Serine proteases increase oxidative stress in lung cells

Kazutetsu Aoshiba, Kimihiko Yasuda, Shuji Yasui, Jun Tamaoki, and Atsushi Nagai

First Department of Medicine, Tokyo Women's Medical University, Shinjuku-ku, Tokyo 162-8666, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Several serine proteases are directly cytotoxic. We investigated whether the cytotoxic effects of proteases are associated with increased levels of reactive oxygen species (ROS) in cells. We found that treatment of lung fibroblasts or bronchial epithelial cells with relatively high concentrations (0.1-100 U/ml) of neutrophil elastase, trypsin, and Pronase increased ROS levels in the mitochondria and cytoplasm. The protease-induced increase in ROS was associated with oxidative cellular injury as determined by generation of 8-hydroxy-2'-deoxyguanosine and malonaldehyde plus 4-hydroxyalkenal. The protease-induced increase in ROS was not merely due to cell detachment because the proteases also caused an increase in ROS in suspended cells, which precluded attachment to the extracellular matrix. The protease-induced increase in ROS appears to contribute to cytotoxicity because cell death induced by proteases was attenuated by treatment with catalase, a decomposer of H2O2, and accelerated by treatment with aminotriazole, a catalase inhibitor. These results suggest that several proteases increase oxidative stress, indicating a direct interaction between proteases and ROS in mediating cytotoxicity.

oxidants; reactive oxygen species; elastase; trypsin; Pronase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BECAUSE OF THEIR ABILITY to cause extensive tissue destruction, proteases are thought to be involved in the mechanism of a variety of lung diseases. Many proteases have been demonstrated to degrade extracellular matrix (ECM) proteins and other substrates such as complement components, proteinase inhibitors, and hemostatic proteins. In particular, the proteolytic degradation of ECM proteins has been established as a mechanism of tissue destructive disorders including pulmonary emphysema, acute respiratory distress syndrome, bronchiectasis, and pulmonary fibrosis (13, 31).

Besides their capacity to degrade noncellular components, serine proteases such as neutrophil elastase, trypsin, and bacterial Pronase are known to mediate direct cytotoxicity (2, 20, 36, 37). They primarily cause cytolysis by inducing necrosis, and some of them have been shown to induce apoptosis of endothelial cells (37), keratinocytes (18), and neutrophils (32). The mechanism of the cytotoxicity of proteases, however, is not fully understood.

Possible mechanisms of protease cytotoxicity include reactive oxygen species (ROS). ROS are produced in response to a variety of cytotoxic stimuli and have been hypothesized to be important mediators of cell death, whether by necrosis or apoptosis (12, 23). However, it is unknown whether ROS are involved in the mechanism of cytotoxicity induced by proteases.

Proteases and ROS are frequent companions at sites of inflammation, and previous studies have pointed to interactions between them. For example, ROS convert the inactive form of procollagenase to its active form (34) and protect proteases by inactivating proteinase inhibitors (6, 21, 33, 35), whereas neutrophil-derived neutral proteases stimulate ROS release from monocytes (28). Although these findings suggest that proteases and ROS may interact with each other under certain circumstances, the role of ROS in protease-induced cytotoxicity is unknown.

In this study, we investigated whether the cytotoxic effects of proteases are associated with increased intracellular levels of ROS within cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Cell culture reagents were obtained from GIBCO BRL (Life Technologies, Gaithersburg, MD), unless otherwise stated. 6-Carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (CDCF) and reduced MitoTracker Red probe (CM-H2XRos) were obtained from Molecular Probes (Eugene, OR). Human neutrophil elastase, trypsin, Pronase, catalase, 3-amino-1,2,4-triazole, o-phenylenediamine, horseradish peroxidase, poly(2-hydroxyethyl methacrylate) (polyHEMA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide, calphostin C, H-89, genistein, herbimycin A, sodium orthovanadate, antimycin A, and pertussis toxin were purchased from Sigma (St. Louis, MO). ONO-6818, a specific inhibitor of neutrophil elastase, was a kind gift from Ono Pharmaceuticals (Osaka, Japan).

