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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3064-3072
Endothelin-1 Induces Production of the Neutrophil Chemotactic Factor
Interleukin-8 by Human Brain-Derived Endothelial Cells
By
F.M. Hofman,
P. Chen,
R. Jeyaseelan,
F. Incardona,
M. Fisher, and
R. Zidovetzki
From the Departments of Pathology, Medicine, and Neurology,
University of Southern California, Los Angeles; and the Departments of
Biology and Neuroscience, University of California, Riverside, CA.
 |
ABSTRACT |
Increased levels of endothelin-1 (Et-1), a potent vasoconstrictor,
have been correlated with hypertension and neuronal damage in
ischemic/reperfusion injury. The presence of polymorphonuclear cells
(PMNs) in the brain has been shown to be directly responsible for this
observed pathology. To address the question of whether Et-1 plays a
role in this process, human brain-derived endothelial cells (CNS-ECs)
were cultured with Et-1. The results demonstrate that Et-1 induces
production of the neutrophil chemoattractant interleukin-8 (IL-8)
twofold to threefold after 72 hours; mRNA was maximal after 1 hour of
stimulation. Conditioned culture medium derived from Et-1-stimulated
CNS-ECs induced a chemotactic response in the PMN migration assay. The
inflammatory cytokines tumor necrosis factor- (TNF) and IL-1
functioned additively with Et-1 in increasing IL-8 production. In
contrast, transforming growth factor- (TGF-), but not IL-10,
completely abolished the effect of Et-1 on IL-8 production. However,
Et-1 did not modulate intercellular adhesion molecule-1 (ICAM-1)
expression. These data demonstrate that Et-1 may be a risk factor in
ischemic/reperfusion injury by inducing increased levels of the
neutrophil chemoattractant IL-8.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ENDOTHELIN-1 (Et-1), a 21-amino acid
peptide originally isolated from endothelial cells, was shown to
mediate vasoconstriction.1 Cerebral microvessels show a
marked sensitivity to Et-1 and are able to produce this
peptide.2,3 Three forms of the peptide have been
characterized and designated Et-1, -2, and -3. Both Et-1 and Et-3 are
present in the brain. Endothelial cells produce only Et-1, the most
potent known vasoconstrictor.4-6 In addition to
vasoconstriction,1 Et-1 peptides have neuroregulatory and physiologic functions.7-9 Increased levels of Et-1 in
plasma and cerebrospinal fluid of patients with hypertension, ischemic stroke, and subarachnoidal hemorrhage have implicated Et-1 as a
possible mediator of cerebrovascular responses in these
disorders.10-13 The elevated levels of Et-1 in ischemic
stroke correlate significantly with the severity of neurologic
deficits.14 However, it is not clear whether increased
levels of Et-1 are the cause or the result of disease processes, and
there is considerable evidence for either situation. Et-1 has been
shown to induce pathology in a variety of experimental protocols.
Tissue levels of Et-1 in rats subjected to either permanent or
transient focal ischemia were shown to be significantly
increased.15 In humans, increased Et-1 tissue levels were
observed during the reperfusion period.16 Elevated levels
of Et-1 are clearly associated with central nervous system (CNS)
pathology in stroke; however, the mechanism of its action has not been
elucidated.
One possible mechanism of Et-1 activity may be involved in affecting
polymorphonuclear cell (PMN) activity during initiation or progression
of ischemia/reperfusion damage. Studies have shown that ischemic brain
injury involves an initial influx or neutrophils followed by
infiltration of monocytes/macrophages into CNS tissue.17 Activated neutrophils are involved in tissue repair, but may also cause
tissue damage through the release of oxygen free radicals, proteinases,
and neurotoxins.18 The significance of PMN accumulation in
ischemia/reperfusion is evident by the impact of PMN depletion on
neuronal damage. Neutropenia induced by antibodies to PMNs decreased
the damage in ischemia19 and improved the neuronal recovery
after complete20 or incomplete21 cerebral
ischemia. Antibodies to neutrophil adhesion molecules CD11/CD18, the
ligand for intercellular adhesion molecule-1 (ICAM-1), decreased
neutrophil binding and infiltration,22,23 leading to
significant reduction in ischemic damage.17 The critical
importance of PMNs to reperfusion injury was further confirmed by
reducing disease with anti-ICAM-1 antibodies or in ischemia induced in
ICAM-1 knockout mice.24,25
The question is then raised as to the mechanism by which the
accumulation of neutrophils in ischemic tissue is achieved. The neutrophil chemotactic factor interleukin-8 (IL-8) was examined, because it is extremely potent in mediating neutrophil migration and
transmigration.26 IL-8 is an -chemokine that activates motility and directional migration, upregulates integrin expression, and increases respiratory burst activity of PMNs.27,28 IL-8 has been shown to be induced by the inflammatory cytokines, tumor necrosis factor- (TNF) and IL-1, and endotoxin.29
The function of IL-8 in the initiation of CNS disease is only now being
examined. A recent study shows that IL-8 is produced during
hypoxia,26 suggesting a possible role for this chemokine in
ischemia/reperfusion damage at the endothelial cell surface. In the
present study, the effect of Et-1 on IL-8 production by CNS endothelial
cells (CNS-ECs) was investigated. The results show that Et-1
upregulates the production of IL-8 by CNS-ECs, and this regulation
occurs at the transcriptional level. Furthermore, TNF and IL-1
enhance this effect, whereas transforming growth factor- (TGF-)
downregulates Et-1-induced IL-8 production.
| |
MATERIALS AND METHODS |
Reagents.
The following reagents were purchased: Et-1 (Peninsula Laboratories,
Belmont, CA), TNF (Boehringer Mannheim, San Diego, CA), IL-1
(Genzyme Corp, Cambridge, MA), TGF-1 (Genzyme), IL-10 (R&D Systems,
Minneapolis, MN), goat polyclonal and mouse monoclonal anti-human IL-8
antibodies (R&D Systems), mouse monoclonal anti-ICAM-1 (Immunotech,
Marseille, France), FITC-goat anti-mouse serum (Becton Dickinson, San
Jose, CA), normal goat serum (Vector Laboratories, Burlingame, CA), and
goat anti-IL-1 and anti-IL-1 (R&D Systems).
