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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3241-3249
NF- B Activation Is Required for C5a-Induced Interleukin-8 Gene
Expression in Mononuclear Cells
By
Matthew H. Hsu,
Meiying Wang,
Darren D. Browning,
Naofumi Mukaida, and
Richard D. Ye
From the Department of Immunology, The Scripps Research Institute, La
Jolla, CA; and the Department of Molecular Pharmacology, Cancer
Research Institute, Kanazawa University, Kanazawa, Ishikawa, Japan.
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ABSTRACT |
C5a, a potent peptide chemoattractant, stimulates interleukin-8
(IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of
IL-8 mRNA in the cytosol and by nuclear expression of a  B DNA
binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding
activity was also detected in C5a-stimulated PBMC; however, its delayed
expression (maximal at 4 hours) suggested a less important role in the
rapid production of IL-8. The correlation between C5a-induced B
binding activity and IL-8 gene expression was examined in the RAW264.7
macrophage cells using reporter genes directed by the B sequence
from IB and IL-8 promoter regions. C5a-induced reporter gene
expression was abolished by introducing mutations into the B sites
and by coexpression of a dominant negative IB construct resistant
to agonist-induced phosphorylation. Pertussis toxin, which
ADP-ribosylates the Gi proteins known to couple to the C5a
receptor, produced minimal inhibition of C5a-induced IL-8 expression
and had little effect on C5a-induced calcium mobilization in RAW264.7
cells. These results suggest that NF-B activation is required for
C5a-induced IL-8 gene expression and that this response is mediated
primarily through a pertussis toxin-insensitive pathway.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
C5a IS A PROINFLAMMATORY peptide
generated through activation of the complement system, which occurs in
the course of trauma, immune complex formation, and bacterial
infection.1,2 C5a is more potent than the other two
activated complement anaphylatoxins, C3a and C4a, in activating
peripheral blood leukocytes.3 The proinflammatory effects
of C5a are mediated through binding of this peptide agonist to specific
receptors expressed on the surface of peripheral blood monocytes and
granulocytes4 (reviewed in Gerard and Gerard5).
More recent studies indicate that the same receptor is also present in
hepatocytes, astrocytes, and glial cells.6-9 Molecular
cloning and characterization of C5a receptors have led to the
determination of its primary structure as a G-protein-coupled receptor
with 7 putative transmembrane domains.10-13 C5a receptor,
together with the receptors for fMet-Leu-Phe, platelet-activating
factor, leukotriene B4, and a growing number of chemokines,
form a subgroup within the large family of rhodopsin-like G-protein-coupled receptors.14 Binding of C5a to its
receptor in phagocytes induces chemotaxis, generation of superoxide
anions, and release of degradative enzymes.2,5 C5a receptor
appears to have a nonredundant function in innate immunity, because
targeted deletion resulted in weakened mocosal host defense against
Pseudomonas aeruginosa infection in the lung.15
Several studies demonstrate that C5a actively regulates local cytokine
networks by stimulating the production of proinflammatory cytokines. It
has been reported that recombinant C5a stimulates transcription of the
interleukin-1 (IL-1) and tumor necrosis factor (TNF)
genes.16 C5a can also enhance the effect of
lipopolysaccharide (LPS) in its stimulation of IL-6
synthesis17 and acts synergestically with  -interferon to
stimulate IL-1 synthesis.18 In addition, C5a has been shown
to induce P-selectin expression in endothelial cells.19 The
role of C5a in regulating gene expression was further suggested by
recent studies of C5a receptor knockout mice, which demonstrate reduced
TNF and IL-6 levels in peritoneal reverse passive Arthus reaction
compared with the wild-type littermates.20 Although these
activities are consistent with the proinflammatory functions of C5a,
the mechanism for C5a stimulated cytokine generation remains
incompletely understood. There have been conflicting reports regarding
whether C5a is able to stimulate transcription and translation of
cytokine gene21,22 or its effect is confined only to
transcriptional activation.16
To better understand the role of C5a in regulating transcription and
translation during inflammation, we investigated the ability of C5a to
stimulate IL-8 synthesis at both protein and nucleic acid levels. IL-8
is a CXC chemotactic cytokine that has been extensively characterized
both structurally and functionally. IL-8 plays an important role in
acute inflammation through binding to and activation of the IL-8
receptors on neutrophils and mononuclear cells.23,24 IL-8
is also angiogenic25 and may stimulate its own biosynthesis
through an autocrine mechanism (unpublished results). The genomic structure of IL-8 and the transcriptional elements regulating its expression have been characterized
previously,26 permitting a detailed study of the effect of
C5a on IL-8 gene expression. Using a combination of immunologic and
recombinant DNA approaches, we investigated IL-8 gene expression and
protein synthesis in peripheral blood mononuclear cells and in a
macrophage-like cell line RAW264.7. Our results indicate that C5a
induces activation of NF-B, which is necessary for a rapid increase
in IL-8 mRNA and protein levels in the stimulated cells.
