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(Journal of Leukocyte Biology. 2001;69:241-247.)
© 2001 by Society for Leukocyte Biology

Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury

Keigo Nakatani, Seiichiro Takeshita, Hiroshi Tsujimoto, Youichi Kawamura and Isao Sekine

Department of Pediatrics, National Defense Medical College, Tokorozawa, Saitama, Japan

Correspondence: Seiichiro Takeshita, M.D., Ph.D., Department of Pediatrics, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama 359-8513, Japan. E-mail: peditake{at}ndmc.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil-mediated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbilical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose-dependently by urinary trypsin inhibitor (ulinastatin, UTI) and ONO-5046, which have the ability to inactivate neutrophil elastase, but not by gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban, which have no ability to inactivate neutrophil elastase. In addition, when UTI and ONO-5046 were added to the tumor necrosis factor {alpha}-primed neutrophils alone, they showed a dose-dependent inhibition of the intracellular elastase activity, but the other SPI did not, for either flow cytometry or confocal microscopy. Therefore, UTI and ONO-5046 may protect EC against the neutrophil-mediated injury not only by inactivating the extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils and suppressing the production and secretion of activated elastase from them.

Key Words: elastase • ulinastatin • ONO-5046


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human neutrophils play a key role in host defense by ingesting and destroying infectious agents. Neutrophils contain a number of proteases in their own granules [1 , 2 ]. Although the physiological functions of proteases have not yet been completely elucidated, they originally demonstrate antimicrobial ability during the process of phagocytosis as a result of their proteolytic activity. The proteases are also involved in the physiological process of matrix proteolysis such as intercellular migration and tissue remodeling and repair [2 ]. However, if the activity of the proteases secreted from neutrophils is uncontrolled and excessive in certain conditions, such as in severe inflammatory diseases, they can degrade the extracellular matrix, thus leading to self-tissue destruction [2 ]. Serine proteases belong to the largest class of mammalian proteases, and elastase in this group is a major protease responsible for extracellular matrix degradation [2 ]. Neutrophil elastase, the strongest serine protease, is stored in an active form within the azurophil granules. Elastase is also reported to play a crucial role in neutrophil-mediated endothelial cell (EC) injury, thus contributing to vascular injury [3 , 4 ].

There are several types of therapeutic serine protease inhibitors (SPI), which have distinct functions. Urinary trypsin inhibitor (ulinastatin, UTI), derived from human urine, inhibits the neutrophil elastase activity [5 ] and trypsin activity [6 ]. UTI has been shown to have a clinical application for the treatment of pancreatitis [6 ] and also has a protective effect on sepsis-induced organ injury in the rat [7 ]. Gabexate mesilate, a synthetic protease inhibitor, inhibits various kinds of plasma proteases, such as trypsin, plasmin, kallikrein, and thrombin in a coagulation cascade [8 ]. This reagent has also been shown to be effective in treating disseminated intravascular coagulation (DIC) [9 ]. Nafamostat mesilate inhibits the enzyme activity of trypsin, thrombin, kallikrein, and plasmin [10 ]. Aprotinin inhibits plasma kallikrein and also has a beneficial effect on septic and endotoxic shock in animals [11 , 12 ]. Argatroban inhibits thrombin and enhances reperfusion induced by tissue plasminogen activator in patients with acute myocardial infarction [13 , 14 ]. ONO-5046 is a specific synthetic inhibitor of neutrophil elastase [15 ]. In addition to SPI, nimesulide, a nonsteroidal anti-inflammatory drug, can also inhibit the elastase activity by rescuing {alpha}1-protease inhibitor (A1PI), thus resulting in tissue protection [16 ].

