Release of a leukocyte activation inhibitor by
staurosporine-treated pulmonary artery endothelial cells
Xilin
Chen and
John D.
Catravas
Vascular Biology Center, Medical College of Georgia, Augusta,
Georgia 30912
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ABSTRACT |
Bovine pulmonary
arterial endothelial cells (BPAE) treated with the protein kinase C
(PKC) inhibitor staurosporine inhibited O
2· generation by neutrophils
exposed to phorbol myristate acetate (PMA) but did not affect
O
2· generated enzymatically by
xanthine/xanthine oxidase (X/XO). Similar results were obtained with
conditioned medium from staurosporine-pretreated BPAE. The inhibitory
effects of staurosporine-treated BPAE on O
2· generation were not altered by
the superoxide dismutase inhibitor diethylcarbamazine. This
BPAE-derived inhibitor was continuously released from
staurosporine-pretreated BPAE for at least 5 h. The exact nature of the
inhibitor remains unknown, but it appears to be a positively charged
molecule with molecular weight <10,000. Treatment of either BPAE or
neutrophils with staurosporine or conditioned medium from
staurosporine-treated BPAE prevented the neutrophil-mediated decrease
in endothelium-bound angiotensin-converting enzyme activity and
cytotoxicity in BPAE. In contrast, staurosporine potentiated the
H2O2-
and X/XO-mediated endothelial cytotoxicity. These data suggest that
staurosporine-treated endothelial cells release a soluble factor that
inhibits neutrophil activation and protects endothelial cells from
neutrophil-mediated injury.
neutrophils; superoxide; cytotoxicity; angiotensin-converting
enzyme activity; protein kinase C
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INTRODUCTION |
CIRCULATING POLYMORPHONUCLEAR neutrophils (PMN)
normally exist in a quiescent state. When neutrophils ingest particles
or react to specific stimuli such as chemotactic peptides, they
interact extensively with the endothelial layer lining the vessel
walls. These interactions include increased adherence to endothelial cells, migration through the endothelium and into extravascular space,
disruption of the plasma membrane, and generation of an array of
reactive oxygen species, including superoxide anion
(O
2·) and hydrogen peroxide
(H2O2;
see Refs. 1, 25, 26). Thus vascular endothelial cells can be injured
indiscriminately by neutrophils proximal to the inflammatory site (20,
25).
Endothelial cells contain various antioxidant mechanisms such as
superoxide dismutase (SOD), catalase, and glutathione reductase. Hoover
and co-workers (14, 15) reported that coincubation of neutrophils with
endothelial cells markedly inhibited the extracellular release of
O
2· from the subsequently
activated neutrophils. Basford et al. (2, 3) have shown that
endothelial cells release a soluble inhibitor that downregulates plasma
membrane receptor-mediated activation of neutrophils. In the present
study, we demonstrate that endothelial cells pretreated with
staurosporine protect against neutrophil-mediated endothelial cell
dysfunction and injury, such as neutrophil-mediated endothelial
angiotensin-converting enzyme (ACE) dysfunction and cytotoxicity. These
protective effects appear to be mediated by a soluble factor released
from the staurosporine-treated endothelial cells that inhibits
neutrophil activation stimulated by either receptor or nonmembrane
receptor-mediated activators.
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METHODS |
Isolation and Culture of Endothelial Cells
Bovine pulmonary arterial endothelial cells (BPAE) were harvested by
the modified method of Ryan (23) and grown in medium 199 (Mediatech,
Washington, DC) with 10% fetal calf serum (Hyclone Laboratories,
Logan, UT), penicillin (100,000 U/l), and streptomycin (100 mg/l) in
T-75 flasks (Corning Glass, Corning, NY). The cultures were
incubated at 37°C in a humidified atmosphere with 5%
CO2. The cells were identified as
endothelial cells by their typical cobblestone morphology under
phase-contrast microscopy, by indirect immunofluorescent staining for
von Willebrand factor, and by the expression of ACE activity. The cells
were grown to confluence in T-75 flasks and then subcultured into
24-well plates, the latter used for enzyme assays and neutrophil
adherence experiments. All experiments with endothelial cells were
performed at 2-3 days after confluence and in three- to seven-
passage cells. Human brain microvascular endothelial cells were
generously provided by Dr. David Hess, Department of Neurology, Medical
College of Georgia (Augusta, GA).
Preparation of Neutrophil Suspensions
Neutrophils were isolated from the peritoneal cavities of adult New
Zealand White rabbits with the method of O'Flaherty et al. (21).
Briefly, neutrophils were obtained from peritoneal exudates 16 h after
the intraperitoneal injection of 0.2% glycogen dissolved in a 0.9%
NaCl solution. They were washed (centrifuged at 100 g) one time with
Ca2+- and
Mg2+-free Hanks' balanced salt
solution (HBSS) and resuspended at a concentration of 40 × 106/ml. Three milliliters of
leukocyte suspension were placed on 3 ml of Ficoll-Histopaque (density
1.077; Sigma) and centrifuged at 270 g
for 20 min to separate neutrophils and contaminating monocytes. The
pellets (containing neutrophils) were collected and washed two times in
Ca2+- and
Mg2+-free HBSS and resuspended in
Earle's salt solution (ESS) to the appropriate neutrophil
concentration. The cells obtained consisted of >95% neutrophils and
were 95% viable as measured by the trypan blue exclusion test.
Neutrophils were not activated by the isolation procedure, as there was
no detectable increase in basal release of
O
2·.