Cell culture and protease treatment. Normal human fetal lung fibroblasts (IMR-90, Clonetics, San Diego, CA) were maintained in Dulbecco's modification of Eagle's MEM (DMEM) containing 10% FCS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Cells were passaged weekly, and cells from passages 8 to 20 were used in the experiments. Normal human bronchial epithelial cells were prepared as described (25) and maintained in bronchial epithelial cell basal medium (modified LHC-9; Clonetics) containing 15 µg/ml of bovine pituitary extract, 0.25 ng/ml of hydrocortisone, 0.25 ng/ml of epidermal growth factor, 0.25 ng/ml of epinephrine, 5 µg/ml of transferrin, 2.5 ng/ml of insulin, 0.05 ng/ml of retinoic acid, 0.25 mg/ml of bovine serum albumin, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Cells from passages 2 to 4 were used in the experiments. Human lymphocytes were isolated from venous blood by dextran sedimentation and centrifugation on a Histopaque gradient (density 1.078). Before being used in the experiments, all cells were washed with PBS twice and reincubated with serum-free DMEM.

Determination of intracellular ROS levels. Intracellular ROS levels were measured with CDCF (1, 10). Briefly, the cells were loaded for 30 min with 5 µM CDCF in a 96-well tissue culture plate or a 6-well tissue culture plate. They were washed with PBS and then exposed to proteases in serum-free DMEM. For mechanical detachment, the cells in a six-well plate were detached with a rubber scraper. Fluorescence was monitored on a Cytofluor II multiplate fluorometer (Perceptive Biosystems, Framingham, MA) by using an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Determination of mitochondrial ROS levels. Mitochondrial ROS levels were determined by using CM-H2XRos (7, 10). Briefly, the cells were incubated for 30 min with 250 nM CM-H2XRos in a 96-well tissue culture plate and then exposed to proteases in serum-free DMEM. Fluorescence was monitored by excitation at 530 nm and emission at 590 nm.

Determination of H2O2 levels. After the cells were treated with neutrophil elastase (100 U/ml) in serum-free DMEM for 3 h, conditioned medium with 5 × 10-4 M ONO-6818 was added to inhibit the resultant enzyme activity. H2O2 levels in the conditioned medium were measured as described (4). Briefly, 50 µl of the medium were combined with 75 µl of 100 mM Tris · HCl buffer, pH 8.0, containing 16 mM o-phenylenediamine and 1 U/ml of horseradish peroxidase in a 96-well microplate and incubated for 30 min at 37°C. The reaction was quenched with 3 N sulfuric acid, and absorbance was measured at 490 nm, with 690 nm as a reference wavelength. H2O2 levels were estimated by comparison with values on a reference curve generated with known amounts of H2O2. We also determined whether cells degrade H2O2 added externally to the medium. Briefly, sufficient H2O2 was added to 100-mm dishes containing cells and serum-free DMEM to produce a final concentration of 20 µM H2O2. H2O2 levels in the medium were monitored before and after the addition of H2O2 at 5- to 10-min intervals for 40 min.

Assay for DNA and lipid oxidation. The cells were exposed to neutrophil elastase (10 or 100 U/ml) for 3 h, and the resultant enzyme activities were inhibited as above. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) levels in the conditioned medium were measured with an ELISA kit (Japan Institute for the Control of Aging, Shizuoka, Japan). Malonaldehyde (MDA) plus 4-hydroxyalkenal levels in the cell extracts were measured by using a colorimetric assay kit (BIOXYTECH LPO-586, OXIS International, Portland, OR) according to the manufacturer's instructions. The assay is based on the reaction between a chromogenic reagent, N-methyl-2-phenylindole, and MDA plus 4-hydroxyalkenal to yield a stable chromophore, with maximal absorbance at 586 nm.

Cell viability assay. The MTT assay, which estimates the number of viable cells, was performed as described (27). MTT (0.5 mg/ml) was added to the cells in serum-free DMEM in a 96-well culture plate during the final 1 h of incubation with the proteases. Cell viability was also determined by quantitating propidium iodide uptake as a measure of cell death (14). Propidium iodide (20 µg/ml) was added to the cells in a 96-well culture plate, and fluorescence was read 10 min later, with excitation at 530 nm and emission at 620 nm. The cells were lysed by a cycle of freezing and thawing, and fluorescence was read to estimate cell numbers. The percent uptake of propidium iodide was calculated according to the formula (fluorescence before freezing and thawing/fluorescence after freezing and thawing) × 100.

