 |
INTRODUCTION |
Chemokines are small molecular weight cytokines involved
in activation of specific subsets of immune cells and their recruitment to the site of injury and inflammation (1-7). They are classified into
C, CC, CXC, and CX3C families (2-4). In the CXC family, the first two
conserved cysteines are separated by one nonconserved amino acid (X in
CXC). CXC chemokines that have a glutamic acid-leucine-arginine (ELR)
sequence immediately preceding the CXC motif are potent neutrophil
chemoattractants (ELR+ CXC chemokines) (2, 8, 9).
Neutrophil migration is stimulated by a gradient of these chemokines
from blood toward the site of inflammation.
We have recently shown up-regulation of the ELR+ CXC
chemokines, LIX1
(lipopolysaccharide-induced CXC chemokine; CXCL5), KC (cytokine-induced neutrophil chemoattractant; CXCL1), and MIP-2 (macrophage inflammatory protein-2; CXCL2) in a rat model of myocardial ischemia/reperfusion injury (10). High levels of myocardial neutrophil infiltration coincided with peak levels of LIX and MIP-2 expression. Neutralization of LIX, KC, and MIP-2 inhibited myeloperoxidase activity, a
measure of neutrophil infiltration, by 79, 28, and 37%, respectively, indicating that LIX may be the predominant neutrophil chemoattractant in this model of reperfusion injury (10). Furthermore, the
proinflammatory cytokine expression preceded the chemokine expression
in this model, suggesting that chemokine expression was a downstream
effect of cytokine production (10). This was confirmed in in
vitro studies where exposure of cardiomyocytes to TNF-
induced
LIX expression via NF-
B activation (10).
NF-
B is a ubiquitous, multisubunit, inducible transcription factor
that regulates the expression of various genes involved in the immune
and inflammatory processes (11, 12). The p50/p65 heterodimer, which has
been most studied, resides in the cytoplasm in an inactive state
because of binding of p65 to an inhibitory subunit I
B. The I
B
family, including I
B-
, I
B-
, I
B-
, I
B-
, all
prevent activation and subsequent nuclear translocation of the
heterodimer. Various stimuli including cytokines, growth factors, and
oxidative stress induce I
B hyperphosphorylation leading to its
selective degradation in the cytoplasm by the ubiquitin-26 S proteasome
system, resulting in NF-
B activation (11, 12).
A multiprotein complex comprised of IKK (I
B kinase)-
, IKK-
,
and a regulatory subunit IKK-
/NEMO was shown to mediate
phosphorylation of I
B by various cytokines (13-17). The
cytokine-initiated signal transduction cascade leading to I
B
phosphorylation has been shown to converge at activation of the IKK by
NF-
B-inducing kinase (NIK). NIK associates with IKK-
and
activates the IKK signalsome. PI 3-kinase, PI-phospholipase C, protein
kinase C, and p38 mitogen-activated protein kinase were implicated as
upstream regulators of NIK and IKK. Furthermore, poly(ADP-ribose)
polymerase 1 (PARP-1), a nuclear protein involved in DNA repair, has
been shown to physically and functionally associate with NF-
B in the
nucleus and modulate NF-
B-dependent cytokine gene
transcription (18, 19). The role of these various regulatory subunits
in chemokine-mediated NF-
B activation and cytokine gene
transcription has not been investigated.
Whereas the agonistic effects of cytokines on chemokine expression are
well described, very little is known about chemokine-mediated cytokine
expression. In the present study we investigated the role of the
ELR+ CXC chemokines, LIX, KC, and MIP-2, in NF-
B
activation and induction of IL-1
and TNF-
expression.
Furthermore, we explored the chemokine receptor usage and signal
transduction pathway involved in chemokine-mediated NF-
B activation.
Our results indicate that the ELR+ CXC chemokines activate
NF-
B, induce proinflammatory cytokine expression, and signal through
CXCR2, and presumably also through CXCR1.
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EXPERIMENTAL PROCEDURES |
Materials and Reagents
Recombinant mouse LIX, KC, and MIP-2 were obtained from
PeproTech, Inc. (Rocky Hill, NJ). Recombinant carrier-free rat IL-1
and rat TNF-
were from R&D Systems (Minneapolis, MN). The
recombinant proteins contained <1 ng of endotoxin per µg of protein.
Polyclonal antibodies against rat IL-1
and TNF-
were from
BIOSOURCE International (Camarillo, CA), and
I
B-
, anti-p50 (sc-1114X), and anti-p65 (sc-372X) subunit-specific
polyclonal antibodies, and anti-
-actin antibodies were obtained from
Santa Cruz Biotechnology, Inc. Phospho-I
B-
(Ser32)
polyclonals, which detect only the phosphorylated form of I
B-
, and not the nonphosphorylated form, were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Normal rabbit IgG (control IgG) was
from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Anti-FLAG, anti-Myc, and anti-HA antibodies were from Sigma, Roche
Applied Biosciences (Indianapolis, IN), and Covance Inc. (Princeton,
NJ), respectively. All tissue culture supplies were from
Invitrogen. Radiochemicals ([
-32P]dCTP,
[
-32P]ATP, and [
-32P]UTP) were
purchased from Amersham Biosciences. SB 447232 (N-[2-hydroxy-3-(N"-isoxazolidinyl sulfonamide)-4-chlorophenyl)-N'-(2,3-dichlorophenyl)urea)
was synthesized in the Department of Medicinal Chemistry at
GlaxoSmithKline (King of Prussia, PA). Wortmannin, LY 294002, chelerythrine chloride, MG-132, 3-aminobenzamide, and pertussis toxin
were obtained from Calbiochem (San Diego, CA). All other chemicals were
purchased from Sigma.
Cell Culture
Nontransformed rat cardiac-derived endothelial cells (rat CDEC;
a generous gift of C. A. Diglio; Ref. 20) and nontransformed mouse
cardiac-derived endothelial cells, described previously (21), were
cultured in medium 199 with 10% fetal calf serum, endothelium growth
supplement (30 mg/liter), heparin (100 mg/liter), penicillin (100,000 units/liter), and streptomycin (100 mg/liter) at 37 °C in a
humidified atmosphere of 95% air, 5% CO2. At
70-80% confluency, the media was replaced with serum-free medium 199 containing 0.5% BSA. After overnight culture, LIX, KC, MIP-2, or PBS
was added and incubated for the indicated time periods. To inhibit
NF-
B DNA binding activity, the cells were pretreated for 1 h
with 3-aminobenzamide (10 mM in ethanol), wortmannin (50 nM), LY 294002 (20 µM), chelerythrine
chloride (60 µM), and MG-132 (5 µM) in
Me2SO, pertussis toxin (100 ng/ml) in PBS or for 4 h with IL-10 (10 ng/ml) or corresponding vehicle before the
addition of LIX.