Monitoring of O
2·
Generation
The concentration of O
2· generated
by neutrophils was monitored by measuring the SOD-inhibitable reduction of ferricytochrome c. Neutrophils
(0.05-1 × 106/ml)
suspended in ESS were incubated with 40 mM ferricytochrome c in the presence or absence of 100 U/ml of SOD for 30 min at 37°C. Phorbol myristate acetate (PMA;
0-20 ng/ml) was used to activate neutrophils. The reduction of
ferricytochrome c was measured from
changes in absorbance at 550 nm (Shimazu, Kyoto, Japan) of the
supernatant after centrifugation (120 g) for 5 min. The amount of
O
2· released was calculated by
dividing the difference in absorbance of the sample with and without
SOD by the extinction coefficient for the change of ferricytochrome c to ferrocytochrome
c
(E550nm = 21.1 mM
1/cm), and
the results are expressed as nanomoles
O
2· per indicated number of
neutrophils (2).
Endothelial ACE Activity Assay
Endothelial ACE assays were performed under first-order reaction
conditions. Postconfluent BPAE were washed two times with ESS and
exposed to PMA, neutrophils, or both for 1-4 h at 37°C in 1.0 ml of ESS. Endothelial monolayers were then washed two times with ESS
and incubated for 30 min at 37°C with the specific ACE substrate
[3H]benzoyl-Phe-Ala-Pro
([3H]BPAP, 0.1 mCi/ml;
22.2 Ci/mmol; Ventrex, Portland, ME). From the culture medium, a 0.4-ml
aliquot was transferred to a 7-ml polyethylene scintillation vial
(Fisher Scientific, Atlanta, GA) containing 6 ml of scintillation
cocktail (Hydrofluor). Total 3H
radioactivity was measured in a Beckman LS 7500 liquid scintillation spectrometer (Beckman Instruments, Irvine, CA).
[3H]BPhe (product of
[3H]BPAP metabolism by
ACE) radioactivity was measured in another 0.4-ml aliquot added to a
7-ml scintillation vial containing 0.12 N HCl. To that, 3 ml of 0.4%
omnifluor (NEN, Boston, MA) in toluene were added.
[3H]BPhe was
preferentially extracted into toluene, and radioactivity was determined
by liquid scintillation spectrometry after the samples were stored in
the dark for 48 h. Because a small amount (5%) of the parent compound
(BPAP) was also extracted into toluene, correction for the extraction
of [3H]BPAP into
toluene was performed, and the true
[3H]BPhe concentration
in toluene was calculated from the solution of the equation
where
fs and
fp represent the fractional
extraction of
[3H]BPAP and
[3H]BPhe,
respectively, into toluene. Enzyme activity was calculated as
where
Vmax is maximal
velocity, Km is
Michaelis constant,
[So] and
[S] are the initial and final (surviving) substrate
concentrations, respectively, and t is
incubation time. Enzyme activity is then expressed in units where one
unit is the
Vmax/Km
value equivalent to 1% substrate metabolism in 1 min under first-order
reaction conditions.
Determination of Neutrophil Adherence to Endothelial Cells
Adherence of neutrophils to cultured endothelial cells was determined
using the modified method of Hoover et al. (15). Briefly, postconfluent
endothelial cells were rinsed two times with ESS; 51Cr-labeled neutrophils (2 mCi/ml
for 1 h at 37°C) were added into each endothelial cell culture well
and allowed to incubate (with or without PMA) for 1-4 h at
37°C in a 5% CO2 incubator.
Unattached neutrophils were then removed by aspiration, and the
monolayers were rinsed one time with 0.5 ml of ESS. Adherent
neutrophils and the endothelial monolayer in each well were lysed with
1 N NaOH, and the lysate plus one 0.5-ml wash were transferred to a
test tube. The radioactivity was measured in a gamma spectrometer with
95% efficiency for 51Cr. The
percentage of adherent neutrophils was calculated as
Cytotoxicity Assay
Cytotoxicity was measured by a standard
51Cr-release assay (25). The
endothelial cell monolayer in a 24-well plate was incubated overnight
with 2 mCi of
Na51CrO4
in culture medium. Cells were then washed two times to remove unincorporated radioactivity. Suspensions of neutrophils in ESS were
added to each well with or without PMA in a final volume of 1 ml. After
an additional incubation at 37°C for the indicated times, the
medium was removed from each well and centrifuged. The supernatant was
transferred to a test tube, and the
51Cr released was quantified in a
gamma spectrometer. Spontaneous release was obtained from wells
receiving medium only, and total release was obtained from wells
exposed to 0.2% Triton X-100. Percent cytotoxicity was calculated by
the following formula
Data Analysis and Statistics
Data are presented as means ± SE of the indicated number of
individual wells or tubes. Statistical comparisons between groups were
performed using one-way analysis of variance followed by the
Newman-Keuls test. Differences among means were considered significant
at P < 0.05.
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RESULTS |
The Observation
The protein kinase C inhibitor staurosporine inhibits
O
2· generation by
activated neutrophils. Coincubation of neutrophils (1 × 106/ml) with PMA (10 ng/ml) for 20 min produced a dramatic increase in
O
2· generation by neutrophils,
from a baseline of 2 to 18 nmol, as determined by the cytochrome
c reduction assay. Treatment with
staurosporine produced a concentration-dependent inhibition in
O
2· generation by PMA-activated neutrophils. There was a 40% inhibition of
O
2· generation at 0.1 µM
staurosporine. Superoxide generation by activated neutrophils was
completely inhibited at a concentration of 1 µM staurosporine (Fig.
1). Because staurosporine was dissolved in dimethyl sulfoxide (DMSO), 0.1% DMSO did not inhibit superoxide generation by activated neutrophils.

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Fig. 1.