Statistical analysis. Data are presented as means ± SE. Comparisons were made with Student's t-test or ANOVA with Fisher's protected least significant difference as a post hoc analysis test. A value of P < 0.05 was accepted as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

As observed previously (2, 20, 36, 37) and described again below, incubation with several serine proteases, i.e., neutrophil elastase, trypsin, and Streptomyces griseus Pronase, which is a mixture of protease A, protease B, and a trypsinlike enzyme, resulted in detachment and death of lung fibroblasts and bronchial epithelial cells.

We investigated whether these proteases increase intracellular ROS by monitoring their levels with the fluorochrome CDCF. As demonstrated in Fig. 1, ROS accumulated within cells maintained in medium alone, suggesting that ROS are produced as by-products of aerobic respiration as previously described (3). When fibroblasts were exposed to neutrophil elastase (1-100 U/ml; Fig. 1A), trypsin (0.1-10 U/ml; Fig. 1B), or Pronase E (0.1 or 10 U/ml; Fig. 1C), the levels of CDCF fluorescence rose twofold or more compared with those in cells maintained in medium alone. The effect of proteases on ROS levels was not cell-type specific because bronchial epithelial cells also showed increased levels of CDCF after exposure to neutrophil elastase (Fig. 1D). We also determined whether these proteases directly oxidize CDCF. Interestingly, we found that Pronase E, but not neutrophil elastase or trypsin, directly oxidized CDCF in the absence of cells (Fig. 1E). Although we removed CDCF from the culture medium by rinsing the cells twice with PBS before exposure to proteases, CDCF that leaked out of cells during exposure to Pronase E may have been directly oxidized by Pronase E. The rise in ROS levels after exposure to proteases appears to require their proteolytic activity because ONO-6818, a specific inhibitor of neutrophil elastase, abolished the elastase-induced rise in CDCF fluorescence (Fig. 2A). ONO-6818 did not inhibit a rise in CDCF fluorescence induced by H2O2 (Fig. 2B), indicating that ONO-6818 does not directly affect H2O2 or CDCF. CDCF fluorescence reflects various ROS including intracellular H2O2 (10, 11), which passes freely through the cell membrane and diffuses readily out of cells. We therefore measured H2O2 concentrations in conditioned medium with a horseradish peroxidase assay with the substrate o-phenylenediamine. We found that neutrophil elastase did not directly oxidize o-phenylenediamine in the absence of cells (data not shown). As demonstrated in Fig. 3A, incubation of fibroblasts with 100 U/ml of neutrophil elastase in serum-free culture medium for 3 h produced a twofold increase in extracellular H2O2 level. We also determined whether cells, which are thought to be equipped with various antioxidants, are capable of decomposing H2O2 added externally to the culture medium. As demonstrated in Fig. 3B, the addition of 20 µM H2O2 to dishes containing fibroblasts bathed in serum-free culture medium produced a step increase in H2O2 level that was sustained for at least 40 min, suggesting that cells are insufficient to decompose H2O2. These results indicate that serine proteases, including neutrophil elastase, trypsin, and bacterial Pronase, increase ROS levels in cells.


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Fig. 1.   Exposure to serine proteases increases intracellular reactive oxygen species (ROS) levels. Fibroblasts (A-C) and bronchial epithelial cells (D) were loaded with 5 µM 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (CDCF) and then exposed to medium alone (open circle ) or neutrophil elastase (A and D), trypsin (B), or bacterial Pronase (C) at concentrations of 0.1 (), 1 (), 10 (), or 100 (black-triangle) U/ml in 96-well culture tissue plates. CDCF fluorescence levels were monitored with a spectrofluorometer. Data are means ± SE from 5 samples. Significantly different from cells incubated with medium alone: * P < 0.05. E: effects of proteases on CDCF fluorescence levels in the absence of cells. Culture medium was added with 5 µM CDCF in the absence of cells and then exposed to medium alone (open circle ), 10 U/ml of neutrophil elastase (), 10 U/ml of trypsin (), or 10 U/ml of bacterial Pronase (). Data are means ± SE from 3 samples. ** P < 0.01 vs. medium alone.