Cell culture.
CNS-ECs were derived from human brain as previously described in
detail.30 Cells were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 100 ng/mL endothelial cell growth factor (Endogro; Vectec, Albany, NY), 2 mmol/L
L-glutamine, 10 mmol/L HEPES, 24 mmol/L sodium bicarbonate,
300 U heparin USP, 1% penicillin/streptomycin, and 10% fetal calf
serum (FCS). Endogro-free medium was used 24 hours before the
experiment. The purity of CNS-ECs (95%) was confirmed by
immunocytochemical staining for von Willebrand factor (vWF), glial
fibrillary acidic protein (GFAP) for astrocytes, and CD11b for
macrophages as previously described.30 The cells were used
until passage four to five only, because it was found that with an
increasing passage number (> seven to 10), the intensity of vWF and
 -glutamyl transpeptidase decreased. Cells were routinely examined
for possible contaminating populations before each experiment using
CD11b and GFAP.
IL-8 assay.
IL-8 production was evaluated using the commercially available
enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). Briefly,
CNS-ECs were grown in culture to confluence in 10% FCS in 60-mm dishes
in a volume of 3 mL; 24 hours before initiation of the experiment, this
medium was replaced by medium containing 2% FCS. The culture
supernatant (100 µL) was removed after 72 hours, unless otherwise
stated, and evaluated for IL-8 content using the ELISA kit. The
experimental groups were set up in triplicate, and ELISA samples were
evaluated in quadruplicate. The data are expressed as nanograms per
106 cells. Cell counts were determined using trypan blue
exclusion. Cell viability was routinely greater than 95%.
Immunocytochemistry.
Cells were treated as already described. At the termination of the
experiment, cell cultures were rinsed with phosphate-buffered saline
(PBS), prepared in suspension, and cytocentrifuged
(5 × 104 cells per slide). Air-dried slides were fixed
in acetone for 5 minutes, again allowed to dry, and then subjected to
the staining procedure.30 Briefly, cell preparations were
treated with the primary monoclonal mouse antibody (18 hours) and
subsequently washed with PBS twice (10 minutes), followed by incubation
with the biotin-labeled secondary antibody, horse anti-mouse Ig (Vector Laboratories) (30 minutes). The slides were then incubated with avidin-biotin-horseradish peroxidase complex (Vector Laboratories) (30 minutes) followed by treatment with aminoethylcarbasole solution (10 minutes), and counterstained with Mayer's hematoxylin (1 minute). Irrelevant isotype-matched antibody was used in place of the primary antibody as the negative control.
RNase protection analysis.
Radioactively labeled RNA antisense probes were prepared following the
manufacturer's protocol. Using the In Vitro Transcription Kit
(Pharmingen, San Diego, CA), 10 µL 32P-UTP (3,000 Ci/mmol, 10 mCi/mL; NEN Research, Wilmington, DE) and 1 µL GACU pool
were added to the RNase protection assay (RPA) template set (HCK-5),
which is a human chemokine multiprobe set including IL-8 and the
housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(Pharmingen). Also included were T7 polymerase, DTT, RNAsin, and
transcription buffer as suggested by the manufacturer. GAPDH expression
may vary somewhat, but the expression level in this culture system
appeared consistent independent of the stimuli used in these
experiments. The reaction was terminated by adding 2 µL DNase for 30 minutes at 37°C. The probe was then extracted using Tris-saturated
phenol: chloroform:isoamylalcohol (25:24:1; GIBCO-BRL) and
chloroform:isoamylalcohol sequentially, and then ethanol-precipitated.
The radiolabeled RNA pellet was air-dried and solubilized with
hybridization buffer.
RNA from 2 to 3 × 106 cultured cells per experimental
group was isolated and prepared according to a modification of the acid phenol method using Trizol reagent (Life Technologies, Gaithersburg, MD) as specified by the manufacturer. Total RNA (10 µg) together with
6 × 105 cpm of probe was heat-denatured at 90°C and
then hybridized overnight at 56°C. Subsequently, the samples were
treated with the RNase cocktail followed by proteinase K cocktails, and
then precipitated using ammonium acetate and ethanol. Air-dried samples
were solubilized in 1× loading buffer, denatured at 90°C for 3 minutes, and then placed on ice. The protected fragments were resolved
in 5% acrylamide/8-mol/L urea gel (24 cm length); the gel was dried
and exposed to Hyper film (Amersham Life Science, Arlington Heights,
IL) at  70°C. The protected bands were observed for IL-8 (181 bp)
and GAPDH (96 bp). Data are presented as the ratio of the
spectrophotometric density of IL-8/GAPDH bands. Differences among the
groups were calculated as the ratio of the band densities of the
experimental/control groups. The manufacturer-recommended yeast tRNA
negative control and a positive control were included in every RPA
experiment.
Flow cytometry.
ICAM-1 expression on CNS-ECs was determined using flow cytometry.
Briefly, cells were incubated overnight in 2% FCS and stimulated with
Et-1 or TNF for 18 hours. Cells were then detached using trypsin
(0.1%) in PBS-EDTA (0.5 µmol/L) and washed with PBS. The primary
antibody was added for 1 hour (ICAM-1, 1:500 dilution) and washed twice
with PBS before application of the secondary FITC-conjugated goat
anti-mouse antibody (1:100; Becton Dickinson) for 30 minutes. Cells
were again washed twice and submitted to flow analysis on a FACstar
(Becton Dickinson) using 5,000 cells per count. ICAM-1 expression is
presented as the percent positive cells. The data are representative of
one of three experiments.
Neutrophil migration assay.