The aim of this study is to investigate whether SPI can inhibit neutrophil-mediated human EC injury in vitro. We first investigated the inhibitory effect of these drugs on the neutrophil-mediated EC cytotoxicity through the use of radiolabeled-human umbilical vein EC (HUVEC). Second, we investigated the inhibitory effect of these drugs on the intracellular elastase activity in neutrophils, using a flow cytometric analysis and confocal laser microscopy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant human TNF-{alpha}, interferon-{gamma} (IFN-{gamma}), and interleukin-1ß (IL-1ß) were purchased from R & D Systems (Minneapolis, MN). LPS derived from Escherichia coli serotype 026:B6, N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), nimesulide, and elastase purified from human leukocytes were purchased from Sigma Chemical (St. Louis, MO). UTI was kindly provided by Mochida Pharmaceutical (Osaka, Japan); gabexate mesilate and ONO-5046 were from Ono Pharmaceutical (Osaka, Japan); nafamostat mesilate was from Torii Pharmaceutical (Tokyo, Japan); aprotinin was from Yoshitomi Pharmaceutical Industries (Osaka, Japan); argatroban was from Daiichi Pharmaceutical (Osaka, Japan). The endotoxin content of the reagents, phosphate-buffered saline (PBS), and medium was <3 pg/mL as determined by Limulus amebocyte lysate assay (Endospecy test, Seikagaku, Tokyo, Japan).

Isolation and culture of HUVEC
HUVEC were isolated according to the methods of Jaffe et al. [17 ]. The cells were suspended in M199 (GIBCO-BRL, Life Technologies, Rockville, MD) containing 10% fetal bovine serum (FBS), 100 units/mL of penicillin and streptomycin, and were cultured in a collagen-type 1-coated plastic dish (Sumitomo Bakelite Medical, Tokyo, Japan) at 37°C. The culture medium was changed the following day and thereafter twice weekly. HUVEC were treated with 0.05% trypsin/0.53 mM EDTA when 70~80% confluent, resuspended in culture medium, plated at 1 x 105/mL in a 100-µL volume in collagen type 1-coated 96-well flat-bottom plates (Iwaki Glass, Tokyo, Japan), and cultured at 37°C in a 5% CO2 atmosphere.

Preparation of neutrophils
Heparinized venous blood was obtained from healthy volunteers. Neutrophils were isolated by the dextran sedimentation method, followed by density gradient centrifugation with Histopaque 1077 (Sigma). Contaminated erythrocytes were removed by hypotonic lysis. Neutrophils were then washed with RPMI 1640 two times and resuspended in RPMI 1640 containing 1% FBS. The purity of the neutrophils was more than 95%, as assessed by a flow cytometer using forward and side scatter. The viability of the neutrophils were more than 98%, as evaluated by trypan blue dye exclusion.

51Cr release cytotoxic assay
The assay was performed as previously described by others [18 , 19 ]. After the culture medium was removed from the 90% confluent HUVEC monolayer, 51Cr (10 µCi/mL) in 200 µL fresh culture medium was added to each well. HUVEC were cultured for 6 h at 37°C and then were further cultured for 12 h in the presence or absence of TNF-{alpha} (100 ng/mL), IFN-{gamma} (100 ng/mL), and/or LPS (1 µg/mL), followed by washing four times with RPMI 1640 containing 1% FBS. After the neutrophils were primed with or without TNF-{alpha} (10 ng/mL), IFN-{gamma} (10 ng/mL), IL-1ß (10 ng/mL), LPS (1 µg/mL), 1 µM fMLP, or 1 µM PMA for 5 min, cells were washed twice with RPMI 1640 containing 1% FBS to remove the effect of cytokine. The cell suspension (2.5 x 106/mL) was added to the monolayer HUVEC (1 x 104 cells) in each well (E/T = 50:1). The cell mixtures were cultured for 12 h at 37°C in the presence or absence of protease inhibitors (UTI, gabexate mesilate, nafamostat mesilate, aprotinin, argatroban, and ONO-5046) and nimesulide, followed by centrifugation at 1500 rpm for 5 min. One hundred microliters of supernatants were carefully aspirated, and the radioactivity was counted by a gamma counter. The neutrophil-mediated HUVEC injury was expressed as percent specific 51Cr release as previously described by others: % lysis = (A - B/C - B) x 100, in which A is the counts/minute in the supernatant of tested wells; B is the mean counts/minute for the spontaneous release of 51Cr in the supernatant of wells with medium alone; and C is the total release of 51Cr in the supernatant of wells in which the HUVEC were completely lysed with 1 N HCl instead of neutrophils. The ratio of the spontaneous 51Cr release against the total release was less than 20% in all experiments. As another indicator of EC injury, the viability of the HUVEC was evaluated by trypan blue dye exclusion, after separate incubation with various inhibitors. The actual cell death was therefore expressed as the proportion (% cell death) of the killed cells.

