Serine protease inhibitors modulate chemotactic cytokine production by human lung fibroblasts in vitro

Hiroki Numanami1, Sekiya Koyama2, Esturo Sato2, Masayuki Haniuda3, Dan K. Nelson1, Jeffrey C. Hoyt1, Jon L. Freels1, Michael P. Habib1, and Richard A. Robbins1

1 Research Service, Southern Arizona Veterans Health Care System, and Arizona Respiratory Center, University of Arizona, Tucson, Arizona 85723; and 2 The First Department of Internal Medicine and 3 The Second Department of Surgery, School of Medicine, Shinshu University, Matsumoto, Japan 390-0802


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha . An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha 1-antitrypsin, or Nalpha -p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha . NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release.

neutrophil; monocyte; interleukin-8; monocyte chemoattractant protein-1; granulocyte/macrophage colony- stimulating factor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A BROAD SPECTRUM OF INFLAMMATORY lung disorders is characterized by tissue destruction and remodeling, caused by an excess of protease activity, e.g., adult respiratory distress syndrome, pulmonary emphysema, bronchopulmonary dysplasia, and cystic fibrosis (2, 3, 48). Protease inhibitors may protect the lung from detrimental destruction by proteases. However, antiproteases may also modulate neutrophil migration (22, 23, 40). This observation suggests that protease inhibitors not only might act directly on proteolysis but may also attenuate inflammation by inhibiting production of chemokines.

The fibroblast is the principal connective tissue cell involved in the synthesis of the collagenous and noncollagenous components of the extracellular matrix. This synthetic activity serves an important structural function by providing a frame network for organ integrity. In addition to this traditionally accepted function, recent studies have demonstrated that fibroblasts may also participate in the orchestration of acute and chronic inflammation. In this context, fibroblasts release monocyte chemoattractant protein-1 (MCP-1), granulocyte/macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-beta , in response to inflammatory cytokines such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha (25, 26, 28, 33). Moreover, fibroblasts secreted a variety of proteases, including plasmin (10), fibroblast activation protein (37), tissue-type plasminogen activator (46), urokinase-type plasminogen activator (16), and a calcium-dependent serine protease (34). Therefore, the fibroblast, because of its anatomical location and protease secretion, is in a pivotal position to participate in and direct communications between interstitial and vascular events in pulmonary inflammation and fibrosis.

On the basis of the rationale above, the purpose of this study was to demonstrate that protease inhibitors modulate the release of proinflammatory cytokines by HFL (human fetal lung)-1 cells. The results demonstrate that the serine protease inhibitor FK-706 inhibited the release of neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) from the lung fibroblast cell line, HFL-1. These results suggest that the interaction between HFL-1 and proinflammatory cytokines involves proteolytic mechanism(s) and that protease inhibitors may have the potential for modulating lung inflammation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cultures. HFL-1 fibroblasts (lung, diploid, human, passage 14) were purchased from American Type 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 (24, 35, 36). After 2-4 days in culture, the cells had reached confluence and were then used for experiments.

Protease inhibitors and stimulants. FK-706 (C26H32F3N4NaO7; Fujisawa Pharmaceutical, Osaka, Japan) was used as a serine protease inhibitor (38). HFL-1 cells were exposed to human recombinant IL-1beta (500, 50, 5, and 0.5 pg/ml; Sigma, St. Louis, MO) or TNF-alpha (100, 10, 1, and 0.1 ng/ml; Sigma) at 37°C in a humidified 5% CO2 atmosphere for 24 h. In some experiments, HFL-1 cells were pretreated with several concentrations of the serine protease inhibitors FK-706 (50, 10, and 5 µg/ml), alpha 1-antitrypsin (200, 100, and 10 µg/ml; Sigma), or Nalpha -p-tosyl-L-lysine chloromethyl ketone (TLCK; 200 and 100 µM; Sigma). In the other experiments, the cysteine protease inhibitor, leupeptin (200 and 100 µM; Sigma), or a metalloprotease inhibitor of neutral endopeptidase 24.11, phosphoramidon (200 and 100 µM; Sigma), were used. The concentrations of FK-706 were based on results showing that neutrophil elastase-induced lung hemorrhage of mice was significantly inhibited by intratracheal treatment with FK-706 at a dose from 1 to 100 µg (38). In addition, the concentrations of other protease inhibitors were based on previous studies (22, 23). In some cultures, neutrophil elastase (Elastin Products, Owensville, MO) was used to reverse the effects of FK-706. IL-1beta and TNF-alpha were tested for LPS contamination, and LPS was shown to be <0.1 ng/ml. These cytokines did not cause HFL-1 cell injury (no deformity of cell shape, no detachment from culture dish, and >98% viability by trypan blue exclusion) after 24 h of incubation at the highest concentration used. The culture supernatant fluid was harvested and frozen at -80°C until assay. At least six separate HFL-1 cell supernatant fluids were harvested from cultures for each experimental condition.