Staurosporine produces a concentration-dependent inhibition of
O 2· generated by polymorphonuclear
neutrophils (PMN). PMN: 1 × 106/ml; phorbol myristate acetate
(PMA): 10 ng/ml; incubation time: 30 min. Values are means ± SE;
n = 6 tubes.
* P < 0.05 from the PMN alone
group; + P < 0.05 from the PMN + PMA 0 staurosporine group.
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Staurosporine-pretreated BPAE inhibit
O
2· generation by
activated neutrophils. As shown in Fig.
2A,
neutrophils (1 × 106/ml)
activated by PMA (10 ng/ml) generated 20 nmol of
O
2·. Coincubation of neutrophils
and BPAE had no effect on the O
2· generation by activated neutrophils. To investigate the effects of
staurosporine-pretreated BPAE on
O
2· generation by activated
neutrophils, BPAE were pretreated with staurosporine (1 µM for 60 min) followed by two careful washes; staurosporine-pretreated BPAE were
then coincubated with neutrophils. Coincubation of
staurosporine-pretreated BPAE with neutrophils completely inhibited
O
2· generation by PMA-stimulated
neutrophils.

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Fig. 2.
Staurosporine-treated bovine pulmonary arterial endothelial cells
(BPAE) or conditioned medium from staurosporine-treated BPAE inhibit
O 2· generated by neutrophils but
not by xanthine (X)/xanthine oxidase (XO).
A: effects of treatment of BPAE with
staurosporine (1 µM for 60 min; ST-EC) or regular medium (SHAM-EC) on
O 2· generated by neutrophils (1 × 106/ml).
B: effects of conditioned medium from
BPAE treated with staurosporine (1 µM for 60 min; ST-M) or vehicle
(SHAM-M) on O 2· generated by
neutrophils (1 × 106/ml).
C: effects of treatment of BPAE with
staurosporine (1 µM for 60 min) or vehicle on
O 2· generation by X (100 mM)/XO
(10 mU/ml). Control, O 2· generated
by activated neutrophils in the absence of BPAE or conditioned medium.
Values are means ± SE; n = 4 tubes. Experiments were repeated three times with similar results.
* P < 0.05 from the
corresponding control group.
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Conditioned medium from staurosporine-treated BPAE
inhibits O
2· generated
by neutrophils exposed to PMA. To assess whether a
soluble inhibitor of neutrophil activation was released by
staurosporine-treated BPAE, we tested the effects of ESS conditioned by
BPAE with or without staurosporine treatment on
O
2· generated by neutrophils
exposed to PMA. BPAE were pretreated with or without staurosporine (1 µM for 60 min) and then carefully washed two times with ESS.
Conditioned ESS was obtained by further incubating fresh ESS with the
BPAE for another hour. Incubation of neutrophils and PMA with normal
medium from BPAE had no effect on
O
2· generation (Fig.
2B). However, incubation of
neutrophils and PMA with conditioned medium from staurosporine-treated BPAE totally inhibited O
2·
generation by PMA-activated neutrophils (Fig.
2B), suggesting that
staurosporine-treated BPAE generate either a soluble inhibitor of
neutrophil activation or a scavenger of
O
2·. If staurosporine-treated BPAE
or conditioned medium could scavenge
O
2· generated by activated
neutrophils, it would similarly inhibit O
2· generated enzymatically by a
cell-free O
2·-generating system.
However, BPAE treated with staurosporine (1 µM for 60 min) had no
effect on the O
2· generated by
xanthine (X; 100 mM) and xanthine oxidase (XO; 10 mU/ml; Fig.
2C). These data suggest that a
soluble inhibitor released from staurosporine-pretreated BPAE inhibits
O
2· generated by activated
neutrophils.
Figure 3 shows the concentration response
of conditioned medium from staurosporine-treated BPAE on
O
2· generation by neutrophils
exposed to PMA. There was a 40% inhibition of
O
2· generation at 5% conditioned
medium and up to 95% inhibition at 20% of the conditioned medium. One explanation of the observed effect of staurosporine may be that BPAE
take up staurosporine during the 60-min incubation time and convert it
into a more potent form of protein kinase C (PKC) inhibitor, which is
then released into the medium. If this were the case, the observed
inhibitory effect on neutrophil activation would be expected to
decrease with time. To test this hypothesis, we treated BPAE with
staurosporine (1 µM) for 60 min followed by two washes and then
collected conditioned medium after each 1-h period of incubation for up
to 5 h. First- hour conditioned medium was obtained by incubating ESS
with staurosporine-pretreated BPAE for 1 h. Second or later hour
conditioned medium was obtained by further incubating fresh ESS with
the cells for another hour. As demonstrated in Fig.
4, staurosporine-conditioned medium
exhibited similar inhibitory effects on neutrophil activation after 5 h, suggesting that the soluble inhibitor is continuously released from
endothelial cells after staurosporine treatment.

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Fig. 3.
Concentration response of conditioned medium from BPAE treated with
staurosporine (1 µM for 60 min) on
O 2· generated by PMA (10 ng/ml)-activated neutrophils (1 × 106/ml). Values are means ± SE; n = 4 tubes.
* P < 0.05 from 0 group.
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Fig. 4.
Time course of release of the inhibitor in conditioned (Cond.) medium
from BPAE treated with staurosporine (STAU; 1 µM for 60 min) as
reflected in the inhibition of O 2·
generated by exposure of neutrophils (1 × 106/ml) to 10 ng/ml PMA. Medium
was harvested and replenished with fresh medium every hour for up to 5 h after staurosporine treatment. Values are means ± SE;
n = 6 tubes.