Assay to determine the intracellular elastase activity in neutrophils by a flow cytometer
After the purified neutrophils were stimulated with TNF-{alpha} for 5 min at 37°C, the cells were washed with PBS alone. The cells were then incubated with RPMI 1640 containing 10% FBS for 1 h at 37°C, in the presence or absence of SPI and nimesulide. After washing three times with PBS, the cells were resuspended with PBS at a concentration of 3 x 106/mL, followed by incubation for 10 min at 37°C. AAPV Elastase CellProbeTM Reagent (Beckman Coulter, Fullerton, CA) was added as a fluorescence probe, and the cells were then incubated for 10 min at 37°C. The reagent translocates into the neutrophils and then reacts with the intracellular elastase, thus resulting in the release of fluorescence (rhodamine 110), based on information provided by the manufacturer. After placing the neutrophils on ice for 10 min, the cells were immediately analyzed by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). After setting the gate around the neutrophil population, the intracellular elastase activity was obtained as the fluorescence intensity through FL1 filter. The data were analyzed by CellQuest soft ware (Becton Dickinson), and the mean fluorescence intensity (MFI) was calculated in each sample.

Confocal laser microscopy
Neutrophils, stained with the intracellular immunofluorescence (AAPV Elastase CellProbe Reagent) as described above, were immediately allowed to adhere to slides through the use of cytospin. To confirm the cellular localization of elastase activity, the cells were then visualized at 525 nm with excitation at 488 nm, using an LSM 410 Laser Scan microscope (Carl Zeiss, Oberkochen, Germany). The fluorescence intensity of each cell was quantified on an arbitrary grayscale (0–255) using the LSM software package (Ver. 3.98, Carl Zeiss). The results were expressed as the MFI ± SD (arbitrary units) obtained from 60 random cells.

Statistical analysis
All data are expressed as the mean ± SD, and the differences analyzed by the Mann-Whitney test. A P value < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of TNF-{alpha}, IFN-{gamma}, and/or LPS on EC injury mediated by TNF-{alpha}-primed neutrophils
Because neutrophil priming with TNF-{alpha} is reported to induce EC injury [19 ], we initially added TNF-{alpha}-primed neutrophils to 51Cr-labeled HUVEC preincubated with TNF-{alpha}, IFN-{gamma}, and/or LPS (Fig. 1 ). The percent lysis was significantly higher in HUVEC preincubated with TNF-{alpha} + IFN-{gamma}, TNF-{alpha} + LPS, IFN-{gamma} + LPS, but not in TNF-{alpha}, IFN-{gamma}, and LPS only, than in unstimulated HUVEC. The maximum degree of EC injury was observed in HUVEC preincubated with TNF-{alpha} + IFN-{gamma}. The data for cell viability (% cell death) showed the same tendencies as those of 51Cr release (% lysis).