Transient Cell Transfections and Reporter Assays
The NF-
B driven luciferase reporter plasmid (pNF-
B-Luc)
was obtained from Stratagene (La Jolla, CA) and contains five copies of
NF-
B consensus sequence linked to the minimal E1B
promoter-luciferase reporter gene. pEGFP-Luc was used as a control. The
phosphorylation-deficient S32A/S36A mutant of I
B-
(pCMX-I
B-
(S32A/S36A)) was a gift from Inder Verma (The
Salk Institute, La Jolla, CA), and the Myc-tagged phosphorylation-deficient S19A/S32A mutant of I
B-
in pCMV-Tag3B (Stratagene) has been described earlier (22). Kinase-deficient NIK
(pRK7-NIK(KK429-430AA)-Flag), IKK-
(pRK5-IKK-
-Flag), and dominant negative IKK-
(pcDNA3-IKK-
-HA) were obtained from
David V. Goeddel (Tularik Inc., South San Francisco, CA), Tom Maniatis (Harvard University, Cambridge, MA), and Gabriel Nunez (University of
Michigan Medical School, Ann Arbor, MI), respectively. Rat CDEC were
plated on six-well tissue culture dishes and transfected the following
day at ~70-80% confluency using LipofectAMINE 2000TM
(Invitrogen, Carlsbad, CA) as described by the manufacturer. pRL
Renilla-luciferase reporter gene (100 ng; pRL-TK vector;
Promega, Madison, WI) was used as an internal control. The empty
vectors pCMX, pCMV-Tag3B, pRK5, pRK7, and pcDNA3 were used as
controls. Data were normalized for transfection efficiency by dividing
firefly luciferase activity with that of corresponding
Renilla luciferase, and expressed as mean relative
stimulation ± S.E. for a representative experiment from three
separate experiments, each performed in triplicate. The amount of DNA
transfected was kept constant (2 µg) in all transfection experiments.
After transfection, the cells were found to be viable (trypan blue dye
exclusion). 24 h after transfection, the media was changed, and
the cells were exposed to LIX, KC, or MIP-2 at the indicated
concentrations and for the specified time periods. Cell extracts were
prepared, and luciferase activity was determined with a TD 20/20
luminometer (Turner Designs, Sunnyvale, CA) using the Promega
BiotechTM dual-luciferase reporter assay system.
Transfection efficiency was determined by transfecting rat CDEC with
pEGFP-N1 vector (Clontech, Palo Alto, CA) that
constitutively expresses the enhanced green fluorescent protein (EGFP)
under the regulation of CMV promoter and enhancer. Once the cells
reached ~70% confluency, the media was replaced with M199 + 0.5%
BSA. After overnight culture, cells were transfected with pEGFP-N1 and
LipofectAMINE 2000. 24 h later, the cells were trypsinized, seeded
onto Lab-Tek II chamber slide (NuncTM), and cultured for an
additional 48 h. Cells were then washed in PBS (pH 7.4), fixed in
4% paraformaldehyde in PBS for 30 min at room temperature. After
washing in PBS, coverslips were mounted using ProLongTM
Antifade kit (Molecular Probes, Eugene, OR). After the mounting media
was dried, the coverslips were sealed with black nail polish, and
stored at 4 °C in the dark. The cells were visualized by a fluorescent microscope (Nikon Eclipse TE200, Nikon Inc., Melville, MA),
and 1,000 cells were counted under ×20 objective, and bright to
very-bright green fluorescent cells were considered positive for the
expression of EGFP, and the others as nontransfected (controls). The
transfection efficiency varied between 37 and 46% with an average of
38.1 ± 2.9%. To determine the role of CXC receptors in
LIX-mediated NF-
B activation, rat and mouse CDEC were treated with
SB447232 (GlaxoSmithKline Beecham), a specific CXCR2 antagonist, for 10 min before the addition of LIX.
Reverse Transcriptase-Polymerase Chain Reaction
To demonstrate expression of CXCR1 and CXCR2, reverse
transcriptase-PCR was performed using total RNA isolated from rat CDEC. The primers were designed based on published sequences for CXCR1 and
CXCR2 in rats (23, 24, 25). In brief, total RNA was isolated with lysis
buffer containing phenol and guanidine isothiocyanate (TRIzol reagent,
Invitrogen). 2 µg of total RNA was reverse transcribed into cDNA
with Moloney murine leukemia virus-reverse transcriptase (Invitrogen)
and random hexamers. Amplification of CXCR1 (183 bp) and CXCR2 (413 bp)
cDNAs was performed using the following primers: CXCR1-sense,
5'-CAGGCTTCTCCAGCACACAAG-3; CXCR1-antisense, 5'-TTGGTCATTGGAACCCTCTTAC-3'; and CXCR2-sense,
5'-GCAAACCCTTCTACCGTAG-3; CXCR2-antisense, 5'-AGAAGTCCATGGCGAAATT-3'.
Amplification was performed with an initial denaturation at 94 °C
for 1 min, followed by 35 cycles of 94 °C, 30 s; 52 °C,
30 s; 72 °C, 1 min with a final 7-min extension. The PCR
products were electrophoresed at 100 volts on a
Tris-acetate-EDTA, 2% agarose gel containing ethidium bromide.
Electrophoretic Mobility Shift Assay
NF-
B DNA binding activity was measured in the nuclear protein
extracts by electrophoretic mobility shift assay (EMSA) as described
earlier (10, 26). In the gel supershift assay, the protein extract (10 µg) was preincubated for 40 min on ice with either anti-p50 or -p65
subunit-specific polyclonal antibodies (1 µg) or control IgG (1 µg)
prior to the addition of 32P-labeled double stranded
NF-
B consensus oligonucleotide
(5'-AGTTGAGGGGACTTTCCCAGGC-3'). Absence of protein extract,
competition with 100-fold molar excess unlabeled consensus NF-
B, and
mutant NF-
B oligonucleotide
(5'-AGTTGAGGCGACTTTCCCAGGC-3'; Santa Cruz Biotechnology, Inc.) served as controls. Levels of Oct-1, a
constitutively expressed transcription factor, were also measured by
EMSA using Oct-1 consensus sequence (5'-TGTCGAATGCAAATCACTAGAA-3'; Santa Cruz Biotechnology, Inc.).
Northern Blot Analysis
Total cellular RNA was isolated using the TRIzol reagent
(Invitrogen). 20 µg of total RNA were resolved on a 0.8%
agarose-formaldehyde gel and electroblotted onto nitrocellulose
membrane. After prehybridization for 4 h, hybridizations were
carried out at 42 °C for 16 h, followed by high stringency
washing at 68 °C in 0.1× SSC, 0.1% SDS. The cDNAs were
amplified using total RNA isolated from rat CDEC and gene-specific
primers (rat IL-1
, GenBankTM accession number NM_031512,
324-bp product, sense, 5'-CTCTGTGACTCGTGGGATGATGAC-3' (bases 383-405)
and antisense, 5'-TCTTCTTCTTTGGGTATTGTTTGG-3' (bases 684-707);
TNF-
, GenBankTM accession number AF329985, 295-bp
product, sense, 5'-TACTGAACTTCGGGGTGATTGGTCC-3' (bases 955-979)
and antisense, 5'-CAGCCTTGTCCCTTGAAGAGAACC-3' (bases 2161-2138).
The PCR products were cloned into
pCRTM2.1-TOPOTM vector (Invitrogen) and
sequenced on both strands for confirmation. The probe for rat LIX
(GenBankTM accession number U90448) was a 329-bp cDNA
cloned into pCRTM2.1-TOPO vector from a reverse
transcriptase-PCR product generated with primers: sense,
5'-GGTCCTGCTCGTCATTCA-3' (bases 41 to 58) and antisense,
5'-CAGTGCAAGTGCATTCCGCT-3' (bases 350 to 369). The cDNAs were
labeled with [
-32P]dCTP (3,000 Ci/mmol; Amersham
Biosciences) using random hexanucleotide primers (Roche Molecular
Biochemicals, Indianapolis, IN). Expression levels were
normalized to 28 S rRNA expression. The 28 S rRNA probe (40 base single
stranded oligonucleotide; Oncogene Science, Uniondale, NY) was 5'
end-labeled with [
-32P]ATP using T4 polynucleotide
kinase (26).