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Fig. 2.   A: protease-induced increases in ROS require proteolytic activity. Fibroblasts were loaded with 5 µM CDCF and then exposed to medium alone (open circle ), 100 U/ml of neutrophil elastase (), or 100 U/ml of neutrophil elastase plus the specific neutrophil elastase inhibitor ONO-6818 (5 × 10-4 M; black-triangle). CDCF fluorescence levels were monitored with a spectrofluorometer. Data are means ± SE from 5 samples. Significantly different from cells incubated with medium alone: dagger  P < 0.05; dagger dagger P < 0.01. Significantly different from cells exposed to neutrophil elastase alone: * P < 0.05; ** P < 0.01. B: effects of ONO-6818 on CDCF fluorescence levels. Culture medium was added with 5 µM CDCF in the absence of cells and then exposed to 100 µM H2O2 or 100 µM H2O2 plus ONO-6818 (5 × 10-4 M). CDCF fluorescence levels were measured after 30 min. Data are means ± SE from 3 samples. dagger dagger P < 0.01 vs. medium alone.



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Fig. 3.   A: neutrophil elastase increases H2O2 production by fibroblasts. Fibroblasts were exposed to serum-free medium alone, 100 U/ml of neutrophil elastase, or 100 U/ml of neutrophil elastase plus 500 U/ml of catalase for 3 h, and the concentration of H2O2 in the medium was determined by a horseradish peroxidase assay with o-phenylenediamine as the substrate. Data are means ± SE from 5 samples. ** P < 0.01 vs. cells exposed to medium alone. B: fibroblasts do not decompose H2O2 added externally to the culture medium. The concentration of H2O2 in culture medium was monitored before and after the addition of 20 µM H2O2 to the medium.

We next measured mitochondrial ROS levels in cells exposed to neutrophil elastase. Fibroblasts and bronchial epithelial cells were loaded with CM-H2XRos, a nonfluorescent compound that enters cells where it can be oxidized at various sites including the mitochondria and then be sequestrated into the mitochondria by virtue of its cationic state. As expected, incubation of fibroblasts with antimycin A, a mitochondrial ROS generator, increased CM-H2XRos fluorescence (Fig. 4A). Incubation with neutrophil elastase resulted in two- to threefold increases in CM-H2XRos fluorescence in fibroblasts (Fig. 4B) and bronchial epithelial cells (Fig. 4C), and treatment with rotenone, a specific inhibitor of mitochondrial electron transport, led to significant inhibition in the rise of CDCF and CM-H2XRos fluorescence in cells exposed to neutrophil elastase (Fig. 4, D and E). Neutrophil elastase did not increase CM-H2XRos fluorescence in the absence of cells (data not shown). These results suggest that the predominant source of ROS in protease-exposed cells is localized within the mitochondria, although some enzymatic mechanisms such as cyclooxygenase, lipoxygenase, monoamine oxidase, xanthine oxidase, NADPH oxidase, and nitric oxide synthase may also be the source of ROS.


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Fig. 4.   Role of mitochondria in the elastase-induced rise in ROS. A: increased mitochondrial ROS levels in cells treated with antimycin A, a mitochondrial ROS generator. Reduced MitoTracker Red probe (CM-H2XRos) fluorescence levels were measured in fibroblasts after 30 min of treatment with and without 10-5 M antimycin A. B and C: increased mitochondrial ROS levels in neutrophil elastase-treated cells. CM-H2XRos fluorescence levels were monitored in fibroblasts (B) and bronchial epithelial cells (C) exposed to medium alone (open circle ) or neutrophil elastase at 1 (), 10 (), or 100 (black-triangle) U/ml. D and E: inhibition of the neutrophil elastase-induced increase in ROS by the mitochondrial electron transport inhibitor rotenone. Levels of CDCF fluorescence (D) and CM-H2XRos (E) were measured in fibroblasts treated with medium alone (CO) or 100 U/ml of neutrophil elastase (NE) for 1 h in the presence and absence of 10-5 M rotenone. Data are means ± SE from 5 samples. Significantly different from cells incubated with medium alone: * P < 0.05; ** P < 0.01. dagger dagger P < 0.01 vs. cells treated with neutrophil elastase alone.

Recent evidence (16) suggests that cell detachment from culture plates increases intracellular levels of ROS. We therefore investigated whether the protease-induced rise in ROS levels was related to cell detachment. The results indicated that the protease-induced rise in ROS levels was not merely due to cell detachment from the ECM because 1) neutrophil elastase increased ROS levels in fibroblasts suspended in polyHEMA-coated plates that preclude cell attachment (Fig. 5A) and in lymphocytes, which do not attach to culture plates (Fig. 5B), and 2) much more ROS were produced in response to neutrophil elastase than to mechanical detachment of the cells (Fig. 5C).


