however, metalloproteinase inhibitors and cysteine proteinase inhibitors were not examined in these studies. In this context, it was consistent that FK706 might attenuate the response of human lung fibroblasts to CSE through suppression of NF-{kappa}B signaling pathway.

Although most serine protease inhibitors cannot penetrate cell membranes, the present study revealed that protease inhibitors affected the responses of HFL-1 cells in response to CSE. The mechanisms of antiprotease inhibition of the inflammatory action, including receptors and signaling pathways are still unclear. Perlmutter and coworkers (29) reported the existence of {alpha}1AT-elastase complex receptors on human hepatoma cells and human monocytes. In recent years, neutrophil elastase has been shown to induce IL-8 gene upregulation in bronchial epithelial cells through a receptor-associated kinase signaling pathway, suggesting that neutrophil elastase stimulates an as yet unidentified receptor (30). Induction of IL-8 has been associated with NF-{kappa}B activation. Although these data are consistent with FK706 blocking NF-{kappa}B signaling pathway through NE-dependent receptor or other unidentified receptor, the mechanism of action is not known.

In summary, a number of protease inhibitors attenuated NCA and MCA in response to CSE on HFL-1. Therefore, protease inhibitors not only have antiproteolytic activity, but also the anti-inflammatory effect for CSE-stimulated HLF-1. In addition, NF-{kappa}B–activated pathway may be concerned with this anti-inflammatory action. Thus, extra cellular protease inhibitors, such as FK706, may be effective in attenuating inflammatory interaction between HFL-1 and CSE.


   Acknowledgments
 
This study was supported by a Merit Review grant from the Veterans' Administration and a grant from the Flight Attendants Medical Research Institute.

Received in original form March 27, 2003

Received in final form May 2, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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