PMNs were isolated from whole blood of healthy human subjects using a
mixture of sodium metrizoate and Ficoll gradient centrifugation (1-step
Polymorphs; Accurate Chemical & Scientific Corp, Westbury, NY).
Approximately 5 mL whole blood was drawn into a syringe containing 0.5 mL 3.8% sodium citrate as an anticoagulant. The blood was then layered
over 3.5 mL Polymorphprep in a 12-mL tube and centrifuged at
500g for 35 minutes at 20°C. After centrifugation, two
leukocyte bands were visible, and the lower band of PMNs was
resuspended in RPMI supplemented with 0.05% FCS and 0.1% bovine serum
albumin (BSA). PMNs were then washed twice for 10 minutes at 400 × g. Cell purity determined by morphology following
hematoxylin staining was 94% to 98%, and viability was greater than
95% as determined by trypan blue dye exclusion.
Migration of PMNs was performed in cell culture inserts (Becton
Dickinson, Frankin Lakes, NJ) using a 3-µm pore PET track-etched membrane and 24-well format. After 72 hours, media from the differently treated CNS-EC groups cultured in RPMI with 0.05% FCS/0.1% BSA were
added to the lower compartment. Media that were not exposed to cells
were used as the control. In the upper compartment, 3 × 105 PMNs in culture media were added. The chambers were
incubated for 30 minutes at 37°C in 5% CO2. At the
conclusion of this period, all liquid in the lower compartments was
individually collected and centrifuged, and the cells were counted on a
hemocytometer. The results are presented as the mean ± SD of three
separate experiments, and are expressed as the increase in the percent
of cells migrating in the experimental groups divided by the number of
cells migrating when exposed to control media multiplied by 100.
Statistics.
Values are presented as the mean ± SEM, unless otherwise stated.
Statistical significance was evaluated using Student's
t-test for paired comparison; P < .05 was
considered significant.
| |
RESULTS |
Et-1 upregulates IL-8 production in CNS-ECs.
To determine whether CNS-ECs respond to Et-1 by producing IL-8, the
cultures were first grown to confluency and then repeatedly treated with Et-1 at 24-hour intervals for the required
experimental period. It was noted that subconfluent cultures were
generally less responsive to Et-1; therefore, cultures used in this
study were greater than 90% confluent. Furthermore, the addition of Et-1 only at initiation of the experiment resulted in a reduced effect.
The results using the ELISA technique on culture supernatants showed
that 24-hour samples expressed little increased IL-8 production compared with control levels (Fig 1). Each
point represents quadruplicate samples; the SEM was usually less than
5%. The concentration of IL-8 in the culture supernatant of 48- and
72-hour cultures increased twofold and threefold, respectively. To
determine whether IL-8 protein was synthesized before 48 hours, the
kinetics of IL-8 production were analyzed on a single cell level.
CNS-ECs were treated with Et-1 for 6, 24, and 48 hours and then
examined using immunocytochemistry. Results in Fig
2A demonstrate that as early as 6 hours,
20% of the cells were IL-8-positive. After 24 hours, 50% to 70% of
the cells stained for IL-8 (Fig 2C). The 48-hour cell preparations
appeared similar to the 24-hour cultures (data not shown). Control
untreated cultures exhibited less than 3% positivity (Fig 2B and D).
Irrelevant isotype-matched monoclonal antibody did not show significant
staining. To determine the optimal concentration of Et-1, a range of
Et-1 concentrations were examined. Results in Fig
3 show that at 10 8 to
106 mol/L Et-1, there was a significant increase of IL-8
production compared with controls; at Et-1 concentrations above
106 mol/L, cell morphology was altered, and adhesion
decreased while IL-8 levels remained constant (data not shown). Based
on these results, all experiments presented here used
107 mol/L Et-1 unless otherwise stated. To confirm that
the observed twofold to threefold increase in IL-8 production with Et-1
stimulation is consistent and reproducible, 11 independent experiments
were performed comparing the supernatants from control and
Et-1-treated cells. Results in Fig 3B demonstrate that Et-1
significantly (P < .001) increased IL-8 production in
CNS-ECs.
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| Fig 1.
Kinetics of IL-8 production by Et-1-stimulated CNS-ECs.
Confluent CNS-ECs were cultured in the absence ( ) or presence ( )
of Et-1 (107 mol/L). At 24-, 48-, and 72-hour intervals,
supernatants were examined for IL-8 production using the ELISA
technique. Data are the mean ± SEM of quadruplicate samples. This is
one of three representative experiments.
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| Fig 3.
(A) Concentration-dependent increase in IL-8 production
stimulated with Et-1. Confluent CNS-ECs were exposed to different
concentrations of Et-1, as well as media alone. After 72 hours,
supernatant samples were analyzed for IL-8 production using the IL-8
ELISA technique. Data are the mean ± SEM of triplicate samples. The
first point is Et-1 at zero concentration. The results represent one of
three experiments performed. (B) Eleven experiments using Et-1 (100 nmol/L)-treated and untreated cell (72 hours) supernatants were
analyzed for IL-8 production. Results for the two groups are
significantly different (P < .001).
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To determine whether Et-1 regulates IL-8 at the level of mRNA, the
multiple RPA was performed, wherein IL-8 and GAPDH gene were present in
the same template set. CNS-EC cultures were exposed to Et-1 for 0.5, 1, and 4 hours. The results showed that Et-1 induced IL-8 mRNA within 0.5 hours (52% increase), with the maximal level of transcription at 1 hour (2.4-fold increase) followed by a decrease in IL-8 mRNA at 4 hours
compared with control levels (Fig 4). GAPDH
levels did not appear to change with activation and therefore were used
to monitor the amount of RNA applied to the gel as a standard. Because
IL-1 has been shown to increase IL-8 production31 and
endothelial cells produce IL-1,32 we performed
experiments to test whether Et-1 induced IL-8 production directly or
via IL-1 production. In Fig 5, CNS-ECs
were stimulated with Et-1 in the presence of anti-IL-1, and then
IL-8 mRNA was analyzed. The results showed that anti-IL-1 does not
affect Et-1-induced IL-8 mRNA. The effect of IL-1 was also
evaluated using anti-IL-1 antibody; the data demonstrate that
anti-IL-1 antibody did not block Et-1-mediated IL-8 production (Fig
5). As a control, anti-IL-1 antibody significantly blocked
IL-1-induced IL-8 mRNA. These data show that Et-1-induced IL-8
production was not mediated through IL-1 or IL-1.