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Fig. 5.   Protease-induced increase in ROS levels is unrelated to cell detachment. Neutrophil elastase increases CDCF fluorescence levels in fibroblasts suspended in poly(2-hydroxyethyl methacrylate)-coated plates (A) and lymphocytes (B). C: neutrophil elastase increases CDCF fluorescence levels much more than mechanical detachment of the cells. open circle , Medium alone; , 100 U/ml of neutrophil elastase; , 10 U/ml of Pronase; black-triangle, cells detached mechanically. Data are means ± SE from 4 samples. Significantly different from cells incubated with medium alone at indicated time points: * P < 0.05; ** P < 0.01. Significantly different from cells incubated with medium alone at all time points: dagger  P < 0.05; dagger dagger P < 0.01.

To investigate whether specific signaling pathways are involved in the protease-induced rise in ROS levels, we evaluated the effect of several inhibitors on the rise in ROS. The results showed that the rise in CDCF fluorescence in fibroblasts exposed to neutrophil elastase (100 U/ml) was not inhibited by prior or simultaneous exposure to the protein kinase C inhibitor calphostin C (10-6 M), the protein kinase A inhibitor H-89 (10-6 M), the broad spectrum tyrosine kinase inhibitors genistein (10-5 M) and herbimycin A (10-6 M), the tyrosine phosphatase inhibitor sodium orthovanadate (10-4 M), or the Gi protein blocker pertussis toxin (400 ng/ml) (data not shown).

Next, to determine whether the protease-induced rise in ROS causes oxidative injury to cells, we measured the level of 8-OHdG as an indicator of DNA oxidation (26), and the level of MDA plus 4-hydroxyalkenal as an indicator of lipid peroxidation (8). As demonstrated in Fig. 6, incubation with neutrophil elastase resulted in two- to fourfold increases in the levels of 8-OHdG and MDA plus 4-hydroxyalkenal, suggesting that elastase induces oxidative injury of cells.


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Fig. 6.   Elastase induces oxidation of DNA and lipids in cells. Fibroblasts were treated for 3 h with elastase in 96-well plates (A) or 100-mm plates (B). A: 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in conditioned medium were measured by ELISA. B: malonaldehyde (MDA) plus 4-hydroxyalkenal levels in cell extracts were measured by colorimetric assay with N-methyl-2-phenylindole as a substrate. Data are means ± SE from 5 samples. ** P < 0.01 vs. cells incubated with medium alone.

Finally, we sought to determine whether the changes in intracellular ROS levels contribute to the cytotoxicity by proteases. As demonstrated in Fig. 7, A-C, catalase, a decomposer of H2O2, significantly inhibited the death of fibroblasts as determined by both the MTT assay and the propidium iodide exclusion test. Because H2O2 is freely permeant in the cell membrane, and, therefore, it readily diffuses out of cells if generated intracellularly, the reduction of extracellular H2O2 by catalase is expected to decrease intracellular H2O2 (1). To further corroborate the role of ROS in protease-mediated cytotoxicity, we evaluated the effect of aminotriazole, a catalase inhibitor, on cell death. The results showed that prior and simultaneous exposure to aminotriazole produced a small but significant rise in the rate of cell death induced by Pronase (Fig. 7D). Aminotriazole did not induce cell death in the absence of Pronase E (Fig. 7D). The above findings suggest that ROS are, at least in part, responsible for protease-mediated cytotoxicity.


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Fig. 7.   Effect of catalase and aminotriazole on protease-mediated cytotoxicity. Fibroblasts were treated with 100 U/ml of neutrophil elastase (A), 10 U/ml of trypsin (B), or 1 U/ml of Pronase (C) in the presence () and absence (open circle ) of 500 U/ml of catalase. Cytotoxicity was evaluated by measuring MTT cell viability and percent uptake of propidium iodide. Data are means ± SE from 4 samples. * P < 0.05 and * P < 0.01 vs. cells treated with the proteases in the absence of catalase. D: fibroblasts were incubated with 1 U/ml of Pronase for 10 h in the presence and absence of 10 mM aminotriazole (AMT). Data are means ± SE from 4 samples. * P < 0.05 vs. cells incubated with Pronase in the absence of aminotriazole.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Growing evidence suggests that production of ROS is triggered by exposure to a variety of stressful and cytotoxic stimuli. In this study, we investigated the effect of several serine proteases on ROS levels of lung fibroblasts and bronchial epithelial cells. The results demonstrated that relatively high concentrations of neutrophil elastase, trypsin, and Pronase induce a rise in ROS, including H2O2. The source of the ROS appears to be the mitochondria, and the contribution of ROS to cytotoxicity has been demonstrated. To our knowledge, this study is the first to demonstrate that proteases directly increase the intracellular levels of ROS in nonphagocytic cells.