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  		Figure 1. Effect of preincubation with TNF-{alpha}, IFN-{gamma}, and/or LPS on the percent lysis and percent cell death of HUVEC mediated by TNF-{alpha}-primed neutrophils. Neutrophils, primed by TNF-{alpha} (10 ng/mL) for 5 min at 37°C, were added to 51Cr-labeled HUVEC preincubated with TNF-{alpha} (100 ng/mL), IFN-{gamma} (100 ng/mL), and/or LPS (1 µg/mL). The EC injury was expressed as the percentage of both the specific 51Cr release (% lysis) and cell death (% cell death) as described in Materials and Methods. Data are expressed as the mean ± SD from five experiments. *P < 0.01 versus unstimulated HUVEC.

 
Effect of neutrophil priming by TNF-{alpha}, IFN-{gamma}, IL-1ß, LPS, fMLP, or PMA on EC injury preincubated with TNF-{alpha} and IFN-{gamma}
After the neutrophils were primed by several cytokines and stimulators (TNF-{alpha}, IFN-{gamma}, IL-1ß, LPS, fMLP, or PMA), they were added to 51Cr-labeled HUVEC preincubated with TNF-{alpha} and IFN-{gamma} (Fig. 2 ). The percent lysis of HUVEC was significantly higher in the neutrophils primed with TNF-{alpha}, LPS, fMLP, and PMA, but not in IFN-{gamma} and IL-1ß, than in unstimulated neutrophils. The maximum EC damage was observed in the neutrophils primed by TNF-{alpha}. Based on these results, we decided to expose the TNF-{alpha}-primed neutrophils to TNF-{alpha} + IFN-{gamma}-preincubated HUVEC in the following experiments. The data for cell viability (% cell death) showed the same tendencies as those of 51Cr release (% lysis).



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  		Figure 2. Effect of neutrophil priming by TNF-{alpha}, IFN-{gamma}, IL-1ß, LPS, fMLP, or PMA on the percent lysis and percent cell death of TNF-{alpha} + IFN-{gamma}-preincubated HUVEC. After the neutrophils were primed with/without TNF-{alpha} (10 ng/mL), IFN-{gamma} (10 ng/mL), IL-1ß (10 ng/mL), LPS (1 µg/mL), 1 µM fMLP, or 1 µM PMA for 5 min at 37°C, they were added to 51Cr-labeled HUVEC preincubated with TNF-{alpha} (100 ng/mL) and IFN-{gamma} (100 ng/mL). The EC injury was expressed as the percentage of both the specific 51Cr release (% lysis) and cell death (% cell death). Data are expressed as the mean ± SD from five experiments. *P < 0.01 versus unstimulated neutrophils.

 
Inhibitory effect of SPI and nimesulide on neutrophil-mediated EC injury
To investigate the inhibitory effect of SPI on neutrophil-mediated EC injury, SPI (UTI, gabexate mesilate, nafamostat mesilate, aprotinin, argatroban, and ONO-5046) and nimesulide in various concentrations were added to TNF-{alpha} + IFN-{gamma}-preincubated HUVEC concurrently with TNF-{alpha}-primed neutrophils (Fig. 3 ). UTI, ONO-5046, and nimesulide decreased the HUVEC cytotoxicity (% of control) in a dose-dependent fashion, but gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban did not. These results indicate that UTI, ONO-5046, and nimesulide reduce the neutrophil-mediated EC injury in vitro.



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  		Figure 3. Inhibitory effect of SPI and nimesulide on percent lysis of 51Cr-labeled HUVEC mediated by neutrophils. When TNF-{alpha} (10 ng/mL) -primed neutrophils were added to the HUVEC preincubated with TNF-{alpha} (100 ng/mL) and IFN-{gamma} (100 ng/mL), UTI (100~5000 U/mL), gabexate mesilate (0.01~50 mM), nafamostat mesilate (0.01~50 mM), aprotinin (100~5000 U/mL), argatroban (0.1~10 mM), ONO-5046 (0.1~100 µg/mL), or nimesulide (0.01~50 mM) was added at the same time. The EC injury was expressed as the cytotoxicity when compared with the controls (cytotoxicity of HUVEC induced by TNF-{alpha}-primed neutrophils in the absence of any inhibitors). Data are expressed as the mean ± SD from five experiments. *P < 0.01 versus controls.