Interleukin-1
and TNF-
Promoter Analyses
Murine IL-1
Promoter--
The murine IL-1
promoter (
4093
to +45) construct in pBluescript vector was a kind gift from Clifford
J. Bellone (St. Louis University School of Medicine, St. Louis, MO;
Ref. 27). This construct contains 4,093 bp of the 5'-flanking sequence
that includes the first exon, first intron, and untranslated region of
the second exon. This promoter construct has been demonstrated to
confer a strong responsiveness to lipopolysaccharide (27). Rat CDEC were transfected with 3 µg of either the IL-1
4093 to +45-CAT (chloramphenical acetyltransferase) or a mock plasmid that contains CAT
reporter gene alone (pFR-CAT; Stratagene). To compensate for variations
in transfection, cells were cotransfected with a
-galactosidase reporter construct (pSV-
-galactosidase control vector, Promega) in
which the SV40 early promoter and enhancer drives transcription of the
lacZ gene, which encodes the
-galactosidase enzyme.
24 h later, the media was changed, and the cells were treated with LIX (100 ng/ml), neutralized LIX, or vehicle. Seven hours later the
cells were processed for CAT levels using a CAT enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) and
-galactosidase levels by using the
-galactosidase assay kit
(Invitrogen) essentially as described by the manufacturers.
Murine TNF-
Promoter--
Rat CDEC were transfected with
pTNF
1080/+138, pTNF
85/+135, or empty vector (pGL3 basic). These
murine TNF-
promoter reporter constructs were described earlier
(28). The 1.1-kb TNF-
promoter construct (
1080/+138 nucleotides;
relative to the transcription start site) contains 4 NF-
B response
elements, and has been demonstrated to confer responsiveness to a
variety of stimuli including lipopolysaccharide. Its deletion mutant
construct (
85/+135) lacks all the four NF-
B response elements but
contains only TATA box and Sp1 site, and responds poorly to
lipopolysaccharide (28). The cells were co-transfected with
pRK-Renilla to compensate for transfection efficiency.
24 h after transfection, the media was changed, and the cells were treated with LIX, neutralized LIX, or vehicle. Seven hours later the
cells were processed for luciferase activity by the dual luciferase assay kit.
mRNA Stability-Actinomycin D Pulse--
Rat CDEC were
cultured in M199 medium containing 10% fetal calf serum. At 70-80%
confluency, the complete medium was replaced with M199 + 0.5% BSA, and
cultured for an additional 16 h. The cells were then treated with
either LIX (100 ng/ml) or vehicle (control) for 4 h. Actinomycin D
(5 µg/ml; Sigma), a potent inhibitor of RNA polymerase
II-dependent transcription, was then added. At the
indicated time periods 1.5, 3, 4.5, and 6 h), cells were harvested
for total RNA isolation. RNA was isolated using TRIzol reagent, and
analyzed by Northern blot hybridization to quantitate IL-1
and
TNF-
mRNA levels as described above.
LIX-mediated IL-1
and TNF-
Transcription (Nuclear
Run-on)
After treating rat CDEC with LIX (100 ng/ml) for 4 h in
M199 medium containing 0.5% BSA, nuclei were isolated, counted in a
hemocytometer, and resuspended (2 × 108/ml) in a
storage buffer (50 mM Tris-HCl, pH 8.0, 5 mM
MgCl2, 0.1 mM EDTA, 2 mM
dithiothreitol, 40% glycerol) as described in detail previously (29).
The nuclei were aliquoted and snap frozen in methanol/dry ice bath and
stored in liquid N2 until further use. For labeling RNA,
nuclei were thawed (100 µl), mixed with equal volumes of labeling
mixture (200 mM KCl, 8 mM MgCl2, 1 mM each of ATP, UTP, and CTP, 100 µM GTP) and
100 µCi of [
-32P]UTP (800 Ci/mmol). The mixture was
incubated at 30 °C for 30 min and 20 µl (4 µg) of RQ1 RNase-free
DNase I (Promega Corp.) was added and incubated for an additional 10 min. After digestion with proteinase K (60 µg in 20 µl) in a buffer
containing 1% SDS, 50 mM Tris-HCl (pH 7.0), 50 mM EDTA for 30 min at 42 °C, it was subjected to
phenol/chloroform/isoamyl alcohol and chloroform extractions. The
aqueous phase was ethanol-precipitated in the presence of 20 µg of
carrier RNA (Escherichia coli transfer RNA, RNase-free,
Roche Molecular Biochemicals). The pellet was dissolved in 80 µl of
STE buffer (100 mM NaCl, 20 mM Tris-HCl, pH
7.5, 10 mM EDTA), and the unincorporated label was removed
using NucTrapTM probe purification columns (Stratagene),
and the incorporated radioactivity was determined in a scintillation counter.
Equal amounts of plasmid vectors containing IL-1
, TNF-
,
glyceraldehyde-3-phosphate dehydrogenase (GenBankTM
accession number M17701; 339-bp product, sense:
5'-TCCGCCCCTTCCGCTGATG-3' (bases 388-406), antisense:
5'-CACGGAAGGCCATGCCAGTGA-3' (bases 707-727)), or empty plasmid
(pCRTM2.1-TOPOTM vector) were alkaline
denatured, applied to nitrocellulose membranes using a slot-blot
apparatus (HYBRI-SLOTTM MANIFOLD, Invitrogen). After fixing
the DNA to the membranes by UV cross-linking, prehybridization was
performed at 52 °C overnight, followed by hybridization for 3 days
with ~106 cpm/ml of labeled RNA. The filters were then
washed three times for 10 min each in 2× SSC plus 0.1% SDS, two times
in 0.1× SSC plus 0.1% SDS at 65 °C for 15 min each, and then
treated with RNase A (10 mg/ml) for 30 min at 37 °C in 2× SSC.
Finally the membranes were washed with 2× SSC at 37 °C for 30 min,
and subjected to autoradiography, and the visualized bands were
semiquantitated by densitometry.
Protein Extraction and Western Blot Analysis
30 µg of cell extract in RIPA buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of
aprotinin, leupeptin, and pepstatin, 1% Nonidet P-40, 1 mM
sodium orthovanadate, and 1 mM NaF) from untreated (control) and treated CDEC were subjected to SDS-PAGE under reducing conditions, and electrotransferred onto polyvinylidene difluoride membranes (Millipore, MA). Nonspecific sites were blocked with 10%
normal goat serum (preimmune; DAKO) for 1 h at room temperature, drained, and incubated overnight at 4 °C with the primary antibody in TBST (Tris-buffered saline containing 0.5% Tween 20) containing normal goat serum, washed in TBST, and incubated further for 1 h
with the secondary antibody conjugated to horseradish peroxidase. After
extensive washings with TBST, the membranes were incubated with an
enhanced chemiluminescence reagent (Amersham Biosciences). The
membranes were then washed, exposed to Kodak X-Omat AR film, and the
autoradiographic bands were semiquantified and normalized to
-actin
levels (10, 26).