In this study, we used CDCF to monitor intracellular ROS levels. A recent study (11) demonstrated that CDCF, which was previously thought to be a specific indicator of H2O2, detects a broad range of ROS that are increased during intracellular oxidative stress. Thus the protease-induced increase in CDCF fluorescence observed in this study may reflect intracellular production of various oxidants including H2O2 and other ROS.

It is currently unknown how proteases increase the level of ROS. Many mechanisms are possible and include detachment of the cells from the culture plates, degradation of the ECM, activation of a proteinase-activated receptor or an ion channel, an increase in membrane permeability, and loss of intracellular antioxidants. A recent report (16) noted that detachment of endothelial cells from the ECM led to a rapid rise in the intracellular level of ROS. Another report (15) demonstrated that disruption of the actin cytoskeleton by an antibody to alpha 5beta 1-integrin led to a change in cell shape and subsequent generation of ROS in fibroblasts. However, the protease-induced rise in ROS observed in our study was not merely due to cell detachment because a rise in ROS induced by proteases was observed in fibroblasts maintained in suspended conditions that preclude cell attachment to culture plates and in lymphocytes, which do not adhere to plates. This view is further supported by the observation that the rise in ROS induced by proteases was much greater than the rise induced by mechanical detachment of cells.

Alternatively, proteases may have activated specific signaling pathways either to increase ROS production or to decrease antioxidant defense levels. In our study, the protease-induced rise in ROS was unaffected by pharmacological inhibition of tyrosine kinases, tyrosine phosphatases, protein kinase C, protein kinase A, and Gi protein. However, proteases may have activated other signaling pathways such as Rac1 and ceramide signaling, both of which have recently been shown to regulate ROS levels in nonphagocytic cells (9, 15, 30). It is well established that serine proteases, including trypsin and thrombin, can stimulate cell proliferation through activation of protease-activated receptors and G proteins (5, 19). However, it is unknown whether protease-activated receptors are involved in ROS production and cell death induced by proteases. Thus whether proteases stimulate specific signaling to increase ROS levels was not confirmed in this study.

Although our results confirmed that neutrophil elastase, trypsin, and Pronase are cytotoxic, we have not yet investigated whether these proteases induce apoptosis or necrosis. Recent reports have documented that serine proteases such as neutrophil elastase and proteinase 3 are capable of inducing apoptosis in endothelial cells (37), keratinocytes (18), and neutrophils (32). Thus the protective effect of catalase on protease cytotoxicity observed in the present study may have been due to inhibition of apoptosis, and the contribution of ROS to apoptosis and necrosis in protease-exposed cells is the focus of ongoing investigations.

Proteases and ROS are frequent companions at sites of inflammation and have been proposed as important mediators of tissue injury. Although previous work (21, 35) has clearly established additive injurious effects of proteases and ROS during inflammation, our finding that proteases directly increase ROS points to a direct link between proteases and ROS in mediating cytotoxicity. This mechanism may be involved in a variety of inflammatory disorders, including pulmonary emphysema and acute respiratory distress syndrome, in which proteases and ROS are believed to act in concert to cause lung injury (17, 22, 29). Besides their injurious effects, ROS have been proposed to serve as signaling and messenger molecules involved in inflammatory processes. For example, ROS, including H2O2, regulate the activation of nuclear factor-kappa B and the production of proinflammatory cytokines such as interleukins 6 and 8 and tumor necrosis factor-alpha (24). Thus ROS released by protease-injured cells may also serve to propagate inflammatory reactions by stimulating immune effector cells in the microenvironment.


    ACKNOWLEDGEMENTS

This work was supported by Grant-in Aid for Scientific Research 12670580 from the Ministry of Education, Science, and Culture, Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: K. Aoshiba, First Dept. of Medicine, Tokyo Women's Medical Univ., 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan (E-mail: kaoshiba{at}chi.twmu.ac.jp).

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 5 September 2000; accepted in final form 30 April 2001.


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RESULTS
DISCUSSION
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