 
Inhibitory effect of UTI and ONO-5046 on purified elastase-mediated EC injury
To investigate whether elastase can actually induce EC injury, purified elastase was added to 51Cr-labeled HUVEC preincubated with TNF-{alpha} and IFN-{gamma} (Fig. 4 ). Elastase increased the percent lysis of HUVEC in a dose-dependent fashion (Fig. 4A) . We next investigated the inhibitory effect of SPI on the purified elastase-mediated EC injury. UTI and ONO-5046 inhibited the HUVEC cytotoxicity (% of control) in a dose-dependent fashion (Fig. 4B and 4C) , but other types of SPI (gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban) did not (data not shown).



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  		Figure 4. Inhibitory effect of UTI and ONO-5046 on percent lysis of 51Cr-labeled HUVEC mediated by purified elastase. (A) Purified elastase (0, 1, 5, 10, 25, and 50 µg/mL) was added to the 51Cr-labeled HUVEC preincubated with TNF-{alpha} (100 ng/mL) and IFN-{gamma} (100 ng/mL), followed by the incubation for 12 h at 37°C. The EC injury was expressed as the percentage of 51Cr release (% lysis). *P < 0.01 versus 0 µg/mL of elastase. (B, C) UTI (100~5000 U/mL) or ONO-5046 (0.1~100 µg/mL) was added to the 51Cr-labeled HUVEC, concomitantly with purified elastase (25 µg/mL). The EC injury was expressed as the cytotoxicity when compared with controls (cytotoxicity of HUVEC induced by purified elastase in the absence of any inhibitors). Data are expressed as the mean ± SD from five experiments. *P < 0.01 versus control.

 
Flow cytometric analysis of inhibitory effect of SPI and nimesulide on intracellular elastase activity in neutrophils
To investigate the drug effect on the intracellular elastase activity in neutrophils, SPI (UTI, gabexate mesilate, nafamostat mesilate, aprotinin, argatroban, and ONO-5046) and nimesulide were added to TNF-{alpha}-primed neutrophils alone (Fig. 5 ). The intracellular elastase activity was determined, through the use of a flow cytometer, to be the fluorescence intensity. The MFI was significantly lower in neutrophils treated with UTI and ONO-5046, but not in those with gabexate mesilate, nafamostat mesilate, aprotinin, argatroban, and nimesulide than in those without these drugs. Furthermore, UTI and ONO-5046 inhibited the intracellular elastase activity in a dose-dependent fashion (Fig. 6 ), whereas other drugs did not (data not shown).



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  		Figure 5. Inhibitory effect of SPI and nimesulide on intracellular elastase activity in TNF-{alpha}-primed neutrophils using a flow cytometric analysis. After neutrophils were preincubated with TNF-{alpha} (10 ng/mL) for 5 min, the cells were treated for 1 h with UTI (1000 U/mL), gabexate mesilate (1 mM), nafamostat mesilate (1 mM), aprotinin (5000 U/mL), argatroban (500 µM), ONO-5046 (100 µg/mL), or nimesulide (1 mM). The intracellular elastase activity was analyzed by a flow cytometer, as described in Materials and Methods. The MFI in each sample was measured and the data expressed as the mean ± SD from three experiments. *P < 0.01 versus TNF-{alpha}-primed neutrophils treated with no drugs.

 


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  		Figure 6. Dose-dependent inhibition of UTI and ONO-5046 for intracellular elastase activity in TNF-{alpha}-primed neutrophils. (A) After neutrophils were incubated with medium alone (thin line) and with TNF-{alpha} (10 ng/mL, bold line), the intracellular elastase activity was analyzed by a flow cytometer. (B) UTI was added to TNF-{alpha}-primed neutrophils at concentrations of 100, 1000, and 5000 U/mL (bold lines). (C) ONO-5046 was added to the TNF-{alpha}-primed neutrophils at concentrations of 1, 10, and 100 µg/mL (bold lines).