Enzyme-linked Immunosorbent Assay
TNF-
(sensitivity 0.7 pg/ml) and IL-1
(sensitivity <3.0
pg/ml) levels in culture supernatants were measured by enzyme-linked immunosorbent assay using commercially available kits
(BIOSOURCE International; Ref. 30). Studies were
performed as per the manufacturer's instructions.
Measurement of PI3K Activity
PI3K lipid kinase assays were performed essentially as described
by Foukas et al. (31). After overnight incubation in 0.5% BSA, M199 media, rat CDEC were treated with rLIX (100 ng/ml) for 5 min
with and without LY 294002. Cleared cell lysates were prepared by
centrifugation at 10,000 × g for 30 min at 4 °C,
and protein concentration was determined. Equal amounts of protein was
immunoprecipitated with affinity purified antibodies against the p85
regulatory subunit of PI3K (Santa Cruz Biotechnology, Inc., number
sc-423) for 2 h followed by protein A-Sepharose (Amersham
Biosciences) for 1 h at 4 °C. After washing the
immunoprecipitates (IP) in Tris-HCl (100 mM, pH 7.4)
containing 0.5 M LiCl and kinase assay buffer (2×, 100 mM HEPES-NaOH, pH 7.4, 200 mM NaCl, 2 mM dithiothreitol), the immunoprecipitates were resuspended
in 50 µl of 1× kinase assay buffer containing 5 mM
MgCl2, 100 µM ATP (plus 0.1 µCi of [
-32P]ATP/assay), and 200 µg/ml phosphatidylinositol
as a substrate. The reaction was incubated at 25 °C for 20 min. The
reaction was stopped by the addition of 100 µl of 0.1 M
HCl and 200 µl of chloroform/methanol (1:1). The lower organic phase
containing phospholipids was recovered and spotted on silica gel
thin-layer chromatography plates (Gel-60, Merck), impregnated with 1%
(w/v) potassium oxalate, 1 mM EDTA in water/methanol (6:4),
and developed in a mixture of chloroform, methanol, 4 M
NH3 (9:7:4). The radioactivity on the dried plate was
visualized and quantified by autoradiography and densitometry.
Measurement of Intracellular Calcium
Intracellular calcium measurements were made in rat CDEC using
the calcium-sensitive probe Fura-2/AM (Molecular Probes). The cells
were loaded with Fura-2/AM pentapotassium salt (5 µM) in M199 medium supplemented with 10% fetal calf serum. After incubation for 45 min at 37 °C, the cells were washed and resuspended at 2 × 106 cells/ml in 137 mM NaCl, 4.5 mM KCl, 1.2 mM
MgCl2·7H2O, 4.9 mM KCl, 1.2 mM NaH2PO4, 20 mM
HEPES, 15 mM D-glucose, 1.8 mM
CaCl2 (pH 7.4). The cell suspension was placed in a
fluorimetry cuvette and stirred continuously at 37 °C. After
equilibrating at 37 °C for 10 min, rLIX (100 ng/ml), neutralized
LIX, or PBS were added, and fluorescence was monitored at excitation
wavelengths of 340 and 380 nm and an emission wavelength of 510 nm on a
Hitachi F-2000 fluorescence spectrophotometer; the results were
calculated as the ratio of emission following excitation at 340 nm with
that produced by excitation at 380 nm. The Fura-2/AM-loaded cells were treated with either Triton X-100 (1%) or 100 mM EGTA to
obtain maximal and minimal fluorescence, and the data were normalized as a percentage of the maximal fluorescence.
Inhibition of Radioligand Binding and Calcium Mobilization
Procedures utilized for 125I-IL-8 binding to
membranes of Chinese hamster ovary cells stably expressing CXCR1 and
CXCR2 were done as previously described (32, 33). Inhibition of 10 nM IL-8-induced calcium mobilization in RBL 2H3 cells
stably expressing CXCR1 or CXCR2 was done as previously described (33).
Inhibition of calcium mobilization with human polymorphonuclear
leukocytes was done using 1 nM IL-8 or 10 nM GRO
as described (32). In addition, the same
procedure was used for inhibition of rat GRO
-induced calcium
mobilization in rat polymorphonuclear leukocytes isolated from
peripheral blood (32, 33).
Statistical Analysis
Comparisons between controls and various treatments were
performed for measures of NF-
B DNA-binding activity,
B-driven
luciferase activity, and cytokine mRNA and protein levels by
analysis of variance with post-hoc Dunnett's t-tests. Error
bars in figures indicate the S.E.
 |
RESULTS |
The Proinflammatory Cytokines IL-1
and TNF-
Induce LIX
Expression--
Both proinflammatory cytokines and chemokines are
induced during inflammation and endotoxemia. We have previously
demonstrated induction of the neutrophil chemoattractants LIX, KC, and
MIP-2, members of the ELR+ CXC chemokines, during
ischemia/reperfusion injury (26). In isolated adult rat cardiomyocytes,
TNF-
induced LIX expression in a NF-
B-dependent
manner (26). In the present study, we investigated whether IL-1
and
TNF-
induce LIX expression in rat cardiac-derived endothelial cells.
EMSA showed rapid and sustained induction of NF-
B DNA-binding
activity by either cytokine in rat CDEC (Fig. 1, A and B). The
induction was observed at 5 min after addition of IL-1
(100 ng/ml)
or TNF-
(10 ng/ml) and persisted up to 48 h. However, no
synergy was observed when the cells were treated with IL-1
and
TNF-
together (Fig. 1C). Furthermore, IL-1
and TNF-
induced LIX mRNA expression in a sustained manner (Fig. 1,
D and E). These results indicate that cytokines
induce chemokine expression, probably through activation of
NF-
B.

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|
Fig. 1.
The proinflammatory cytokines
IL-1 and TNF-
activate NF- B and induce LIX
expression. Rat CDEC were treated with recombinant IL-1 (100 ng/ml), TNF- (10 ng/ml), IL-1 (100 ng/ml) + TNF- (10 ng/ml)
for the indicated time periods. Nuclear protein extracts were prepared
and analyzed for NF- B DNA-binding activity by EMSA (A-C)
as described under "Experimental Procedures." Total RNA was
isolated and LIX mRNA and 28 S RNA expression was analyzed by
Northern blot analyses (D and E). After rat CDEC
reached 70-80% confluency in M199 + 10% fetal calf serum, the media
was replaced with M199 + 0.5% BSA. After overnight culture, the cells
were treated with rLIX (100 ng/ml). At the indicated time periods, the
media was removed and the cells were rinsed with ice-cold PBS. The
cells were then stored at 80 °C until further analysis. Total RNA
was isolated from frozen cells, and quantitated at 260 nm. 20 µg of
total RNA was electrophoresed in 0.8% agarose-formaldehyde gels,
electroblotted onto nitrocellulose membrane, fixed by UV irradiation,
and analyzed for cytokine mRNA expression by Northern blot
analysis. The same membrane was used after stripping off its previous
label. 28 S rRNA was used as an internal control. Our results indicate
that both IL-1 and TNF- are potent inducers of NF- B
(A and B). Both cytokines rapidly increased
NF- B DNA-binding activity that sustained up to 48 h. However,
when IL-1 and TNF- were added together, no further increase in
NF- B levels (C) was detected. Similarly, IL-1 -induced
LIX mRNA persisted up to 24 h, and that induced by TNF-
remained high up to 48 h (D and E).
