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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3064-3072
By
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.
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- © 1998 by The American Society of Hematology.
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 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 Reagents.
The following reagents were purchased: Et-1 (Peninsula Laboratories,
Belmont, CA), TNF (Boehringer Mannheim, San Diego, CA), IL-1 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
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.
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.
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.
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
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-
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.
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.
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 10 Submitted December 22, 1997;
accepted June 12, 1998.
The authors thank MoLi Chen and Donald Krasniak for expert technical
assistance, and Myrna Cisneros for secretarial assistance.
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Abstract
Introduction
(TNF) and IL-1
functioned additively with Et-1 in increasing IL-8 production. In
contrast, transforming growth factor-
Abstract
-glutamyl transpeptidase decreased. Cells were routinely examined
for possible contaminating populations before each experiment using
CD11b and GFAP.
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.
8 to
10
) or presence (
)
of Et-1 (10