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| Fig 4.
Kinetics of IL-8 mRNA expression with Et-1 treatment by
RPA. Confluent CNS-EC cultures were treated with Et-1
(107 mol/L) for 0.5, 1, or 4 hours or left untreated.
Subsequently, total RNA was isolated from the cells and probed with
32P-labeled riboprobes for IL-8 or GAPDH. The results were
visualized by autoradiography. The protected size corresponding to IL-8
(181 bp) and GAPDH (96 bp) is shown.
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| Fig 5.
Et-1-induced IL-8 mRNA expression is independent of
IL-1 mRNA synthesis. Confluent CNS-EC cultures were treated with
either Et-1 (107 mol/L) or IL-1 (10 pg/mL) in the
absence or presence of anti-IL-1 (200 µg/mL) or anti-IL-1 (200 µg/mL) for 1 hour. Control cultures included untreated CNS-ECs and
cultures exposed only to anti-IL-1. Total RNA was isolated from the
different treatment groups and probed with 32P-labeled IL-8
or GAPDH riboprobes. Specific bands for IL-8 and GAPDH were visualized
using autoradiography. The bands were identified by their appropriate
size (IL-8, 181 bp; GAPDH, 96 bp). Results are calculated as the ratio
of the spectrophotometric density measurement of the IL-8 band divided
by the corresponding GAPDH band, followed by a comparison of
experimental group and control values.
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Cytokines modulate Et-1-induced IL-8 production.
A series of experiments were performed to determine whether
Et-1-induced production of IL-8 at the protein level can be modulated by proinflammatory or antiinflammatory cytokines. Results in Fig 6 demonstrate that either Et-1 treatment
alone or TNF (10 pg/mL) treatment alone increased IL-8 protein
production (2.9-fold and 4.5-fold, respectively), and these reagents
together had an additive (8.4-fold) effect. In contrast, IL-10 (10 ng/mL), an antiinflammatory cytokine known to downregulate immune
function,33 did not affect Et-1-induced IL-8 protein
production (Fig 6). IL-10 itself did not regulate IL-8 production. The
effects of TGF- on Et-1-induced IL-8 secretion were examined.
TGF- (10 ng/mL) did not affect IL-8 production by CNS-ECs, but
abolished Et-1-induced IL-8 production (Fig
7). IL-1 (1 pg/mL) was also a potent
inducer of IL-8 (fivefold). IL-1 and Et-1 together had an additive
effect on IL-8 production (Fig 7).
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| Fig 6.
TNF modulates Et-1-induced IL-8 production. Confluent
CNS-ECs were exposed to Et-1 (107 mol/L), TNF (10 pg/mL), or IL-10 (10 ng/mL) separately or in the combinations of TNF + Et-1 or IL-10 + Et-1. After 72 hours, the culture supernatant was
examined for IL-8 production using the ELISA technique. Results are the
mean ± SEM of triplicate cultures. The data represent one of three
experiments.
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| Fig 7.
TGF- and IL-1 regulate Et-1-induced IL-8
production. CNS-EC cultures were left untreated or treated with Et-1
(107 mol/L), TGF- (10 ng/mL), or IL-1 (1 pg/mL)
separately or in the combinations of TGF- + Et-1 or IL- + Et-1. After 72 hours of culture, the supernatants were analyzed for
IL-8 production using the ELISA. Results are the mean ± SEM of
triplicate cultures. This represents one of three experiments
performed.
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Et-1 produces a functional chemoattractant.
To determine whether Et-1 induced production of functional IL-8,
CNS-ECs were treated with Et-1 as previously described. The supernatants from 72-hour cultures were added to the lower compartment as described in the methods. The results are expressed as the ratio of
the number of cells migrating in the presence of culture supernatants
from the different experimental groups to the number of cells present
when medium alone was added. The results (Fig 8) show that Et-1 increased PMN migration
by sevenfold compared with untreated cell cultures. Supernatants from
24- and 48-hour cultures were less effective (data not shown). To
determine whether the effect of Et-1 and TNF (1 ng/mL) together had a
similar functional effect on PMNs, supernatants from cultures
stimulated with both reagents were tested. The results show that
supernatant from cultures exposed to Et-1 and TNF together induced
greater migration (75%) compared with Et-1 (48%) or TNF (45%) alone.
To determine whether IL-8 was responsible for this migration,
polyclonal goat anti-IL-8 antibody (200 µg/mL) was added to the
Et-1-induced supernatant for 1 hour before the migration assay was
initiated. The results demonstrated that anti-IL-8 antibody blocked
PMN migration by Et-1-treated culture supernatant by 60% compared
with non-antibody-treated culture supernatants. Anti-IL-8 antibody
also decreased the activity in Et-1 + TNF supernatants by 70% (Fig 8).
Control goat antibody had no effect on cell migration using the
different supernatants.
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| Fig 8.
Et-1-treated CNS-ECs produce functional neutrophil
chemotaxis factor. Confluent cultures of CNS-ECs were left untreated or
treated with Et-1 (107 mol/L), TNF (1 ng/mL), or both
Et-1 + TNF for 72 hours. Supernatants were then collected, and Et-1
or Et-1 + TNF supernatants were exposed to either goat anti-IL-8
antibody (200 µg/mL) or goat serum (200 µg/mL). The experimental
supernatants were then placed in the lower compartment of the migration
chamber. Freshly isolated PMNs were placed in the upper chamber. After
30 minutes, cells in the lower compartment were collected and counted
using the trypan blue technique. The data are accumulated from 5 experiments and presented as the mean ± SEM. The data were calculated
as the ratio of the number of cells migrating when exposed to
experimental supernatants compared with media alone (media that had not
been in contact with cells) times 100.