* P < 0.05 from control (no
medium) group.
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Sources and Targets of the Soluble Inhibitor
The conditioned medium from staurosporine-treated BPAE also inhibited
O
2· generated by mononuclear leukocytes (monocytes/macrophage 70%, lymphocytes 30%; Table
1). Furthermore, treatment of human brain
microvascular endothelial cells with staurosporine (1 µM for 60 min)
also inhibited superoxide generated by PMA-exposed neutrophils from
26.5 ± 8.5 to 2.5 ± 2.1 nmol.
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Table 1.
Effects of conditioned medium from staurosporine-treated endothelial
cells on superoxide generation by neutrophils or mononuclear leukocytes
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We also tested the effects of conditioned medium from
staurosporine-treated BPAE on two other neutrophil activators, A-23187 (2 µM), a calcium ionophore, and formyl-Met-Leu-Phe (FMLP; 100 nM), a
plasma membrane receptor-mediated agonist. As shown in Table 1,
O
2· production by FMLP- or
A-23187-activated neutrophils was also inhibited by conditioned medium
from staurosporine-treated BPAE.
Effects of other PKC inhibitors on
O
2· generation by
neutrophils. Two other PKC inhibitors, H-7 and
sphingosine, were tested to determine whether they also might have
effects similar to those of staurosporine. As shown in Fig.
5, H-7 (10-200 µM; see Ref. 4)
produced a dose-dependent inhibition of
O
2· by neutrophils exposed to PMA.
However, conditioned medium from H-7 (200 µM for 60 min)-treated BPAE
had no effect on O
2· generated by
PMA-exposed neutrophils. Sphingosine (1-20 µM; see Ref. 22) also
produced a dose-dependent inhibition in
O
2· by PMA-exposed neutrophils;
however, conditioned medium from sphingosine (1-20 µM for 60 min)-pretreated BPAE had no effect on
O
2· generated by PMA-exposed
neutrophils (Fig. 5).

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Fig. 5.
A: effects of the protein kinase C
(PKC) inhibitor H-7 on O 2·
generated by PMA-activated neutrophils (1 × 106/ml).
* P < 0.05 from the PMN group.
B: effects of treatment of BPAE with
H-7 (EC-H7; 100 µM for 60 min) or staurosporine (EC-STAU; 1 µM for
60 min) on O 2· generated by
PMA-activated neutrophils (1 × 106/ml). PMA concentration: 10 ng/ml; incubation time: 30 min. Values are means ± SE;
n = 6 tubes.
* P < 0.05 from the control
group (CTL: neutrophils and BPAE in the absence of PMA).
+ P < 0.05 from
the EC group (untreated BPAE in the presence of PMN + PMA).
C: effects of coincubation with the
PKC inhibitor sphingosine on O 2·
generated by PMA-activated neutrophils (1 × 106/ml).
* P < 0.05 from the
PMN group. D: effects of treatment of
BPAE alone with sphingosine on O 2·
generated by PMA-activated neutrophils (1 × 106/ml); PMA concentration: 10 ng/ml; incubation time: 30 min. Values are means ± SE;
n = 6 tubes.
* P < 0.05 from the PMN + PMA
group.
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Effects of inhibition of endothelial
SOD. To further eliminate the possibility that the
soluble factor inhibiting neutrophil superoxide production might be
SOD, BPAE were treated with the SOD inhibitor diethyldithiocarbamate
(DTCA; see Ref. 11). As shown in Table 2,
treatment of BPAE with DTCA (1 mM, 30 min before adding staurosporine;
see Ref. 2) had no effect on the inhibitory effects of
staurosporine-treated BPAE on the levels of
O
2· generated by neutrophils
exposed to PMA. These data suggest that the inhibitor of neutrophil
activation released from staurosporine-treated BPAE is not SOD.
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Table 2.
Effects of different treatments on superoxide generation by neutrophils
coincubated with BPAE monolayers
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Characterization of the Soluble Inhibitor
Inhibition of arachidonate and adenosine
metabolism. The inhibition of neutrophil activation by
staurosporine-pretreated BPAE was not affected by the cyclooxygenase
inhibitor indomethacin (0.1 mM, 30 min before adding staurosporine and
allowed to remain in the medium throughout the treatment phase), nor
was it affected by diethylcarbamazine (250 µM), an inhibitor of
lipoxygenase pathways (19; Table 2). Adenosine released from
endothelial cells or fibroblasts has been reported to inhibit
O
2· generation by FMLP-activated
neutrophils (12, 13). To investigate whether the soluble
inhibitor might be adenosine, BPAE were exposed to staurosporine
in the presence of adenosine deaminase. The addition of 1-5 U/ml
of adenosine deaminase to BPAE treated with staurosporine for 30 min
had no effect on the inhibition of neutrophil activation by
staurosporine-treated BPAE (Table 2). In a separate experiment, it was
also shown that adenosine (1 mM) had no effect on
O
2· generated by PMA-activated
neutrophils.
Effects of protein synthesis
inhibitors. To examine whether the soluble inhibitor
might be a newly synthesized protein, the protein synthesis inhibitor
cycloheximide (10 µM) was added to BPAE 30 min before staurosporine
and remained in the medium for the entire treatment phase. As shown in
Table 2, cycloheximide had no effect on the inhibition of neutrophil
activation by staurosporine-treated BPAE.