 
Cellular localization and quantification of the elastase activity in neutrophils through the use of a confocal laser scanning microscope
The neutrophils stained with fluorescence for the intracellular elastase activity were also observed under confocal laser scanning microscopy (Fig. 7 ). Because the dye stained the inside of the neutrophils aside from the nuclear portion, the elastase activity was confirmed to be localized at the cytoplasm (Fig. 7E) . It seemed that the cells treated with TNF-{alpha} and UTI or ONO-5046 had a lower fluorescence intensity than the cells treated with TNF-{alpha} alone (Fig. 7A 7B 7C 7D) . To investigate any significant differences in the fluorescence intensity, the degree of intracellular staining was next traced and scanned in each cell, followed by the calculation of the MFI (Fig. 8 ). The MFI in the TNF-{alpha}-stimulated neutrophils was significantly higher than that in the unstimulated neutrophils. When the TNF-{alpha}-stimulated neutrophils were treated with UTI and ONO-5046, the MFI decreased significantly, compared with TNF-{alpha} alone. These results thus confirm that UTI and ONO-5046 inhibit the intracellular elastase activity of neutrophils.



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  		Figure 7. Fluorescence staining of neutrophils for intracellular elastase activity using a confocal laser microscope. After neutrophils were incubated with medium alone (A), TNF-{alpha} (10 ng/mL) (B), TNF-{alpha} (10 ng/mL) + UTI (1000 U/mL) (C), or TNF-{alpha} (10 ng/mL) + ONO-5046 (100 µg/mL) (D), the fluorescence for elastase activity was observed under a confocal laser microscope. Fluorescence for elastase activity was stained in the cytoplasm of neutrophils except for the nucleus (E). The pseudocolor scales (0–255) refer to the fluorescence intensity values._art>

 


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  		Figure 8. Inhibitory effect of UTI and ONO-5046 on the intracellular elastase activity in TNF-{alpha}-primed neutrophils with a confocal scan laser microscope. The fluorescence staining for elastase activity in the cytoplasm was traced and scanned in 60 cells under a confocal laser microscope. MFI was calculated and expressed as the mean ± SD. *P < 0.01 versus TNF-{alpha}-primed neutrophils.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human neutrophils have been implicated as mediators of tissue-destructive events in several inflammatory diseases such as rheumatic diseases, adult respiratory distress syndrome (ARDS), reperfusion disorders, skin diseases, and ulcerative colitis [20 ]. The EC is an initial and important potential target because of its close association with neutrophils during the inflammatory process. EC injury mediated by activated neutrophils has been seen in systemic inflammatory response syndrome (SIRS), ARDS, and multiple organ failure (MOF) [21 , 22 ]. The neutrophil-mediated EC injury is reported to be mediated by a synergistic interaction of reactive oxidants and proteases derived from neutrophils [23 , 24 ]. Catalase, a radical scavenger, inhibits the neutrophil-mediated EC injury in vitro [25 ], but it has not yet been used in a clinical setting. On the other hand, several types of SPI have been developed for therapeutic use, and their ability to inhibit the protease activity has led investigators to explore its applications for the treatment of pancreatitis, DIC, and cardiovascular diseases [6 , 9 , 13 , 14 ]. Although UTI is reported to inhibit the neutrophil-mediated injury of EC derived from bovine carotid artery [26 ], so far no one has investigated whether SPI inhibit human EC injury mediated by neutrophils. To investigate the therapeutic potential, we therefore focused on the inhibitory effect of SPI on human EC injury mediated by neutrophils in the present study.

