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
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-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
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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.
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, interferon-
(IFN-), 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- (100
ng/mL), IFN- (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- (10 ng/mL), IFN- (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- 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.
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, IFN-, and/or LPS on EC injury mediated by
TNF--primed neutrophils is reported to induce EC
injury [19
], we initially added TNF--primed
neutrophils to 51Cr-labeled HUVEC preincubated with
TNF-, IFN-, and/or LPS (Fig. 1
). The percent lysis was significantly higher in HUVEC preincubated
with TNF- + IFN-, TNF- + LPS, IFN- + LPS,
but not in TNF-, IFN-, and LPS only, than in unstimulated HUVEC.
The maximum degree of EC injury was observed in HUVEC preincubated with
TNF- + IFN-. The data for cell viability (% cell death)
showed the same tendencies as those of 51Cr release (%
lysis).
 View larger version (12K): [in a new window] |
Figure 1. Effect of preincubation with TNF-, IFN-, and/or LPS on the
percent lysis and percent cell death of HUVEC mediated by
TNF--primed neutrophils. Neutrophils, primed by TNF- (10 ng/mL)
for 5 min at 37°C, were added to 51Cr-labeled HUVEC
preincubated with TNF- (100 ng/mL), IFN- (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.
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, IFN-, IL-1ß, LPS,
fMLP, or PMA on EC injury preincubated with TNF- and IFN-, IFN-, IL-1ß, LPS, fMLP, or PMA), they were
added to 51Cr-labeled HUVEC preincubated with TNF- and
IFN- (Fig. 2
). The percent lysis of HUVEC was significantly higher in the
neutrophils primed with TNF-, LPS, fMLP, and PMA, but not in IFN-
and IL-1ß, than in unstimulated neutrophils. The maximum EC damage
was observed in the neutrophils primed by TNF-. Based on these
results, we decided to expose the TNF--primed neutrophils to
TNF- + IFN--preincubated HUVEC in the following experiments.
The data for cell viability (% cell death) showed the same tendencies
as those of 51Cr release (% lysis).
 View larger version (13K): [in a new window] |
Figure 2. Effect of neutrophil priming by TNF-, IFN-, IL-1ß, LPS, fMLP,
or PMA on the percent lysis and percent cell death of TNF- +
IFN--preincubated HUVEC. After the neutrophils were primed
with/without TNF- (10 ng/mL), IFN- (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- (100 ng/mL) and IFN- (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.
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+ IFN--preincubated HUVEC concurrently with
TNF--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.
 View larger version (28K): [in a new window] |
Figure 3. Inhibitory effect of SPI and nimesulide on percent lysis of
51Cr-labeled HUVEC mediated by neutrophils. When TNF-
(10 ng/mL) -primed neutrophils were added to the HUVEC preincubated
with TNF- (100 ng/mL) and IFN- (100 ng/mL), UTI (1005000
U/mL), gabexate mesilate (0.0150 mM), nafamostat mesilate (0.0150
mM), aprotinin (1005000 U/mL), argatroban (0.110 mM), ONO-5046
(0.1100 µg/mL), or nimesulide (0.0150 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--primed
neutrophils in the absence of any inhibitors). Data are expressed as
the mean ± SD from five experiments.
*P < 0.01 versus controls.
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and IFN- (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).
 View larger version (13K): [in a new window] |
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- (100 ng/mL)
and IFN- (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 (1005000 U/mL) or ONO-5046
(0.1100 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.
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-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).
 View larger version (16K): [in a new window] |
Figure 5. Inhibitory effect of SPI and nimesulide on intracellular elastase
activity in TNF--primed neutrophils using a flow cytometric
analysis. After neutrophils were preincubated with TNF- (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--primed neutrophils treated with no drugs.
|
 View larger version (28K): [in a new window] |
Figure 6. Dose-dependent inhibition of UTI and ONO-5046 for intracellular
elastase activity in TNF--primed neutrophils. (A) After neutrophils
were incubated with medium alone (thin line) and with TNF- (10
ng/mL, bold line), the intracellular elastase activity was analyzed by
a flow cytometer. (B) UTI was added to TNF--primed neutrophils at
concentrations of 100, 1000, and 5000 U/mL (bold lines). (C) ONO-5046
was added to the TNF--primed neutrophils at concentrations of 1, 10,
and 100 µg/mL (bold lines).
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and UTI or ONO-5046 had a lower fluorescence intensity
than the cells treated with TNF- 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--stimulated neutrophils was significantly
higher than that in the unstimulated neutrophils. When the
TNF--stimulated neutrophils were treated with UTI and ONO-5046, the
MFI decreased significantly, compared with TNF- alone. These results
thus confirm that UTI and ONO-5046 inhibit the intracellular elastase
activity of neutrophils.
 View larger version (99K): [in a new window] |
Figure 7. Fluorescence staining of neutrophils for intracellular elastase
activity using a confocal laser microscope. After neutrophils were
incubated with medium alone (A), TNF- (10 ng/mL) (B), TNF- (10
ng/mL) + UTI (1000 U/mL) (C), or TNF- (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>
|
 View larger version (11K): [in a new window] |
Figure 8. Inhibitory effect of UTI and ONO-5046 on the intracellular elastase
activity in TNF--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--primed
neutrophils.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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-, the transendothelial migration of neutrophils
[27
], and the TNF--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.
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