Other characteristics. The soluble
inhibitor was stable for at least 3 mo at
20°C and was
resistant to protease treatment (0.1 mg/ml trypsin for 30 min), acid
treatment (pH 1 for 1 h), alkaline treatment (pH 12 for 1 h), and
heating (boiling for 1 h). The inhibitor was not retained by passage
through an anion-exchange column [Dowex 1 × 8, 400-mesh
(Cl
) anion-exchange
resin] but was retained after passage through a cation- exchange
column (Bio-Rad 70) and was lost from dialysis in a membrane with a
molecular weight of 10,000 lower retention limit, suggesting that it is
a positively charged molecule with molecular weight
<10,000.
Effects of the Soluble Inhibitor of Leukocyte Activation on
BPAE Function
Pretreatment of neutrophils or endothelial cells with
staurosporine inhibits the neutrophil-mediated decrease in endothelial ACE activity. We have reported previously that
PMA-activated neutrophils decrease endothelial ACE activity by
generating oxygen free radicals (7-9). We thus investigated the
effects of staurosporine on neutrophil-mediated endothelial ACE
dysfunction. Staurosporine alone (1 µM) had no effect on endothelial
ACE activity. When BPAE were coincubated with PMA (10 ng/ml)-activated
neutrophils (1 × 106/ml),
endothelial ACE activity was almost totally abolished. Treatment with
staurosporine produced a dose-dependent protection; at 1 µM
staurosporine, neutrophil-mediated ACE dysfunction was completely prevented (Fig.
6A).
These data suggest that activation of PKC may play an
important role in neutrophil-mediated endothelial ACE dysfunction. To
determine the cellular source of PKC responsible for the
neutrophil-mediated decrease in ACE activity, we treated either
neutrophils or endothelial cells with staurosporine. Treatment of
neutrophils with staurosporine for 1 h and then carefully washing out
the staurosporine and coincubating treated neutrophils with PMA
attenuated the neutrophil-mediated decrease in ACE activity in a
dose-dependent manner similar to cotreatment of neutrophils and
endothelial cells with staurosporine (Fig.
6B). However, preincubation of BPAE
with staurosporine for 1 h followed by two careful washes also
prevented the neutrophil-mediated decrease in endothelial ACE activity
(Fig. 6C).

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Fig. 6.
Treatment of BPAE or neutrophils with staurosporine prevents
neutrophil-mediated decrease in endothelial-bound
angiotensin-converting enzyme (ACE) activity.
A: coincubation of staurosporine with
neutrophils and BPAE. B: pretreatment
of neutrophils with staurosporine for 1 h followed by 2 washes before
incubation with BPAE and PMA. C:
pretreatment of endothelial cells with staurosporine for 1 h followed
by 3 washes before incubation with neutrophils. Control ACE activity:
A, 17.6 ± 1.1 mU;
B, 21.4 ± 1.9 mU;
C, 17.6 ± 1.1 mU. PMN: 1 × 106/ml; PMA: 10 ng/ml. Values are
means ± SE; n = 6 tubes.
* P < 0.05 from either PMN
alone group; + P < 0.05 from the PMN + PMA 0 staurosporine group.
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Staurosporine-treated endothelial cells inhibit
neutrophil-mediated endothelial cytotoxicity. Because
treatment of BPAE with staurosporine prevented the neutrophil-mediated
decrease in endothelial ACE activity, we investigated whether it would
also attenuate the activated neutrophil-mediated endothelial
cytotoxicity. As shown in Fig.
7A, BPAE
exposed to PMA (10 ng/ml)-activated neutrophils (4 × 106/ml) for 4 h exhibited 30%
cytotoxicity. Cotreatment of cultures with staurosporine (1 µM)
nearly abolished the neutrophil-mediated endothelial cytotoxicity (Fig.
7A). However, pretreatment of BPAE only with staurosporine (1 µM for 60 min) followed by two washes also
significantly attenuated the neutrophil-mediated endothelial cytotoxicity (Fig. 7A).

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Fig. 7.
A: treatment of BPAE with
staurosporine prevents PMN-mediated endothelial cytotoxicity. EC-ST,
incubation of staurosporine (1 µM) with BPAE followed by 3 washes and
exposure to either vehicle or PMA-activated neutrophils; vehicle,
incubation of BPAE with vehicle followed by 3 washes and exposure to
PMA-activated neutrophils; (PMN + EC)-ST, incubation of BPAE and
neutrophils with staurosporine followed by exposure to PMA. Neutrophil
concentration: 4 × 106/ml;
PMA concentration: 10 ng/ml; incubation time: 1 h; exposure time: 4 h.
B and
C: effects of treatment of BPAE with
staurosporine (1 µM) on vehicle,
H2O2
(100 mM, 2 h, B)-, and X (100 mM)-XO
(10 mU/ml, C)-induced endothelial
cytotoxicity. Values are means ± SE;
n = 6 tubes.
* P < 0.05 from the
corresponding group without staurosporine.
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Staurosporine-treated endothelial cells potentiate
H2O2- and
X/XO-mediated endothelial cytotoxicity.
In contrast to neutrophil-mediated endothelial injury, treatment of
BPAE with staurosporine dramatically potentiated
H2O2- or X/XO-induced endothelial cytotoxicity. As shown in Fig.
7B, exposure of BPAE to 0.1 mM
H2O2
for 2 h produced 20% cytotoxicity. Treatment of BPAE with
staurosporine (1 µM for 60 min) caused a threefold increase in
H2O2-mediated
cytotoxicity. Similarly, treatment of BPAE with staurosporine also
potentiated the X/XO-induced endothelial cytotoxicity (Fig.
7C). These data suggest that
inhibition of endothelial PKC potentiates
H2O2-
and X/XO-induced endothelial cytotoxicity.
Effects of staurosporine on adherence of PMA-activated
neutrophils to endothelial cells. Neutrophil adherence
to endothelial cells is an important mechanism mediating cytotoxicity.