Lanes 1-3 in panels A-C: lane 1,
competition with mutant NF- B oligonucleotide. Protein extract from
CDEC treated with cytokine for 30 min was preincubated with 100-fold
molar excess of unlabeled double-stranded mutant NF- B
oligonucleotide followed by the addition of 32P-labeled
consensus B probe. Lane 2, competition with consensus
NF- B oligonucleotide. Protein extract from CDEC treated with
cytokine for 30 min was preincubated with 100-fold molar excess of
unlabeled double-stranded consensus NF- B oligonucleotide followed by
the addition of 32P-labeled consensus B probe.
Lane 3, no protein extract but contains
32P-labeled consensus B probe.
|
|
ELR+ CXC Chemokines Activate NF-
B DNA-binding
Activity--
To determine whether cytokine-chemokine cross-talk
amplifies the proinflammatory cytokine cascade, we investigated whether ELR+ CXC chemokines induce cytokine expression, and the
role of NF-
B in chemokine-mediated cytokine expression. EMSA showed
that unstimulated rat CDEC contained low levels of NF-
B in the
nuclear protein extracts. Treatment with all three ELR+ CXC
chemokines increased NF-
B activity in a dose-dependent
manner (Fig. 2A). LIX-induced
NF-
B activity increased to near peak levels at 100 ng/ml, with a
slight further increase when the concentration was increased to 1000 ng/ml. Even at 1000 ng/ml, KC- and MIP-2-induced NF-
B activity in
rat CDEC did not reach levels comparable with that induced by LIX. To
confirm the EMSA results we performed transient transfections with a
pNF-
B luciferase reporter vector. LIX, KC, and MIP-2 induced
B-driven luciferase activity (Fig. 2B), and LIX was the
most potent inducer of
B activation. Neutralization of LIX with
anti-LIX antibodies completely blocked LIX-induced
B-driven
luciferase activity. Semiquantitative analysis by densitometry of
autoradiographic bands indicated that LIX induced a rapid increase in
NF-
B activation (15 min; 2.6-fold, versus untreated
controls) that persisted up to 48 h (Fig. 2C). Whereas
increased NF-
B activity was readily detected upon treatment of rat
CDEC with LIX, LIX treatment had no effect on the expression of Oct1, a
constitutively expressed transcription factor (Fig. 2D).

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Fig. 2.
The ELR+ CXC chemokines LIX, KC,
and MIP-2 activate NF- B and induce
B-driven luciferase activity in rat cardiac-derived
endothelial cells. Rat CDEC were treated with LIX, KC, and MIP-2
(0, 1, 10, 100, or 1000 ng/ml) for 1 h, and nuclear protein
extracts were analyzed by EMSA. Our results indicate that while all
three chemokines activated NF- B, LIX was the most potent
(A). Similar to the EMSA results, LIX (100 ng/ml) was also
found to be the most potent inducer of B-driven luciferase activity
in rat CDEC, and neutralizing LIX with anti-LIX antibodies abrogated
LIX-induced B-activation (B). C1,
untransfected and untreated cells; C2, cells transfected
with the control vector pEGFP-Luc; C3, cells transfected
with pEGFP-Luc and treated with LIX. LIX, anti-LIX
neutralizing antibodies. **, p < 0.01; *,
p < 0.05 (versus C1); , p < 0.01 (versus LIX). A rapid and persistent increase in
NF- B activation was detected upon LIX treatment (C).
However, LIX treatment had no effect on the basal expression of the
constitutively expressed transcription factor Oct1 (D).
LIX-induced I B- degradation was associated with transient
increase in the phosphorylated form of I B- (P-I B- ;
E). However, -actin levels showed no variations between
samples. Furthermore, LIX-induced NF- B contained both p50 and p65
complexes as assessed by gel supershift assay (F).
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Activation of NF-
B results from phosphorylation and dissociation of
I
B from the NF-
B complex. The phosphorylated I
B is then
polyubiquitinated and degraded in the cytoplasm by the 26S proteasome. Therefore, we examined the levels of I
B-
and the phosphorylated form of I
B-
in the nucleus-free cellular extracts from rat CDEC treated with LIX. Treatment with LIX rapidly but transiently induced phosphorylation of I
B-
as seen by increased P-I
B-
levels at 10 min with a corresponding decrease in I
B-
levels (Fig. 2E).
Because the subunit composition of NF-
B determines in part the
binding affinity to various promoters, we next determined the
composition of NF-
B by gel supershift assay using subunit-specific polyclonal antibodies. The results show a supershift in NF-
B binding
when both p50 and p65, but not control, antibodies were used,
indicating the presence of both p50 and p65 subunits in the nuclear
protein extracts of rat CDEC treated with LIX (Fig. 2F). Because LIX was the most potent inducer of
NF-
B activation, in all subsequent experiments we used LIX at a
concentration of 100 ng/ml.
LIX Induces Proinflammatory Cytokine Expression--
The
promoter/enhancer regions of proinflammatory cytokines contain binding
elements for various stress-responsive transcription factors that are
regulated by oxidative stress and proinflammatory stimuli. Therefore,
we assessed the effects of LIX on IL-1
and TNF-
expression in rat
CDEC. The results are shown in Fig.
3. IL-1
and TNF-
mRNA were detected at low levels under basal conditions, and were
up-regulated by LIX, with peak levels detected around 4 h (Fig.
3A). Whereas IL-1
expression returned to near basal level
by 48 h, LIX-induced TNF-
expression remained high. In addition
to IL-1
and TNF-
gene transcription, LIX treatment also increased
cytokine protein levels in the culture supernatants (Fig.
3B). Both cytokines were induced at high levels by LIX at 4 h, and their expression was blocked when rat CDEC were treated with LIX after antibody neutralization. Thus, LIX induces both transcription and translation of IL-1
and TNF-
.

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Fig. 3.
LIX treatment up-regulated the
B-responsive proinflammatory cytokines
IL-1 and TNF- ,
increased transcription rate and mRNA half-life, and induced
cytokine promoter activity in rat CDEC. After rat CDEC reached
70-80% confluency, the complete media was replaced with M199
containing 0.5% BSA. After overnight culture, LIX (100 ng/ml) was
added and the incubation continued up to 48 h. At the end of
experimental period, media was separated and the cells were rinsed with
ice-cold PBS. Cells were then processed for mRNA expression by
Northern blotting. Culture supernatants were assayed for cytokine
protein levels by enzyme-linked immunosorbent assay. Our results
indicate that LIX up-regulated both IL-1 and TNF- mRNA
expression in a time-dependent manner with significant
increases detected around 4 h post-treatment (A).