Measurement of cytokines in the supernatant fluids. The concentrations of IL-8, MCP-1, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and GM-CSF were measured in the cell supernatant fluids using commercially available ELISAs (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, 9.0 pg/ml for M-CSF, 20 pg/ml for G-CSF, and 3.0 pg/ml for GM-CSF.

Effects of protease inhibitors on NCA and MCA by HFL-1 supernatant fluids. Polymorphonuclear leukocytes were purified from heparinized normal human blood by the method of Böyum (4). 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 Gey's balanced salt solution (GIBCO, Grand Island, NY) containing 2% bovine serum albumin (BSA; Sigma) at pH 7.2 to give a final concentration of 3.0 × 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 Gey's balanced salt solution containing 2% BSA at pH 7.2 to give a final concentration of 5.0 × 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, Cabin John, MD), as has been described previously (13). A 10-mm-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 top pieces of the chamber were applied, and 50 µl of the cell suspension was placed into the top 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, McGaw Park, IL), and mounted on a glass slide. Cells that completely migrated through the filter were counted by using light microscopy in 10 random high-power fields per well.

Evaluation of mRNA expression. Cytokine mRNA was analyzed by RT-PCR. HFL-1 cells were incubated with FK-706 and cytokines for 12 h, and total cellular RNA was extracted from adherent cells using a modification of the methods of Chomczynski and Sacchi (7). 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, using a commercially available primer pair (R&D Systems), were added at 0.3 µM final concentration. PCR was performed in a Perkin Elmer 480 thermal cycler using 94°C for 2 min and 26 cycles consisting of 94°C for 45 s, primer annealing at 55°C for 45 s, and primer extension at 72°C for 45 s, followed by 72°C for an additional 7 min. beta -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 beta -actin.

Statistical analysis. Data were analyzed by Dunnett's one-way analysis of variance with a Fisher's protected least significant differences test. In all cases, P < 0.05 was considered significant. The data are expressed as means ± SD.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

Release of NCA and MCA from HFL-1 cells in response to IL-1beta and TNF-alpha . IL-1beta or TNF-alpha stimulated the release of NCA and MCA from HFL-1 in a dose-dependent fashion (Fig. 1, A-D, n = 6). The release of NCA and MCA was observed after 24 h of incubation in response to 100 pg/ml of IL-1beta and 1 ng/ml of TNF-alpha . IL-1beta and TNF-alpha induced the release of significant NCA at 5 pg/ml and 0.1 ng/ml, and MCA at 0.1 pg/ml and 0.1 ng/ml, respectively.


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Fig. 1.   Neutrophil chemotactic activity (NCA; A and C) and monocyte chemotactic activity (MCA; B and D) in response to IL-1beta (A and B) or TNF-alpha (C and D) from human fetal lung fibroblast (HFL-1) monolayers after 24 h of incubation (n = 6). Chemotactic activities are on the ordinate, and the concentration of IL-1beta and TNF-alpha is on the abscissa. Values are expressed as means ± SD. *P < 0.05 compared with supernatant fluids without stimuli. **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured without stimuli. ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured without stimuli.

Effects of FK-706 on cytokine production from HFL-1. HFL-1 spontaneously released IL-8, MCP-1, M-CSF, G-CSF, and GM-CSF, but the inflammatory cytokines IL-1beta and TNF-alpha stimulated the release of these cytokines from HFL-1 (Table 1, n = 4).

                              
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Table 1.   Concentration of cytokines in HFL-1 culture supernatant fluids after 24 h

The release of IL-8 and MCP-1 was dose dependently inhibited by FK-706 (Fig. 2, n = 4). FK-706 had no stimulating effect on release of these chemokines from HFL-1 (Table 1).


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Fig. 2.   Effects of FK-706 on IL-8 (A and B) or monocyte chemoattractant protein-1 (MCP-1; C and D) stimulated with IL-1beta (100 pg/ml, A and C) or TNF-alpha (1,000 pg/ml, B and D) from HFL-1 monolayers after 24 h of 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 means ± SD. **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha . ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha .

FK-706 also inhibited the release of M-CSF, GM-CSF, and G-CSF in response to IL-1beta and TNF-alpha from HFL-1 cell monolayers (Table 1, n = 4).