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MATERIALS AND METHODS |
Materials.
Human C5a was obtained from two sources: natural C5a purified from
human serum (kindly provided by Dr T.E. Hugli, The Scripps Research
Institute, La Jolla, CA) and recombinant human C5a (purchased from
Sigma Chemical Co, St Louis, MO). The recombinant C5a was tested
negative for the presence of LPS with a Limulus amebocyte lysate assay that detects LPS at a concentration of 25 pg/mL. Human
IL-8 enzyme-linked immunosorbent assay (ELISA) kit was purchased from
Biosource (Camarillo, CA). Pertussis toxin was obtained from Calbiochem
(San Diego, CA). Actinomycin D, cyclohexamide, and pyrrolidine
dithiocarbamate were purchased from Sigma. Oligonucleotides with the
consensus sequences of NF-B, AP-1, and NF-IL-6 binding sites were
obtained from Promega (Madison, WI). Rabbit antibodies against p50 and
p65, and their neutralizing peptides, were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
Preparation of peripheral blood mononuclear cells (PBMC).
Human PBMC were prepared from fresh, heparinized venous blood by
Ficoll-Hypaque (Sigma) density gradient centrifugation. Platelets were
separated from PBMC by washing with Hank's balanced salt solution
(GIBCO-BRL, Gaithersburg, MD) and by centrifugation through a fetal
bovine serum (FBS) cushion gradient that allows penetration of
mononuclear cells but not platelets. The PBMC pellet was resuspended in
RPMI 1640 culture medium (Irvine Scientific, Santa Ana, CA) and
cultured in round-bottom culture tubes for induction of IL-8 synthesis
at 37°C in a humidified atmosphere containing 5% CO2. All of the solutions used in these studies were either subjected to
ultrafiltration for elimination of microbial products or treated with
an endotoxin removal kit (Associates of Cape Cord, Inc, Woods Hole, MA).
Determination of immunoreactive IL-8 by ELISA.
Approximately 0.5 × 106 PBMC were cultured in 1 mL
RPMI 1640 medium and stimulated with C5a for various time periods. The
supernatants were collected and assayed for IL-8 secretion by ELISA
according to instructions from the manufacturer (Biosource).
RNA isolation and Northern blot analysis.
PBMC (0.5 × 106) were preincubated with or without
various inhibitors or bacterial toxins for 1 to 4 hours, followed by
C5a stimulation for 2 hours. Cells were harvested, and pellets were resuspended in 1 mL TRI-reagent (Molecular Research Center, Inc, Cincinnati, OH) for total RNA isolation. For Northern blot, 10 µg of
RNA was subjected to electrophoresis in a gel containing 1.2% agarose
and 6.6% formaldehyde as denaturing agent. The RNA was then
transferred to nylon membrane and fixed by short-wave UV radiation. The
Northern blot was prehybridized for 1 hour in the ExpressHyb solution
(Clontech, Palo Alto, CA) and hybridized with a
[-32P]dCTP-labeled IL-8 DNA fragment at 1 × 106 cpm/mL. Hybridization was performed at 65°C for 3 hours in the ExpressHyb solution. The blot was washed twice in
0.5× standard sodium citrate solution with 0.1% sodium dodecyl
sulfate at 65°C for 15 minutes. The blot was then stripped and
rehybridized with a [-32P]dCTP-labeled  -actin cDNA
for equal loading control.
Preparation of nuclear extracts.
Freshly isolated PBMC were placed at a density of 5 × 106 in round-bottom culture tubes and stimulated with C5a.
After stimulation, cells were washed 3 times with ice-cold
phosphate-buffered saline (PBS), harvested, and resuspended in 0.4 mL
of buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA,
0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, and 0.5 mmol/L
phenolmethylsulfonyl fluoride (PMSF). After 10 minute, 23 µL of 10 % Nonidet P-40 was added and mixed for 2 seconds. Nuclei were separated
from cytosol by centrifugation at 13,000g for 10 seconds and
resuspended in 50 µL of buffer B (20 mmol/L HEPES, pH 7.9, 0.4 mol/L
NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 0.1 mmol/L PMSF). After 30 minutes at 4°C, lysates were separated by centrifugation at
13,000g for 30 minutes, and supernatant containing nuclear
proteins was aliquoted and stored at  80°C. The protein
concentration of extracts was measured using the BCA protein dye
reagent (Pierce, Rockford, IL) with bovine serum albumin as standard.