To examine whether adherence of neutrophils to endothelial cells was
affected by the soluble inhibitor, we measured neutrophil adherence to staurosporine-treated BPAE or to naive BPAE in the presence of conditioned medium from staurosporine-treated BPAE. When untreated neutrophils were exposed to BPAE for 30 min, 25% of neutrophils adhered to endothelial cells (Fig. 8); this
increased to 51.5% in the presence of PMA (10 ng/ml; Fig. 8). BPAE
pretreated with staurosporine (1 µM for 60 min) did not affect the
adherence of PMA-activated neutrophils (Fig. 8). Conditioned medium
from staurosporine-treated BPAE significantly decreased adherence of
PMA-activated neutrophils to BPAE (Fig. 8). Again, this conditioned
medium was obtained by incubating ESS for 1 h with BPAE that had been
treated with staurosporine (1 µM for 60 min) and then carefully
washed two times with ESS. Exposure of BPAE to PMA and neutrophils in
the presence of staurosporine (1 µM) prevented the increased
adherence of activated neutrophils to BPAE (Fig. 8).

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Fig. 8.
Effects of vehicle-treated BPAE (vehicle), staurosporine (1 µM for 60 min)-treated BPAE (EC-STAU), conditioned medium from BPAE treated with
staurosporine (1 mM, 1 h; STAU-M), or staurosporine (1 mM; STAU) on
adherence of PMA (10 ng/ml)-activated neutrophils to endothelial cells.
Values are means ± SE; n = 6 tubes. * P < 0.05 from the
control group (PMN without PMA);
+ P < 0.05 from
the PMA group.
|
|
 |
DISCUSSION |
PMA, a strong PKC activator, is an agent frequently used to activate
neutrophils and to induce acute endothelial cell injury (18, 25). We
have reported previously that PMA-activated neutrophils produced a
profound decrease in endothelial ACE activity in perfused rabbit lung
preparation and cultured endothelial cells (7, 9). PMA-activated
neutrophils produced ACE dysfunction by generating oxygen free
radicals,
H2O2,
and its conversion to hydroxyl radicals (8). While investigating the
role of PKC in the neutrophil activation and neutrophil-mediated ACE
dysfunction using the PKC inhibitor staurosporine, we made the
following unexpected observations: 1) treatment of BPAE with
staurosporine, like treatment of neutrophils or cotreatment of BPAE and
neutrophils with staurosporine, prevents neutrophil-mediated
endothelial ACE dysfunction and neutrophil-mediated endothelial
cytotoxicity; 2) treatment of BPAE
with staurosporine dramatically potentiated
H2O2-
or X/XO-mediated endothelial cytotoxicity; and
3) conditioned medium from
staurosporine-treated BPAE inhibited O
2· generation by PMA-activated
neutrophils and monocytes and decreased neutrophil adherence to
endothelial cells.
Our previous data suggest that neutrophil-mediated endothelial ACE
dysfunction depends on PKC activity of neutrophils, since 1) preincubation of neutrophils with
PMA produced a decrease in ACE activity, whereas pretreatment of
endothelial cells with PMA followed by incubation with neutrophils had
no effects on ACE activity, and 2)
inhibition of neutrophil PKC activity by staurosporine prevented
neutrophil-mediated endothelial ACE dysfunction (7). However, we were
surprised to find that inhibition of endothelial cell PKC activity by
staurosporine also prevented the neutrophil-mediated endothelial ACE
dysfunction and cytotoxicity. One possible explanation is that
endothelial cell PKC activity is required for neutrophil-mediated endothelial injury. Johnson et al. (17) reported that, in isolated guinea pig lungs, the PKC inhibitor H-7 prevented pulmonary edema induced by
H2O2,
suggesting a role for PKC in
H2O2-induced
pulmonary edema. Because oxygen metabolites such as
O
2· or
H2O2
are important mediators of neutrophil-mediated endothelial ACE
dysfunction and cytotoxicity, we tested whether inhibition of
endothelial PKC activity attenuates
H2O2-
or X/XO-induced cytotoxicity. Contrary to this hypothesis, inhibition
of endothelial PKC activity dramatically potentiated the
H2O2-
or X/XO-induced cytotoxicity, suggesting that inhibition of endothelial
PKC increases the vulnerability of endothelium to oxidant injury. The
mechanism for this potentiation of oxidant-induced endothelial injury
is not clear. One possible explanation is that inhibition of
endothelial PKC activity prevents phosphorylation of certain proteins
and decreases the activity of key enzymes such as antioxidant or
mitochondrial enzymes, thus rendering endothelial cells more
susceptible to oxidant injury.
The major discovery of this study is that endothelial cells treated
with the PKC inhibitor staurosporine generate a soluble neutrophil
activation inhibitor. There are several reports that have shown that
coincubation of neutrophils with endothelial cells markedly inhibited
the extracellular release of O
2· in vitro after activation of neutrophils by membrane receptor-mediated activators such as FMLP, opsonized zymosan, or heat-killed
staphylococci (2, 3, 14, 15) but not by non-plasma membrane
receptor-mediated activators, e.g., PMA (2). Basford et al. (2) showed
that this soluble inhibitor released by unstimulated endothelial cells appears to be a polypeptide and is not adenosine, an arachidonate metabolite, or SOD. Unlike the soluble inhibitor reported by Basford and co-workers, the inhibitor reported in this study is released only
from endothelial cells treated with staurosporine and inhibits O
2· generated by neutrophils
activated either by a membrane receptor-mediated activator (FMLP) or a
non-plasma membrane-mediated activator (PMA).