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ET-1 does not affect ICAM-1 expression.
Because PMN binding to ECs is crucial in ischemic/reperfusion injury,
the role of Et-1 in modulating ICAM-1 was examined. The results
demonstrate that in one of three representative experiments, Et-1 did
not modulate ICAM-1 expression after 18 hours of treatment (2%
positive; Fig 9), whereas TNF (0.1 ng/mL)
significantly upregulated this activity (34% positive). Et-1-treated
CNS-EC cultures were also examined after 24, 28, and 72 hours, with no
observed increase in ICAM-1 (data not shown). To determine whether Et-1
can modify TNF-induced expression of ICAM-1, both Et-1 and TNF together
were used to treat CNS-ECs. The results show that TNF-induced ICAM-1 expression was not modified by Et-1 (27% positive; Fig 9). TNF at
concentrations of 1 pg/mL to 1 ng/mL did not exhibit an additive effect
with Et-1 (data not shown). These data show that Et-1 does not affect
ICAM-1 expression and that endotoxin-like impurities are not
responsible for the observed Et-1 activity.
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| Fig 9.
ICAM-1 expression is not modulated by Et-1. CNS-EC
cultures were exposed to Et-1 (107 mol/L) or TNF (0.1 ng/mL) or left untreated for 18 hours. The cells were subsequently
resuspended and incubated with anti-ICAM-1 and FITC-conjugated
anti-mouse antibody consecutively. Labeled cells were analyzed in a
FACS sorter, and the number of FITC-positive cells was determined. Data
are the number of positive cells divided by the total number of cells
counted (5,000 cells per sample).
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| |
DISCUSSION |
Numerous studies have implicated neutrophils in the development of
neuronal damage during and after ischemia in the
brain.34-36 Therefore, it is important to understand by
what mechanism PMNs are recruited to the damaged site in CNS tissue and
to identify factors that regulate this activity. The present study has
demonstrated that Et-1 stimulates CNS-ECs to produce IL-8, thereby
linking hypertension with ischemia and reperfusion injury. The increase in protein production detected here is twofold to threefold at Et-1
concentrations of 109 to 107 mol/L,
suggesting that low levels of Et-1 may have significant impact on the
local microenvironment. Furthermore, these data demonstrate that
increased Et-1 levels are likely associated with consistently higher
basal levels of IL-8 at the tissue level.
Et-1 rapidly induced an increase in IL-8 mRNA within 30 minutes
following stimulation of CNS-ECs, with a maximum value at 1 hour. By 4 hours, IL-8 mRNA was reduced to levels similar to the controls. This
pattern of relatively short-lived mRNA may account for the need to
continually restimulate the CNS-ECs with Et-1 every 24 hours for
maximal IL-8 production. A similar kinetic pattern was observed in
thrombin-induced IL-8 production in human umbilical vein endothelial
cells.37 Based on immunocytochemistry, IL-8 protein was
shown to be expressed within 6 hours of stimulation, with an increasing
number of positive cells at 24 hours. The detection of IL-8 only after
48 hours using the ELISA is likely due to the lack of sensitivity of
this technique at the high media volume to cell ratio in this culture
system. The rapid increase in IL-8 production supports a physiologic
role for this chemokine because in clinical observations PMN
infiltration is already apparent at 4 hours postreperfusion, with
monocytes detected at the ischemic site only after 18 to 24 hours
postinjury. IL-8 production is also detected at the tissue level at
this early time without being detected in the plasma.38
Endothelial cells are a source of IL-1, and this cytokine has been
shown to effectively upregulate IL-8 production.39,40 In
the case of hypoxia, IL-1 mRNA is upregulated after 16 hours, late
compared with IL-8, whereas other systems show IL-1 production early
in the response.41 Our results support the notion that Et-1
acts directly on endothelial cells and IL-1 is not an intermediary. The present data show that anti-IL-1 antibody did not block
Et-1-induced IL-8 mRNA transcription while blocking IL-1-induced
IL-8 mRNA synthesis.
We have found that Et-1 and IL-1 or TNF function in an additive
manner. The additive effect of Et-1 with TNF or IL-1 on IL-8
production, particularly at low concentrations of either TNF or IL-1
(10 pg/mL), suggests these proinflammatory cytokines have significant
impact on PMN recruitment. TNF, a cytokine produced by
monocytes/macrophages and T cells during immune activation or
infection, has been shown to upregulate IL-8 mRNA production within 1 hour, with maximum expression at 6 hours.27 IL-1, produced by endothelial cells as well as leukocytes, also induces IL-8
production.32 During bacterial or viral infection, plasma levels of inflammatory cytokines increase, with pathology observed at
high concentrations.42 The results presented here suggest that TNF and IL-1 at low concentrations causing relatively low levels of responsiveness can, in the presence of Et-1, produce significantly higher levels of IL-8, thereby increasing the possible disease progression.
Our results demonstrate that although Et-1 induced IL-8 production, it
did not increase ICAM-1 expression on CNS-ECs. However, PMN damage in
ischemic/reperfusion pathology involves both recruitment and adhesion
of endothelial cells,17 with ICAM-1 being critical for PMN
binding and subsequent transmigration into tissue.43 Neutrophil activation is induced by the binding of CD11b/CD18 (MAC-1)
integrins to ICAM-1, the ligand expressed on endothelial cells.17 IL-8 has been shown to upregulate PMN expression
of the 2 integrins CD11b and CD11c.40 Thus,
the first stage in CNS damage is PMN recruitment mediated by IL-8, and
the second stage is PMN-EC adhesion mediated by upregulation of
integrins on PMNs and upregulation of ICAM-1 adhesion molecules on
endothelial cells. As we have shown here, Et-1 does not upregulate
ICAM-1; therefore, further activation would likely require the presence of proinflammatory cytokines, particularly TNF or IL-1. TNF and IL-1 are known to increase ICAM-1 expression on endothelial
cells.44-46 Further interaction between IL-8 and TNF or
IL-1 should occur during transmigration. The net effect of the
simultaneous presence of Et-1 and TNF or IL-1 would be a significant
enhancement of ischemia/reperfusion injury.