Similarly, treatment with LIX, but not LIX after neutralization,
significantly increased cytokine protein levels in culture supernatants
at 4 h (B; *, p < 0.001 (versus control); , p < 0.001 (versus LIX)). Because increased transcription and/or
mRNA stability contribute to mRNA expression, we then studied
the effects of LIX on cytokine transcription by nuclear run-on assay
and mRNA stability by actinomycin D pulse. Our results indicate
that LIX treatment significantly increased transcripts for IL-1
(C) and TNF- (D) in nuclei isolated from
LIX-treated rat CDEC. Actinomycin D pulse following LIX treatment
showed increased stability of IL-1 mRNA as compared with control
(E). However, mRNA half-life for TNF- and controls
was similar (F). To demonstrate the effects of LIX on
cytokine promoter activity, rat CDEC were transiently transfected with
3 µg of IL-1 promoter construct (IL-1 -4093 to +45-CAT) or a
mock plasmid (pFR-CAT). Cells were cotransfected with
pSV- -galactosidase vector to compensate for variability in
transfection efficiency. CAT and -galactosidase levels were
measured, and CAT expression was normalized to that of
-galactosidase, and represented as normalized CAT expression. The
results indicate that LIX significantly induced IL-1 promoter
activity (G), and neutralizing LIX with anti-LIX antibodies
prevented its stimulatory effects on the promoter activity. To
demonstrate LIX effects on TNF- promoter activity, rat CDEC were
transfected with a 1.1-kb TNF- promoter that contained 4 NF- B
response elements (TNF- 1080/+138), its deletion mutant that lacks
all NF- B response elements (TNF- 85/+138), or the empty vector
(pGL3 basic). Cells were cotransfected with pRK-Renilla to
compensate for variability in transfection efficiency. The results are
represented as a ratio of firefly luciferase to Renilla
luciferase. The results indicate that LIX significantly increased
TNF- promoter activity (H), and lack of B-response
elements abrogated LIX-mediated luciferase activity. *,
p < 0.01 (versus LIX-treated empty vector
transfected cells).
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Increased mRNA levels reflect enhanced transcription and/or
increased mRNA half-life. To determine whether increase in cytokine mRNA expression is because of increased gene transcription, we performed nuclear run-on analyses. As illustrated in Fig. 3,
C and D, low levels of IL-1
and TNF-
transcripts were detected in control rat CDEC, but were significantly
increased at 4 h after LIX treatment (Il-1
, 4.8-fold; TNF-
,
11.3-fold; p < 0.001). These results indicate that
LIX-induced cytokine expression is regulated at the transcriptional level.
To determine mRNA half-life, cells were treated with LIX for 4 h, followed by actinomycin D pulse for up to 6 h. Fig. 3,
E and F, shows that the half-life of LIX-induced
IL-1
mRNA was twice that of untreated controls
(t1/2; control, 2.5 h, IL-1
, ~4.25 h).
However, the half-life of TNF-
mRNA was similar in untreated and
LIX-treated cells (t1/2, control ~2.5 h, TNF-
,
~2.75 h). These results indicate that while increased transcription
and mRNA stability contributed to LIX-induced IL-1
induction,
increased transcription contributed to LIX-induced TNF-
expression.
Because LIX up-regulated cytokine mRNA expression, we next
determined whether LIX regulates cytokine promoter activity. Rat CDEC
were transiently transfected with IL-1
or TNF-
promoter-reporter constructs. Fig. 3G shows that LIX, but not neutralized LIX,
significantly increased IL-1
promoter activity, as seen by increased
CAT expression (p < 0.01). To determine TNF-
promoter activity, we used a 1.1-kb TNF-
promoter (TNF-
(
1080/+135)) that contains 4 NF-
B sequence and a deletion
construct (TNF-
(
85/+135)) that lacks all four NF-
B response
elements (Fig. 3H). Treatment with LIX, but not neutralized
LIX, induced a 3.5-fold increase in luciferase activity as compared
with control (untreated and untransfected cells) and LIX-treated empty
vector-transfected rat CDEC (p < 0.01). In contrast, cells transfected with the TNF-
promoter construct that lacks all
four NF-
B sites failed to respond to LIX (Fig. 3H).
LIX-induced Cytokine Expression Is Dependent on Activation of
NF-
B, and Involves NIK, IKK, and I
B--
The signaling cascade
initiated by free radicals and proinflammatory cytokines resulting in
NF-
B activation has previously been shown to converge at IKK. The
NIK associates with IKK-
, and activates IKK-
and IKK-
of the
IKK signalsome resulting in phosphorylation and degradation of I
B.
Because LIX activated NF-
B (Fig. 1, A and B)
and induced proinflammatory cytokines (Fig. 3, A and
B) in rat CDEC, we next determined if LIX-mediated cytokine
expression was dependent on activation of NF-
B, and whether
LIX-mediated NF-
B activation proceeds via NIK, IKK, and I
B. We
used a series of vectors that expressed dominant negative I
B-
,
I
B-
, or IKK-
or kinase-deficient NIK or IKK-
. We confirmed that the dominant negative or kinase-deficient vectors expressed the
appropriate protein (Fig. 4, A
and B). We then studied the effects of LIX on rat CDEC that
had been transiently transfected with the above expression vectors. The
results are shown in Fig. 4. LIX-mediated NF-
B activation was
significantly inhibited by overexpression of dnI
B-
, dnI
B-
,
kdNIK, kdIKK-
, and dnIKK-
but not by the corresponding empty
vectors (Fig. 4C). LIX-mediated cytokine mRNA (Fig.
4D) and protein (Fig. 4E) was also similarly inhibited by these dominant negative and kinase-deficient expression vectors as compared with empty vector-transfected cells.

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Fig. 4.
LIX-induced NF- B
activation involves NIK, IKK, and I B. Rat CDEC were
transiently transfected with either empty vectors or dominant negative
I B- , I B- , IKK- , kinase deficient NIK or IKK- .
Expression of I B- mutant (pCMX-I B- (S32A/S36A)) was
confirmed in an immunoblotting by its slower mobility as compared with
the wild type I B- (61), and also by its nondegradability after
LIX treatment (A). Expression of dnI B-
(pCMV-Tag3B-I B- (S19A/S23A)), kinase-deficient IKK-
(pRK5-IKK- -Flag), kdNIK (pRK7-NIK(KK429-430AA)-Flag), and dnIKK-
(pcDNA3- IKK- -HA) was confirmed by immunoblotting using
anti-Myc, -FLAG, and -hemagglutinin antibodies. B, -actin
levels demonstrated similar amounts of protein loading. Cells
transfected with empty vectors (pCMX, pCMV-Tag 3B, pRK5, pRK-7, and
pcDNA3) were used as controls. 24 h after transfection, the
media was changed, and the cells were treated with LIX (100 ng/ml) for
either 1 (C) or 4 h (D and E).
The results indicate that pretreatment with the anti-inflammatory
cytokine IL-10 (10 ng/ml for 4 h) and overexpression of
dnI B- or dnI B- inhibited LIX-induced NF- B activation
(C), and cytokine mRNA (D) and protein levels
(E). Similar results were obtained by the overexpression of
kdNIK, kdIKK- , and dnIKK- . *, p < 0.001 (versus empty vector transfected cells).
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In addition, we have also tested the effects of IL-10, an
anti-inflammatory cytokine, on LIX-mediated NF-
B activation. Our results indicate that pretreatment with IL-10 for 4 h
significantly inhibited LIX-induced NF-
B activation and cytokine
expression (Fig. 4, B-D).