Effects of protease inhibitors on NCA and MCA. IL-1beta or TNF-alpha stimulated NCA and MCA from HFL-1. FK-706 inhibited, in a dose-dependent manner, NCA and MCA from HFL-1 stimulated with IL-1beta or TNF-alpha (Fig. 3, n = 6). FK-706 alone had no effect on baseline release of NCA and MCA under control conditions (P > 0.05). The other serine protease inhibitors alpha 1-antitrypsin and TLCK reduced the IL-1beta - or TNF-alpha -induced NCA and MCA (Fig. 4, n = 6). None of these inhibitors altered baseline NCA and MCA release (P > 0.05, Fig. 4).


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Fig. 3.   Effects of FK-706 on NCA (closed bars) and MCA (open bars) in response to IL-1beta (A) or TNF-alpha (B) from HFL-1 monolayers after 24 h of incubation (n = 6). NCA and MCA are on the ordinate, and the experimental groups are on the abscissa. Values are expressed as means ± SD. **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha . ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha .



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Fig. 4.   Effects of alpha 1-antitrypsin (alpha 1-AT; A and B) and Nalpha -p-tosyl-L-lysine chloromethyl ketone (TLCK; C and D) on NCA (closed bars) and MCA (open bars) in response to IL-1beta (A and C) or TNF-alpha (B and D) from HFL-1 monolayers after 24 h of incubation (n = 6). NCA and MCA are on the ordinate, and the experimental groups are on the abscissa. Values are expressed as means ± SD. **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha . ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha .

IL-1beta - and TNF-alpha -induced release of NCA and MCA from HFL-1 monolayers was not modulated by phosphoramidon at all concentrations tested (P > 0.05, Fig. 5, A and B, n = 6). However, leupeptin significantly inhibited the release of NCA and MCA (Fig. 5, C and D, n = 6).


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Fig. 5.   Effects of leupeptin (A and B) and phosphoramidon (C and D) on NCA (closed bars) and MCA (open bars) in response to IL-1beta (A and C) or TNF-alpha (B and D) from HFL-1 monolayers after 24 h of incubation (n = 6). NCA and MCA are on the ordinate, and the experimental groups are on the abscissa. Values are expressed as means ± SD. *P < 0.05 compared with supernatant fluids with IL-1beta or TNF-alpha . **P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha . ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta or TNF-alpha .

Effect of elastase on the anti-inflammatory action of FK-706. Neutrophil elastase attenuated the anti-inflammatory action of FK-706. In the sample of preincubated FK-706 and 50 µg/ml of neutrophil elastase, the inhibition for releases of NCA, MCA, and IL-8 in response to IL-1beta decreased compared with FK-706 (Fig. 6, n = 4). The preincubated reagents alone had no effect on baseline releases of NCA, MCA, and IL-8 under control conditions (P > 0.05).


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Fig. 6.   Effects of neutrophil elastase (NE) on the anti-inflammatory action of FK-706 on IL-8 expression (hatched bars), NCA (closed bars), and MCA (open bars). Values are expressed as means ± SD, n = 4. ** P < 0.01 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta . ***P < 0.001 compared with supernatant fluids from HFL-1 cells cultured with IL-1beta .

Effects of FK-706 on mRNA expression from HFL-1. Semiquantitative RT-PCR was performed to evaluate the effect of FK-706 on cytokine mRNA expression in HFL-1 (n = 3). IL-1beta - or TNF-alpha -induced IL-8 and MCP-1 mRNA expressions were suppressed by pretreatment with FK-706 (Fig. 7).


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Fig. 7.   Effects of FK-706 on IL-8 and MCP-1 mRNA expression from HFL-1 stimulated with IL-1beta or TNF-alpha . Representative results from 3 experiments with RT-PCR for IL-8, MCP-1, and beta -actin.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrated that HFL-1 released NCA, MCA, and cytokines, including IL-8, MCP-1, G-CSF, M-CSF, and GM-CSF, in response to proinflammatory cytokines. Several structurally different serine protease inhibitors attenuated the release of NCA and MCA by the HFL-1 in response to IL-1beta or TNF-alpha . FK-706 suppressed the expression of these cytokines. These protease inhibitors had no effect on NCA or MCA by unstimulated HFL-1. Consistent with these results, FK-706 treatment of HFL-1 also showed the suppressive effect on cytokine production and expression of IL-8 and MCP-1 mRNA. These results suggest that HFL-1 interaction with IL-1beta or TNF-alpha may involve proteolytic mechanisms, and serine protease inhibitors may modulate the interaction between proinflammatory cytokines and HFL-1.