We have further investigated the role of Et-1 interaction with other
regulatory cytokines by including TGF-, which, in contrast to TNF or
IL-1, is often associated with downregulation of inflammatory processes.47-53 The in vivo relevance of
TGF- has been documented with its decline in experimental allergic
encephalomyelitis49 and its upregulation in HIV
infection.54 Both of these pathologic processes support the
function of TGF- as an immune suppressor in the CNS. Furthermore,
there is a suggestion that TGF- may be important in stroke, with
evidence that serum levels of this growth factor are decreased in
patients with ischemic stroke in the acute stage compared with
age-matched normal controls.36 Our results show that
TGF- is a potent inhibitor of IL-8 production. Based on the results
presented here, the mechanism by which TGF- regulates IL-8
production in CNS-ECs may be, in part, at the level of transcription,
but more likely at other processes. Thus, in ischemic stroke, TGF-
may function as an endogenous immune-suppressive agent and is therefore
potentially useful therapeutically to inhibit IL-8 production by
endothelial cells. In contrast to TGF-, IL-10, also known to have
immune-suppressive effects on endothelial cells, astrocytes, and
lymphocytes,33,55,56 did not affect IL-8 production. These
data emphasize the selective effect of TGF- in downregulating IL-8
synthesis.
In conclusion, the present study demonstrates that CNS-ECs, upon
stimulation with Et-1, produce functional IL-8. This Et-1-induced IL-8
synthesis can be upregulated by the proinflammatory cytokines TNF or
IL-1 and downregulated by TGF-. These data suggest that circulating Et-1, even at low levels, is likely a contributing factor
to PMN recruitment. The presence of low levels of the inflammatory cytokines TNF and IL-1 together with Et-1 may play a significant role in the enhanced IL-8 production and subsequent neutrophil activation and eventual neuronal damage observed in
ischemia/reperfusion injury.
| |
FOOTNOTES |
Submitted December 22, 1997;
accepted June 12, 1998.
Supported by National Institutes of Health Grant No. P01-NS31946.
Address reprint requests to F.M. Hofman, PhD, Department
of Pathology, University of Southern California School of Medicine, 2011 Zonal Ave, HMR 312, Los Angeles, CA 90033.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank MoLi Chen and Donald Krasniak for expert technical
assistance, and Myrna Cisneros for secretarial assistance.
| |
REFERENCES |
1.
Yanagisawa M,
Kurihara H,
Kimura S,
Tomobe Y,
Kobayashi M,
Mitsui Y,
Yazuki Y,
Goto K,
Masaki T:
A novel potent vasoconstrictor peptide produced by vascular endothelial cells.
Nature
332:411,
1988[Medline]
[Order article via Infotrieve]
2.
Hardebo JE,
Kahrstrom J,
Ownan C,
Salford LG:
Endothelin is a potent constrictor of human intracranial arteries and veins.
Blood Vessels
26:249,
1989[Medline]
[Order article via Infotrieve]
3.
Yoshimoto S,
Ishizaki Y,
Kurihara H,
Sasaki T,
Yoshizumi M,
Yanagisawa M,
Yazaki Y,
Masaki T,
Takakura K,
Murata S:
Cerebral microvessel endothelium is producing endothelin.
Brain Res
508:283,
1990[Medline]
[Order article via Infotrieve]
4.
Matsumoto H,
Suzuki N,
Onda H,
Funino M:
Abundance of endothelin-3 in rat intestine, pituitary gland and brain.
Biochem Biophys Res Commun
164:74,
1989[Medline]
[Order article via Infotrieve]
5.
Shinmi O,
Kimura S,
Sawamura S,
Sugita T,
Yoshizawa Y,
Uchiyama Y,
Uchiyama Y,
Yanagisawa M,
Goto T,
Masaki T,
Kanazawa I:
Endothelin-3 is a novel neuropeptide: Isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain.
Biochem Biophys Res Commun
164:587,
1989[Medline]
[Order article via Infotrieve]
6.
Takahashi K,
Ghatei M,
Jones PM,
Murphy JK,
Lam H-C,
Halloran DJ,
Bloom SR:
Endothelin in human brain and pituitary gland: Presence of immunoreactive endothelin, endothelin mRNA and endothelin receptors.
J Clin Endocrinol Metab
72:693,
1990[Abstract/Free Full Text]
7.
Yanagisawa M,
Masaki T:
Molecular biology and biochemistry of the endothelins.
Trends Neurosci
101:374,
1989
8.
Gulati A,
Srimal RC:
Endothelin mechanisms in the central nervous system: A target for drug development.
Drug Dev Res
26:361,
1992
9.
Greenberg DA,
Chan J,
Sampson HA:
Endothelins and the nervous system.
Neurology
42:25,
1992[Free Full Text]
10.
Kohno M,
Yasunari K,
Murakawak K:
Plasma immunoreactive endothelin in essential hypertension.
Am J Med
88:614,
1990[Medline]
[Order article via Infotrieve]
11.
Yasuda M,
Kohno M,
Tahara A:
Circulating immunoreactive endothelin in ischemic heart disease.
Am Heart J
119:801,
1990[Medline]
[Order article via Infotrieve]
12.
Masaoka H,
Suzuki R,
Hirata Y,
Emon T,
Marumo F,
Harakawa K:
Raised plasma endothelin aneurysmal subarachnoid hemorrhage.
Lancet
2:1402,
1989[Medline]
[Order article via Infotrieve]
13.