Hoover and co-workers (15) reported that
O
2· released at the
neutrophil-endothelial interface was scavenged by endothelial SOD,
accounting for a spurious "inhibition" of
O
2· production. Three lines of
evidence suggest that scavenging did not account for our observation.
First, using the X/XO system to generate
O
2· at levels comparable to those
of PMA-activated neutrophils, we found no evidence of scavenging
O
2· by staurosporine-treated endothelial cells. Second, treatment of BPAE with the SOD inhibitor DTCA did not decrease the observed inhibitory effects. Third, conditioned medium from staurosporine-treated endothelial cells had
qualitatively similar inhibitory effects on
O
2· production by neutrophils.
Taken together, these observations strongly suggest that regulation of
activation of neutrophils, rather than scavenging of
O
2· by SOD, appears to account for
the inhibitory effects of conditioned medium from staurosporine-treated
endothelial cells.
Two other PKC inhibitors, H-7 and sphingosine, failed to produce the
same effects as staurosporine, suggesting that staurosporine's effects
may not be due to direct inhibition of PKC but instead due to some as
yet unidentified mechanism. In addition to inhibiting PKC activity,
staurosporine has been reported to inhibit protein tyrosine kinase
activity (16, 24). Because H-7 and sphingosine do not inhibit protein
tyrosine kinase, it is possible that staurosporine's effects may be
due to its ability to block protein tyrosine kinase activity.
The soluble inhibitor only partly prevented PMA-treated PMN from
adhering to endothelial cells. Similarly, whereas treatment of
endothelial cells with staurosporine prevented superoxide release or
decrease in endothelial ACE activity from PMA-exposed PMN, it did not
alter the PMA-induced increased PMN adherence to endothelial cells. One
explanation is that, since staurosporine caused endothelial cell
retraction, it is possible that a fraction of the PMN adhered to the
subendothelial matrix rather than to endothelial cells. Although these
mechanisms remain unclear, these findings suggest that the adherent PMN
were not significantly activated, since they were unable to elicit
either superoxide release or ACE dysfunction.
The identity of the soluble inhibitor remains elusive. Cyclooxygenase
inhibitors, lipoxygenase inhibitors, adenosine deaminase, and protein
synthesis inhibitors had negligible effects on its activity. It is
still not clear whether or not this inhibitor is released under any
physiological or pathophysiological conditions, nor do we know the mode
of action of this inhibitor. Nevertheless, characterization and
purification of this inhibitor may lead to discovery of a novel agent
that can be used in the treatment and/or prevention of
inflammatory processes.
In summary, treatment of endothelial cells with staurosporine, like
treatment of neutrophils, prevented neutrophil activation as well as
neutrophil-mediated endothelial ACE dysfunction and endothelial
cytotoxicity. Treatment of endothelial cells with staurosporine
potentiated the
H2O2-
and X/XO-mediated endothelial cytotoxicity. Endothelial cells or
conditioned medium from staurosporine-treated BPAE inhibited
O
2· generation by activated neutrophils and increased neutrophil adherence to endothelial cells.
Endothelial cells treated with staurosporine do not inhibit O
2· generated by X/XO. The
inhibitory effects of endothelial cells treated with staurosporine on
O
2· generation were not affected
by the SOD inhibitor DTCA. Thus these data suggest that a soluble
factor is released by endothelial cells treated with staurosporine,
which can inhibit neutrophil activation and neutrophil-mediated
endothelial injury.
 |
ACKNOWLEDGEMENTS |
We are very thankful to Dr. James Ryan (Vascular Biology Center,
Medical College of Georgia) and Dr. Pradyumna E. Tummala (Division of
Cardiology, Emory University) for critical reading of the manuscript
and to Connie Snead for expert technical assistance.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-31422.
Present address of X. Chen: Div. of Cardiology, Emory University School
of Medicine, 1639 Pierce Dr., WMB319, Atlanta, GA 30322.
Address for reprint requests: J. D. Catravas, Vascular Biology Center,
Medical College of Georgia, Augusta, GA 30912-2500.
Received 28 January 1997; accepted in final form 31 March 1998.
 |
REFERENCES |
1.
Babior, B. M.
Oxygen-dependent microbial killing by phagocytes (Abstract).
N. Engl. J. Med.
298:
659,
1978[Medline].
2.
Basford, R. E.,
R. L. Clark,
R. A. Stiller,
S. S. Kaplan,
D. B. Kuhns,
and
J. E. Rinaldo.
Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor.
Am. J. Respir. Cell Mol. Biol.
2:
235-243,
1990[Medline].
3.
Basford, R. E.,
S. S. Kaplan,
R. L. Clark,
D. B. Kulns,
and
J. E. Rinaldo.
Endothelial cells produce a soluble inhibitor of superoxide production by neutrophils (Abstract).
Blood
68:
79a,
1986.
4.
Berkow, R. L.,
R. W. Dodson,
and
A. S. Kraft.
The effect of a protein kinase C inhibitor, H7, on human neutrophil oxidative burst and degranulation.
J. Leukoc. Biol.
41:
441-446,
1987[Abstract].
5.
Catravas, J. D.,
J. S. Lazo,
K. D. Dobular,
L. R. Mills,
and
C. N. Gillis.
Pulmonary endothelial dysfunction in the presence or absence of interstitial injury induced by intratracheally injected bleomycin in rabbits.
Am. Rev. Respir. Dis.
128:
740-746,
1983[Medline].
6.
Catravas, J. D.,
and
R. E. White.
Kinetics of pulmonary angiotensin-converting enzyme and 5'-nucleotidase in vivo.