Protease imbalance has been proposed in the pathogenesis of several acute and chronic inflammatory diseases, including pulmonary emphysema (5, 12), adult respiratory distress syndrome (27, 31), cystic fibrosis (17, 32), chronic bronchitis (29), septic shock (44), and other inflammatory states (1, 14). Antiproteases have been reported to modulate neutrophil migration in response to several stimuli (22, 23, 40). Furthermore, Churg and coworkers (8) noted that alpha 1-antitrypsin suppressed silica-induced neutrophil influx and MCP-1 gene expression. Kikuchi et al. (19) reported that secretory leukocyte protease inhibitor (SLPI) and alpha 1-antitrypsin augmented hepatocyte growth factor production in human lung fibroblasts. SLPI has also been suggested to suppress prostaglandin E2 and metalloproteinase production in monocytes (49). In this context, protease inhibitors may reduce lung injury directly by preventing destruction of connective tissue, but also indirectly by attenuating recruitment of neutrophils and monocytes to sites of inflammation. Activated inflammatory cells release a variety of degradative enzymes, oxygen metabolites, and cytokines, which may lead to further tissue damage.

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 cytokines during inflammation (24). Moreover, fibroblasts have been reported to produce large amounts of the chemotactic cytokines, IL-8, MCP-1, G-CSF, and GM-CSF, in response to various stimuli (41, 42). In the present study, IL-1beta or TNF-alpha stimulated the release of these cytokines 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 antiprotease activity 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.

In this study we used phosphoramidon as a matrix metalloprotease inhibitor, but it did not inhibit either NCA or MCA. Other reports indicate that matrix metalloprotease inhibitors reduce inflammation in vivo and in vitro (18, 43). Phosphoramidon is a selective inhibitor of neutral endopeptidase (NEP), a metalloprotease on the surface membrane of fibroblasts (15). Moreover, NEP expression from lung fibroblasts was enhanced by IL-1beta and TNF-alpha (21). In contrast, other reports suggest that NEP may reduce inflammation by enzymatic cleavage of inflammatory substances, such as bradykinin (45), substance P (39), and the chemotactic peptide formyl-Met-Leu-Phe (11). These later observations would suggest that matrix metalloproteinase inhibitors are unlikely to work as anti-inflammatories by inhibition of NEP, whose activity may reduce inflammation.

FK-706 is a water-soluble, chloromethyl ketone derivative that inhibits serine proteases (38). The 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 and 100 nM. FK-706 acted against elastase-induced lung hemorrhage and elastase-induced skin edema in animal models (38). FK-706 consists of a trifluoromethyl ketone motif, as an active site, with a molecular mass of 0.59 kDa. Although it is not fully understood which signaling pathways were stimulated and/or inhibited in response to protease inhibitors, chloromethyl ketone derivatives reduce activation of NF-kappa B, a major nuclear factor inducing the release of several inflammatory cytokines (6, 20). In this study, we demonstrated that FK-706 blocked the release of inflammatory cytokines and suppressed the expression of IL-8 and MCP-1 mRNA. These data indicate that FK-706 may attenuate the response of lung fibroblasts to IL-1beta and TNF-alpha upstream at or before transcription, possibly 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 to proinflammatory cytokines. The mechanisms of antiprotease inhibition of the inflammatory action, including receptors and signaling pathways, are still unclear. In recent years, protease-activated receptors have been described that are proteolysis-activated G protein-coupled receptors (9, 30). Neutrophil elastase induces IL-8 gene upregulation in bronchial epithelial cells through an IL-1beta receptor-associated kinase signaling pathway, suggesting that neutrophil elastase stimulates an as yet unidentified receptor (47). In this study, neutrophil elastase attenuated the anti-inflammatory action of FK-706. In this context, FK-706 may act through protease-activated receptors or other unidentified mechanisms.

In conclusion, a number of protease inhibitors attenuated NCA and MCA in response to proinflammatory cytokines on HFL-1. Therefore, proteolytic activity appears to play a critical role in the release of NCA and MCA. Antiprotease attenuated this activity at the transcriptional level. Thus extracellular protease inhibitors, such as FK-706, may be effective in attenuating inflammatory interaction between HFL-1 and proinflammatory cytokines.


    ACKNOWLEDGEMENTS

This work was supported by a Merit Review grant from the Veterans Administration.


    FOOTNOTES

Address for reprint requests and other correspondence: R. A. Robbins, Research Service Line, Southern Arizona Veterans Health Care System, 3601 S. 6th Ave., Tucson, AZ 85723 (E-mail: Richard.Robbins2{at}med.va.gov).

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.

First published December 13, 2002;10.1152/ajplung.00211.2002

Received 3 July 2002; accepted in final form 10 December 2002.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 284(5):L882-L890




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