Suzuki H,
Sato S,
Suzuki Y,
Takekoshi K,
Ishihara N,
Shimodin S:
Increased endothelin concentration in CSF from patients with subarachnoid hemorrhage.
Acta Neurol Scand
81:553,
1990[Medline]
[Order article via Infotrieve]
14.
Estrada V,
Tellez MJ,
Moya J,
Fernandez-Durango R,
Egido J,
Cruz AF:
High plasma levels of endothelin-1 and atrial natriuretic peptide in patients with acute ischemic stroke.
Am J Hypertens
7:1085,
1994[Medline]
[Order article via Infotrieve]
15.
Fuxe K,
Kurosawa N,
Cinfra A,
Hallstrom A,
Goliny M,
Rosen L,
Agnati LF,
Ungerstedt U:
Involvement of local ischemia in endothelin-1 induced lesions of the neostriatum of the anesthetized rat.
Exp Brain Res
88:131,
1992[Medline]
[Order article via Infotrieve]
16.
Barone FC,
Globus MYT,
Price WJ,
White RF,
Storer BL,
Feuerstein GZ,
Busto R,
Holstein EH:
Endothelin levels increase in rat focal and global ischemia.
J Cereb Blood Flow Metab
14:337,
1994[Medline]
[Order article via Infotrieve]
17.
Chopp M,
Zhang RL,
Chen H,
Li Y,
Jiang N,
Rusche JR:
Postischemic administration of an anti-MAC-1 antibody reduces ischemia cell damage after transient middle cerebral artery occlusions in rats.
Stroke
25:869,
1994[Abstract]
18.
Ward PA,
Warren JS,
Johnson KJ:
Oxygen radicals, inflammation and tissue injury.
Free Radic Biol Med
5:403,
1988[Medline]
[Order article via Infotrieve]
19.
Simpson PJ,
Todd RF III,
Fantone JC,
Nickelson JK,
Griffin JD,
Lucchese BR:
Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-MO1, anti CD11b) that inhibits leukocyte adhesion.
J Clin Invest
81:624,
1988
20.
Vasthare US,
Heinel LA,
Rosenwasser RH,
Tuma RF:
Leukocyte involvement in cerebral ischemia and reperfusion injury.
Surg Neurol
33:261,
1990[Medline]
[Order article via Infotrieve]
21.
Gragaard B,
Shurer L,
Gerdin B,
Arfors KE:
Delayed hypoperfusion after incomplete forebrain ischemia in the rat: The role of polymorphonuclear leukocytes.
J Cereb Blood Flow Metab
9:500,
1989[Medline]
[Order article via Infotrieve]
22.
Clark WM,
Madden KP,
Rothlein R,
Zivin JA:
Reduction of central nervous system ischemic injury in rabbit using leukocyte adhesion antibody treatment.
Stroke
22:877,
1991[Abstract/Free Full Text]
23.
Arnould T,
Michiels C,
Remacle J:
Increased PMN adherence on endothelial cells after hypoxia: Involvement of PAF, CD18/CD11b and ICAM-1.
Am J Physiol
264:C1102,
1993[Abstract/Free Full Text](suppl)
24.
Matsuo Y,
Onodero H,
Shiga Y,
Shozuhara H,
Ninomiya M,
Kihara T,
Tamatani T,
Miyasaka H,
Kogure K:
Role of cell adhesion molecules in brain injury after transient middle cerebral artery occlusion in the rat.
Brain Res
656:344,
1994[Medline]
[Order article via Infotrieve]
25.
Soriano SG,
Lipton SA,
Wang YF,
Xiao M,
Springer TA,
Gutierrez-Ramos JC,
Hickey PR:
Intercellular adhesion molecule deficient mice are less susceptible to cerebral ischemia-reperfusion injury.
Neurology
39:619,
1996
26.
Karakurum M,
Shreeniwas R,
Chen J,
Pinsky D,
Yan SD,
Anderson M,
Sunouchi K,
Major J,
Hamilton T,
Kuwabara K,
Rot A,
Nowyrod R,
Stern D:
Hypoxic induction of interleukin-8 gene expression in human endothelial cells.
J Clin Invest
93:1564,
1994
27.
Baggiolini M,
Walz A,
Kunkel SH:
Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils.
J Clin Invest
84:1045,
1989
28.
Oppenheim JJ,
Zachariae COC,
Mukaida N,
Matsushima K:
Properties of the novel proinflammatory supergene "intercrine" cytokine family.
Annu Rev Immunol
9:617,
1991[Medline]
[Order article via Infotrieve]
29.
Huber AR,
Kunkel SL,
Todd RF III,
Weiss SJ:
Regulation of transendothelial neutrophil migration by endogenous interleukin-8.
Science
254:99,
1991[Abstract/Free Full Text]
30.
Hofman FM,
Dohadwala MH,
Wright AD,
Hinton DR,
Walker SM:
Exogenous tat protein activates central nervous system-derived endothelial cells.
J Neuroimmunol
54:19,
1994[Medline]
[Order article via Infotrieve]
31.
Gimbrone MA,
Orin MS,
Brock AF,
Lus EA,
Hass PE,
Hebert CA,
Yip YK,
Leung DW,
Lowe DG,
Kohr WJ,
Darbonne WC,
Bechtol KB,
Baker JB:
Endothelial interleukin-8: A novel inhibitor of leucocyte-endothelial interactions.
Science
246:1601,
1989[Abstract/Free Full Text]
32.
Miossec P,
Cavender D,
Ziff M:
Production of interleukin 1 by human endothelial cells.
J Immunol
136:2486,
1986[Abstract]
33.
Frei K,
Lins H,
Schwerdel C,
Fontana A:
Antigen presentation in the central nervous system.
J Immunol
152:2720,
1994[Abstract]
34.
Okada Y,
Copeland BR,
Mori E,
Tung M-M,
Thomas WS,
del Zoppo GJ:
P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion.