LIX-mediated NF-
B Activation Involves PI3K, PKC, PARP-1, and
Proteasome--
The signal transduction pathway(s) initiated by
ELR+ CXC chemokines in the activation of NF-
B are not
fully known. To determine the role of PI3K, PKC, PARP-1, and proteasome
in LIX-mediated activation of NF-
B, rat CDEC were pretreated with
selective inhibitors. Inhibition of PI3K with the selective inhibitor
LY 294002 significantly inhibited LIX-mediated
B activation and
cytokine expression (Fig. 5A).
Similar results were obtained with wortmannin. Furthermore, 3-aminobenzamide, a specific PARP-1 inhibitor, and MG-132, a proteasome inhibitor, also inhibited LIX-mediated
B activation and cytokine expression (Fig. 5, B and C). On the other hand,
inhibition of PKC by chelerythrine chloride partially, but
significantly, attenuated
B activation and cytokine expression.

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Fig. 5.
LIX-induced NF- B
activation involves PARP-1, PI3K, and PKC. Rat CDEC were treated
with LIX (100 ng/ml) for either 1 (A) or 4 h
(B and C) with and without pretreatment with
inhibitors of PARP-1 (3-aminobenzamide, 10 mM, 1 h),
PI3K (LY 294002, 20 µM; wortmannin, 50 nM),
PKC (chelerythrine chloride, 60 µM), or 26 S proteasome
(MG-132, 5 µM). Control, untreated cells. Our
results indicate that LIX-mediated NF- B activation is abrogated by
inhibiting PI3K and attenuated by PKC inhibition. Inhibition of PARP-1
as well as 26 S proteasome similarly prevented LIX-mediated NF- B
activation. *, p < 0.01 (versus control).
Because LY 294002 and wortmannin inhibited LIX-induced NF- B
activation and cytokine expression, we further confirmed the effects of
LIX on activation of PI3K. We performed PI3K lipid kinase assays, and
our results demonstrate that treatment with LIX for 10 min
significantly increased PI3K-mediated phosphatidylinositol
1,4,5-trisphosphate (PIP3) formation (D).
Pretreatment with LY 294002, but not Me2SO, inhibited
LIX-mediated PI3K activation as seen by reduced formation of PIP3
(D).
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To confirm activation of PI3K by LIX, we performed PI3K lipid kinase
assays. Fig. 5D shows that LIX, but not neutralized LIX, activates PI3K (2.4-fold increase, p < 0.01). Whereas
Me2SO and LY 294002 had no effect on basal levels of PI3K,
pretreatment with LY 294002 significantly inhibited LIX-induced
activation of PI3K (p < 0.01; Fig. 5D).
LIX-mediated NF-
B Activation Is G
Protein-dependent--
The ELR+ CXC chemokines
signal through the 7 transmembrane domain G protein-coupled receptors
CXCR1 (R1) and CXCR2 (R2). Damaj et al. (34) have shown that
addition of IL-8 to cell lines that specifically express either R1 or
R2, or to neutrophils that express both R1 and R2 resulted in the
formation of immunoprecipitable complexes containing the receptors and
the
subunits of Gi2 proteins. In addition,
IL-8-mediated increase in cytosolic-free calcium was inhibited by
pertussis toxin indicating a G protein-coupled signal transduction
(35). Therefore, we determined the role of G proteins in LIX-mediated
cellular and NF-
B activation. Rat CDEC were pretreated with
pertussis toxin for 1 h followed by LIX stimulation. G-protein
function was determined by measuring intracellular calcium levels in
Fura-2/AM-loaded rat CDEC. Fig. 6
illustrates that treatment with LIX (100 ng/ml), but not PBS, increased
intracellular calcium levels rapidly but transiently (compare Fig. 6,
A and B). Pretreatment with pertussis toxin
significantly attenuated LIX-induced calcium transient (Fig.
6C). Furthermore, LIX-mediated NF-
B activation and
cytokine expression were also blocked by pertussis toxin (Fig. 6,
D and E) indicating the role of inhibitory G
proteins in LIX-mediated NF-
B activation.

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Fig. 6.
The LIX-mediated increase in intracellular
Ca2+i and NF- B activation
was sensitive to pertussis toxin. Rat CDEC loaded with Fura-2/AM
were treated with pertussis toxin (PTx; 100 ng/ml) for
1 h prior to the addition of LIX (100 ng/ml). Intracellular
calcium levels were determined in a spectrophotofluorometer. Our
results indicate that whereas PBS had no effect (A),
treatment with LIX increased a rapid but transient increase in
Ca2+i (B), pretreatment with PTx
significantly attenuated LIX-mediated increase in intracellular
Ca2+i (C) indicating that the inhibitory
G proteins are involved in LIX signaling. In the next series of
experiments we determined the effects of PTx on LIX-mediated NF- B
activation. Rat CDEC were treated with pertussis toxin (100 ng/ml) for
1 h prior to the addition of LIX (100 ng/ml) either for 1 (D) or 4 h (E). Our results indicate that
pretreatment with PTx inhibits LIX-mediated NF- B activation
(D) and B-responsive cytokine mRNA expression
(E). C1, PBS-treated; C2,
untreated.
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LIX-mediated NF-
B Activation Involves Both CXCR1 and
CXCR2--
CXCR1 and -R2 have a 78% sequence homology within the
transmembrane domains, but differ in the extracellular, intracellular, and NH2- and COOH-terminals, leading to distinct ligand
specificity and signaling (36-39). Therefore, we determined the role
of CXCR1 and -R2 in LIX-mediated NF-
B activation. Rat CDEC that
express both R1 and R2 (Fig.
7A), and mouse CDEC that
express only R2 were pretreated with SB447232, a CXCR2-specific
antagonist (32), and LIX-induced NF-
B activation was determined. SB
447232 is a potent CXCR2 antagonist with similar selectivity to SB
225002 (32) and SB 265610 (33), i.e. >100-fold higher
affinity for human CXCR2 versus CXCR1 (binding
IC50 values of 26.8 ± 3.5 (n = 6) and
5,610 ± 1,433 nM, respectively (Table
I). In addition, SB 447232 was a potent
inhibitor of CXCR2 (0.1 nM rat GRO
-induced calcium
mobilization) on rat neutrophils with an IC50 of 16 nM (Table I). The advantage of SB 447232 over SB 225002 and
SB 265610 is that the former compound has much better bioavailability
in rodents and will be useful for in vivo studies in the
future. In mouse CDEC, CXCR2 blockade completely abrogated NF-
B
activation and
B-driven luciferase activity. Blockade of R2 in rat
CDEC attenuated LIX-induced NF-
B activation and
B-driven
luciferase activity by 50% (Fig. 7, B and C)
indicating that LIX signals through CXCR2, and presumably CXCR1.

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Fig. 7.
CXCR2-specific blockade inhibits LIX-mediated
NF- B activation and
B-driven luciferase activity. Reverse
transcriptase-PCR was performed to demonstrate expression of CXCR1 and
CXCR2 in rat CDEC using primers designed based on published sequences
for CXCR1 and CXCR2 in rats. Our results show that rat CDEC expresses
both CXCR1 and CXCR2 at basal conditions (A). B,
NF- B DNA binding activity by EMSA; C, B-driven
luciferase activity. Treatment with a specific CXCR2 antagonist
(SB447232, 10 nM, 10 min) followed by LIX treatment for
1 h attenuated NF- B activation in rat CDEC that express both
CXCR1 and -R2, and abrogated LIX-mediated NF- B activation in mouse
CDEC that express only R2, indicating that LIX signals via both CXCR1
and -R2.