J. Appl. Physiol.
57:
1173-1181,
1984[Abstract/Free Full Text].
7.
Chen, X.,
and
J. D. Catravas.
PMA-activated neutrophils decrease endothelial ectoenzyme activities in perfused rabbit lung.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L657-L663,
1992[Abstract/Free Full Text].
8.
Chen, X.,
and
J. D. Catravas.
Neutrophil-mediated endothelial angiotensin-converting enzyme dysfunction: role of oxygen-derived free radicals.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L243-L249,
1993[Abstract/Free Full Text].
9.
Chen, X.,
M. Tzanela,
M. K. Baumgartner,
J. R. McCormick,
and
J. D. Catravas.
PMA-activated neutrophils decrease endothelial ectoenzyme activities in cultured aortic endothelial cells.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L650-L656,
1992[Abstract/Free Full Text].
10.
Cronstein, B. N.,
M. A. Eberle,
H. E. Gruber,
and
R. I. Levin.
Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells.
Proc. Natl. Acad. Sci. USA
88:
2441-2445,
1991[Abstract].
11.
Frank, L.,
D. L. Wood,
and
R. J. Roberts.
Effect of diethyldithiocarbamate on oxygen toxicity and lung enzyme activity in immature and adult rats.
Biochem. Pharmacol.
27:
251-254,
1978[Medline].
12.
Gunther, G. R.,
and
M. B. Herring.
Inhibition of neutrophil superoxide production by adenosine released from vascular endothelial cells.
Ann. Vasc. Surg.
5:
325-330,
1991[Medline].
13.
Hardart, G. E.,
G. W. Sullivan,
H. T. Carper,
and
G. L. Mandell.
Adenosine and 2-phenylaminoadenosine (CV-1808) inhibit human neutrophil bactericidal function.
Infect. Immun.
59:
885-889,
1991[Medline].
14.
Hoover, R. L.,
J. M. Robinson,
and
M. J. Karnovsky.
Superoxide production by polymorphonuclear leukocytes is inhibited by contact with endothelial cells (Abstract).
J. Cell Biol.
95:
L10A,
1982.
15.
Hoover, R. L.,
J. M. Robinson,
and
M. J. Karnovsky.
Adhesion of polymorphonuclear leukocytes to endothelium enhances the efficiency of detoxification of oxygen-free radicals.
Am. J. Pathol.
126:
258-268,
1987[Abstract].
16.
Imbert, V.,
R. A. Rupec,
A. Livolsi,
H. L. Pahl,
E. B. Traenckner,
C. Mueller-Dieckmann,
D. Farahifar,
B. Rossi,
P. Auberger,
P. A. Baeuerle,
and
J.-F. Peyron.
Tyrosine phosphorylation of I
B-
activated NF-
B without proteolytic degradation of I
B-
.
Cell
86:
787-789,
1996[Medline].
17.
Johnson, A.,
P. Phillips,
D. Hocking,
M.-F. Tsan,
and
T. Ferro.
Protein kinase inhibitor prevents pulmonary edema in response to H2O2.
Am. J. Physiol.
256 (Heart Circ. Physiol. 25):
H1012-H1022,
1989[Abstract/Free Full Text].
18.
Johnson, K. L.,
and
P. A. Ward.
Acute and progressive lung injury after contact with phorbol myristate acetate.
Am. J. Pathol.
107:
29-35,
1982[Abstract].
19.
Koyama, S.,
S. I. Rennard,
G. D. Leikauf,
and
R. A. Robbins.
Bronchial epithelial cells release monocyte chemotactic activity in response to smoke and endotoxin.
J. Immunol.
147:
972-979,
1991[Abstract/Free Full Text].
20.
Martin, W. J., II.
Neutrophils kill pulmonary endothelial cells by hydrogen peroxide dependent pathway. An in vitro model of neutrophil-mediated lung injury.
Am. Rev. Respir. Dis.
130:
209-213,
1984[Medline].
21.
O'Flaherty, J. T.,
D. L. Kreuttzer,
H. J. Showell,
and
P. A. Ward.
Influence of inhibitors of cellular function on chemotactic factor-induced neutrophil aggregation.
J. Immunol.
119:
1751-1756,
1977[Abstract].
22.
Rao, G. N.,
and
B. C. Berk.
Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression.
Circ. Res.
70:
593-599,
1992[Abstract].
23.
Ryan, U. S.
Isolation and culture of pulmonary endothelial cell.
Environ. Health Perspect.
56:
103-114,
1984[Medline].
24.
Secrist, J. P.,
I. Sehgal,
G. Powis,
and
R. T. Abraham.
Preferential inhibition of the platelet-derived growth factor receptor tyrosine kinase by staurosporine.
J. Biol. Chem.
265:
20394-20400,
1990[Abstract/Free Full Text].
25.
Varani, J.,
S. E. G. Fligiel,
G. O. Till,
R. G. Kunkel,
U. S. Ryan,
and
P. A. Ward.
Pulmonary endothelial cell killing by human neutrophils: possible involvement of hydroxyl radical.
Lab. Invest.
53:
656-663,
1985[Medline].
26.
Weiss, S. J.,
J. Yong,
A. F. LoBuglio,
A. Slivka,
and
N. F. Nimeh.
Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells (Abstract).
J. Clin. Invest.
68:
714,
1981[Medline].
27.
Wheeler, M. E.,
F. W. Luscinskas,
M. P. Bevilacqua,
and
M. A. Gimbrone, Jr.
Cultured human endothelial cells stimulated with cytokines or endotoxin produce an inhibitor of leukocyte adhesion.
J. Clin. Invest.
82:
1211-1218,
1988[Medline].
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