Stroke
25:202,
1994[Abstract]
35.
Matsuo Y,
Kihara Y,
Ikeda M,
Ninomiya M,
Onodera H,
Kogure K:
Role of neutrophils in free radical production during ischemia and reperfusion of the rat brain: Effect of neutrophil depletion on extracellular ascorbyl radical formation.
J Cereb Blood Flow Metab
15:941,
1995[Medline]
[Order article via Infotrieve]
36.
Kim JS:
Cytokines and adhesion molecules in stroke and related diseases.
J Neurol Sci
137:69,
1996[Medline]
[Order article via Infotrieve]
37.
Kaplanski G,
Fabrigoule M,
Boulary V,
Dinarello CA,
Bongrand P,
Kaplanski S,
Farnaner C:
Thrombin induces endothelial type II activation.
In Vitro
158:5435,
1997
38.
Harada A,
Sekido N,
Kahashi TA,
Wada T,
Mukoidu N,
Matsushima K:
Essential involvement of interleukin-8 (IL-8) in acute inflammation.
J Leukoc Biol
56:559,
1994[Abstract]
39.
Pober JS,
Cotran RS:
The role of endothelial cells in inflammation.
Transplantation
50:537,
1990[Medline]
[Order article via Infotrieve]
40.
Baggiolini M,
Dewald B,
Moser B:
Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines.
Adv Immunol
55:97,
1994[Medline]
[Order article via Infotrieve]
41.
Shreenwias R,
Koga S,
Karakurum M,
Pursky D,
Kaiser E,
Brett J,
Walitzky BA,
Norton C,
Polanski J,
Benjamin W,
Burns DK,
Goldstein A,
Stern D:
Hypoxia-mediated induction of endothelial cell interleukin-1a. An autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface.
J Clin Invest
90:2333,
1992
42.
Le J,
Vilcek J:
Tumor necrosis factor and interleukin 1: Cytokines with multiple overlapping biological activities.
Lab Invest
56:234,
1987[Medline]
[Order article via Infotrieve]
43.
Connolly ES,
Winfree CJ,
Springer TA,
Naka Y,
Liao H,
Yan SD,
Stern DM,
Solomon RA,
Gutierrez-Ramos JC,
Pinsky JC:
Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion.
J Clin Invest
97:209,
1996[Medline]
[Order article via Infotrieve]
44.
Dusin ML,
Springer TA:
Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells.
J Cell Biol
107:321,
1988[Abstract/Free Full Text]
45.
Pober JS,
Slowik MR,
DeLuca LG,
Ritchie AJ:
Elevated cyclic AMP inhibits endothelial cell synthesis and expression of TNF-induced endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1, but not intercellular adhesion molecule-1.
J Immunol
150:5114,
1993[Abstract]
46.
McCarron RM,
Wang L,
Racke MK,
McFarlin DE,
Spatz M:
Cytokine regulated adhesion between encephalitogenic T lymphocytes and cerebrovascular endothelial cells.
J Neuroimmunol
43:23,
1993[Medline]
[Order article via Infotrieve]
47.
Zauli G,
Vitale M,
Gibellini D,
Capitani S:
Inhibition of purified CD34+ hematopoietic progenitor cells by human immunodeficiency virus 1 or gp 120 mediated by endogenous transforming growth factor 1.
J Exp Med
183:99,
1996[Abstract/Free Full Text]
48.
Mooradian DL,
Digluo CA:
Effects of epidermal growth factor and transforming growth factor 1 and rat heart endothelial cell anchorage-dependent and independent growth.
Exp Cell Res
186:122,
1990[Medline]
[Order article via Infotrieve]
49.
Fabry Z,
Topham DJ,
Fee D,
Herlein J,
Carlino JA,
Hart MN,
Sriram S:
TGF-B2 decreases migration of lymphocytes in vitro and homing of cells into the central nervous system in vivo.
J Immunol
155:325,
1995[Abstract]
50.
Lee YJ,
Lu HT,
Nguyen V,
Qin H,
Howe PH,
Hocevar BA,
Boss JM,
Ransohoff RM,
Benveniste EN:
TGF-2 suppresses IFN- induction of class II MHC gene expression by inhibiting class II transactivation messenger RNA expression.
J Immunol
158:2065,
1997[Abstract]
51.
Chen TC,
Hinton D,
Yong VW,
Hofman FM:
TGF-2 and soluble p55 TNF R modulate VCAM-1 expression in glioma cells and brain derived endothelial cells.
J Neuroimmunol
73:155,
1997[Medline]
[Order article via Infotrieve]
52.
Penttinen RP,
Kobayashi S,
Bornstein P:
Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability.
Proc Natl Acad Sci USA
85:1105,
1988[Abstract/Free Full Text]
53.
Roberts AB,
Sporn MB,
Assoian RK,
Smith JM,
Roche NS,
Wakefield LM,
Mune UI,
Liotta LA,
Falanga V,
Kehrl JH:
Transforming growth factor beta rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro.
Proc Natl Acad Sci USA
83:4167,
1986[Abstract/Free Full Text]
54.
Hu R,
Oyaizu N,
Than S,
Kalyanaraman VS,
Wang X-P,
Pahwa S:
HIV-1 gp 160 induces transforming growth factor-beta production in human PBMC. I.
Clin Immunol Immunopathol
80:283,
1996[Medline]
[Order article via Infotrieve]
55.
Wogenson L,
Huang X,
Sauvetnick N:
Leukocyte extravasation into pancreatic tissue in transgenic mice expressing interleukin 10 in the islet of Langerhans.
J Exp Med
178:175,
1993[Abstract/Free Full Text]
56.
Koedel U,
Bernatowicz A,
Frei K,
Fontana A,
Pfister HW:
Systemically (but not intrathecally) administered IL-10 attenuates pathophysiologic alterations in experimental pneumococcal meningitis.
J Immunol
157:5185,
1996[Abstract]
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Abstract
Introduction
Abstract