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Table I
SB 447232 is a potent selective CXCR2 antagonist
Comparison of SB 447232 with SB 225002 as selective CXCR2 antagonists.
CXCR1 and CXCR2 binding was done using membranes from chinese hamster
ovary cells stably expressing the individual receptors and
125I-labeled IL-8. Calcium mobilization was done with RBL 2H3
cells stably expressing the individual receptors with IL-8 being used
as the agonist. Human (hPMN) and rat (rPMN) peripheral blood
neutrophils were evaluated as suspensions in calcium mobilization using
ligands defined in the table.
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 |
DISCUSSION |
Our results indicate for the first time that the ELR+
CXC chemokines LIX, KC, and MIP-2 up-regulate the proinflammatory
cytokines IL-1
and TNF-
in cardiac-derived endothelial cells via
activation of NF-
B. LIX-mediated NF-
B activation and
B-responsive gene transcription involves I
B hyperphosphorylation
leading to its selective degradation in the cytoplasm by the proteasome
system. The LIX signaling was inhibited by IL-10 and NF-
B
pathway-specific mutant expression vectors. LIX signals via both CXCR1
and -R2 in inducing NF-
B activation. Specific blocking of CXCR2
attenuated NF-
B activation in rat cardiac-derived endothelial cells
that express both R1 and R2, and completely abrogated LIX-induced
NF-
B activation and
B-driven luciferase activity in mouse
cardiac-derived endothelial cells that express only R2.
The ELR+ CXC chemokines primarily attract and activate
neutrophils to the site of injury/inflammation (1-7). We have
previously shown that LIX, KC, and MIP-2 are expressed in the
post-ischemic myocardium, and most notably LIX is expressed by all
myocardial constituent cells (10). Although activated neutrophils at
the site of myocardial ischemic injury play a role in scavenging
damaged tissue and subsequent remodeling, at least initially they
exacerbate tissue injury through generation of free radicals, and
secretion of various proteolytic enzymes and proinflammatory cytokines
(40-48). The results presented here demonstrate that chemokines (LIX)
in addition to the recruitment of neutrophils to the site of myocardial ischemic injury may contribute to myocardial inflammation by the direct
induction of cytokine expression.
Interleukin-1
and TNF-
are
B-responsive proinflammatory
cytokines with known negative myocardial inotropic effects. In isolated
cardiomyocytes, papillary muscles, myocardial segments, Langendorff
preparations, and in whole animals addition/infusion of TNF-
has
been shown to depresses contractile function via induction of the
inducible form of nitric-oxide synthase and sustained generation of
high levels of nitric oxide (49-54). High levels of TNF-
expression
have also been shown to induce cell death by apoptosis (49). Both
TNF-
and IL-1
are known free radical generators and NF-
B
activators (11). We have previously shown that TNF-
induces LIX via
activation of NF-
B in isolated cardiomyocytes (10). Furthermore, we
demonstrated activation of NF-
B and induction of LIX by IL-1
and
TNF-
in rat CDEC (Fig. 1). In the present study, we describe
the converse, that is, the induction of proinflammatory cytokines by
chemokines via NF-
B activation. Together, these observations
indicate that NF-
B activation plays a central role in regulating
cross-talk between chemokines and cytokines in myocardial cells.
The ELR+ CXC chemokines bind and exert their biological
effects via the seven-transmembrane heterotrimeric G protein-coupled receptors CXCR1 and CXCR2. The sequences of CXCR1 and -R2 within the
seven-transmembrane domains and the connecting loops are homologous, but differ in the N and C-terminal domains, leading to overlapping as
well as distinct ligand-binding and selective signal transduction pathways (36-39). Whereas all ELR+ CXC chemokines bind
with high affinity to CXCR2, IL-8, because of the presence of
Tyr13 and Lys15 in the N terminus has been
shown to also bind R1 with high affinity (55). Recently, granulocyte
chemotactic protein-2 has also been shown to bind R1 with high affinity
because of the presence of Arg20 (56) indicating that other
ELR+ CXC chemokines may bind to both R1 and R2 with high
affinity. In the present study, we demonstrated that LIX-induced
NF-
B activation is mediated in part through R2 in rat CDEC that
express both R1 and R2, and fully through R2 in mouse CDEC that express
only this receptor. Presumably, in the rat CDEC that express both
receptors, the R2-independent signaling occurs through R1.
Pretreatment of endothelial cells with pertussis toxin, which
specifically blocks the coupling of CXC receptor to Gi
proteins, attenuated the LIX-induced increase in intracellular calcium
levels and completely inhibited LIX-induced NF-
B activation.
Similarly, treatment with LY 294002, a specific PI3K inhibitor,
inhibited LIX-induced activation of PI3K activity and completely
blocked NF-
B activation and
B-responsive cytokine gene
transcription. In contrast, chelerythrine chloride, a PKC inhibitor,
partially inhibited LIX-mediated NF-
B activation and cytokine
expression. Collectively, these data indicate that LIX signals via
inhibitory G proteins and PI3K, and partially via PKC. Although other G
proteins may be involved in chemokine-mediated cell signaling (57, 58), our results exclude this probability because LIX-mediated NF-
B activation was completely blocked by pertussis toxin.
Interleukin-10 is an anti-inflammatory cytokine, and has been shown to
block expression of various proinflammatory cytokines via inhibition of
NF-
B activation (59). In the present study we demonstrate that IL-10
blocked LIX-induced NF-
B activation and
B-responsive gene
transcription. It has been previously shown that IL-10 could block
NF-
B activation by inhibiting IKK-mediated I
B phosphorylation and
degradation (55). Because the inhibitory effects of IL-10 are not
cell-specific, and can inhibit activation of NF-
B in response to
various proinflammatory stimuli, IL-10 may have a therapeutic potential
in ischemia/reperfusion injury by blocking induction of proinflammatory
cytokines and chemokines (60).
In the present study, we demonstrate for the first time that inhibition
of PARP-1 activation prevents LIX-mediated NF-
B activation and
IL-1
and TNF-
expression. It has been demonstrated recently that
PARP-1, a nuclear protein involved in repairing DNA strand breaks, has
also been shown to activate NF-
B (18). PARP-1 activation has
been described during endotoxemia and inflammation (62-66). Administration of lipopolysaccharide to mice activated PARP-1 and
resulted in PARP-1-dependent
B-responsive IL-1, IL-6,
TNF-
, iNOS gene expression, and iNOS-mediated NO generation (63). Furthermore, in the murine system PARP-1 gene disruption or
pharmacological inhibition of PARP-1 activation has been shown to
reduce free radical generation, attenuate
B-responsive gene
transcription, and reduce neutrophil infiltration in the lungs (64).
Whether PARP-1 may be a logical target for inhibition to attenuate
post-ischemic myocardial injury will require further study.
Taken together, our results indicate that the ELR+ CXC
chemokines, besides being potent neutrophil chemoattractants, also
induce proinflammatory cytokine expression via activation of NF-
B.
Blunting the activation of NF-
B or other components of the signaling
cascade, rather than targeting inhibition of individual cytokines,
chemokines, or adhesion molecules, may be a valid strategy to attenuate
myocardial tissue injury during various inflammatory conditions.