The present results revealed that UTI and ONO-5046 inhibited neutrophil-mediated EC injury in vitro, whereas other SPI did not. UTI and ONO-5046 have a common pharmacological function that inactivates neutrophil elastase, but other SPI do not [5 6 7 8 9 10 11 12 13 14 15 ]. Nimesulide, an anti-inflammatory drug with the ability to inhibit neutrophil elastase [16 ], is also known to reduce neutrophil-mediated EC injury. Neutrophil elastase out of serine proteases plays a major role in the EC injury mediated by stimulated neutrophils [3 , 4 ]. Therefore, these findings indicate that UTI and ONO-5046 inhibit EC injury by inactivating the elastase secreted from stimulated neutrophils. UTI is also reported to reduce the EC-neutrophil adhesion mediated by IL-1 and TNF-{alpha}, the transendothelial migration of neutrophils [27 ], and the TNF-{alpha}-induced expression of ICAM-1 on HUVEC [28 ]. It is therefore possible that UTI inhibits neutrophil-mediated EC injury by reducing the neutrophil-EC adhesion induced by cytokines.

In the human body, there are several types of endogenous protease inhibitors, out of which A1PI forms complexes with elastase and inactivates the free elastase activity [2 , 20 ]. Nevertheless, activated neutrophils release the reactive oxidants, which consequently oxidize and inactivate the A1PI. The elastase-A1PI balance is maintained in physiological conditions, while the balance is occasionally impaired in pathological conditions with an abnormal activation of neutrophils, which thus allows for the uncontrolled digestive activity of elastase [2 , 20 ]. Because UTI is reported to reduce the extracellular release of reactive oxidants from neutrophils [29 ], UTI may protect EC from damage in part by rescuing the endogenous A1PI. However, once neutrophils adhere to EC and extracellular matrix, A1PI cannot diffuse into the zones of the tight adherence, accelerating the tissue digestion and destruction induced by elastase [2 , 20 ]. It is therefore unlikely that the exogenous SPI such as UTI and ONO-5046 diffuse the tight space and inactivate the elastase. The present study demonstrated that UTI and ONO-5046 inhibited the intracellular elastase activity in neutrophils by acting directly on these cells. These drugs are thus suggested to reduce the production and secretion of the active form of elastase from neutrophils, thus resulting in protection from EC injury even after the occurrence of adherence between neutrophils and EC.

The administration of UTI has been reported to suppress the release of elastase from circulating neutrophils in patients treated by cardiopulmonary resuscitation [30 ]. UTI suppresses the increased mRNA expression of prostaglandin H2 synthetase-type 2 in neutrophils during the acute phase of Kawasaki disease, suggesting that UTI treatment may be a beneficial therapeutic approach to the disease [31 ]. UTI also suppresses the Ca2+ influx into neutrophils by a mechanism in which the trypsin inhibitor region of UTI acts on the cell membrane of neutrophils [32 , 33 ]. ONO-5046 is reported to reduce chemoattractant production from cytokine-stimulated neutrophils [34 ]. Therefore, UTI and ONO-5046 may attenuate the function of activated neutrophils, but this mechanism was not elucidated in this study. Kobayashi et al. reported that receptors for UTI are present on the surface of tumor cell lines [35 ]. Because exogenous UTI is reported to be internalized by culture cells through the endocytic pathway [36 ], it is possible that the uptake of UTI also may take place in neutrophils. The functional mechanisms of UTI and ONO-5046 on neutrophils should therefore be further investigated in the future.

In summary, SPI such as UTI and ONO-5046 were observed to inhibit the neutrophil-mediated EC injury in vitro by suppressing the activity of either extracellular elastase secreted by the neutrophils or intracellular elastase in them. It is therefore possible that these drugs may reduce the degree of EC damage mediated by neutrophils in vivo and may also be clinically beneficial for such inflammatory diseases with activated neutrophils as SIRS and ARDS.

Received June 17, 2000; revised October 10, 2000; accepted October 16, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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