Electrophoretic mobility shift assays (EMSA).
EMSA were performed by incubating 2.5 µg of nuclear extract in 12 µL of binding buffer [5 mmol/L HEPES, pH 7.9, 5 mmol/L
MgCl2, 50 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.4 mg/mL
poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ), 0.1 mg/mL sonicated
double-stranded salmon sperm DNA, and 10% glycerol] for 10 minutes at
room temperature. 32P-labeled oligonucleotide probe (30,000 cpm) was then added, and the reaction mixture was incubated for another
10 minutes at room temperature. For reactions involving competitive
oligonucleotides, the unlabeld competitor, at 100-fold molar excess,
and the labeled probe were premixed before addition to the reaction
mixture. For supershift assays, the reaction mixture minus the probe
was incubated with 2 µL of specific antibodies to NF-B proteins
for 30 minutes at room temperature. The 32P-labeled
oligonucleotide probe was then added and incubation was continued for
15 minutes. The samples were analyzed on 6% acrylamide gels. The gels
were pre-electrophoresed at 12 V/cm for 1.5 hours at room temperature
and 0.5 hours in a cold room, and the electrophoresis was continued at
the same voltage for another 2 hours at room temperature. Gel contents
were transferred to Whatman chromatography paper (Markson Lab Sales,
Hillsboro, OR), dried, and exposed to a PhosphorImage
screen (Molecular Dynamics, Sunnyvale, CA) for 3 hours.
Cell culture and transfection.
The murine RAW264.7 macrophage cell line was maintained in RPMI 1640 culture medium containing 2 mmol/L L-glutamine, 100 IU/mL penicillin,
50 µg/mL streptomycin, and 10% fetal bovine serum at 37°C in a
humidified 5% CO2 environment. RAW264.7 cells were cotransfected, by electroporation, with reporter gene plasmid DNA and
the control plasmid pCMV. For each transfection, a total of 10 µg
supercoiled DNA was added to 2 × 107 cells
resuspended in 0.2 mL of fresh growth medium in an electroporation cuvette with 0.4-cm gap and incubated at room temperature for 5 minutes. Electroporation was performed using a Bio-Rad Gene Pulser
(Bio-Rad, Hercules, CA), with voltage and capacitance
settings of 300 V (750 V/cm) and 960 µF, respectively. The cells were
collected immediately after electroporation, seeded in 2 mL growth
medium, and cultured overnight in a 6-well plate until starvation and stimulation.
Chloramphenicol acetyltransferase (CAT) assay.
Eight micrograms of each CAT reporter construct DNA and 2 µg of the
control plasmid pCMV were cotransfected into 2 × 107 RAW264.7 cells by electroporation. In experiments with
a dominant negative IB construct (kindly provided by Dr D. Van
Antwerp and I. Verma, The Salk Institute, La Jolla,
CA), 4 µg of the plasmid DNA was cotransfected with a
CAT reporter. Sixteen hours after transfection, the cells were washed
with serum-free medium, replaced with starvation medium (RPMI 1640 medium containing 2 mmol/L L-glutamine, 100 IU/mL penicillin, 50 µg/mL streptomycin, and 0.5% fetal bovine serum), and cultured for 4 hours. Cells were then stimulated with C5a (100 nmol/L) for 2 to 6 hours in the starvation medium and collected in 100 µL of 0.25 mol/L
Tris (pH 7.8) followed by 3 cycles of freeze-thaw. CAT activities were
measured in crude cellular extracts using
[14C]chloramphenicol (Amersham, Arlington Heights, IL) as
substrate and thin-layer chromatography to separate the native form
from acetylated form. Relative transfection efficiency was determined by normalizing the CAT activity to -galactosidase activity measured in cell extracts from the cotransfected pCMV.
Calcium mobilization assay.
Cells were treated with or without pertussis toxin (100 ng/mL, or as
indicated in the figures) for 16 hours, harvested with trypsin-free
cell dissociation buffer (GIBCO-BRL), and loaded with Indo-1 (5 µmol/L; Molecular Probes, Eugene, OR) by incubation at 37°C for
30 minutes with gentle rocking. Agonist-induced calcium mobilization
was measured in an SLM 8000C spectrofluorometer
(SLM-Aminco, Urbana, IL) detecting at 400 and 490 nm, with an
excitation wavelength of 340 nm.
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RESULTS |
C5a stimulates synthesis of immunoreactive IL-8 from PBMC.
Freshly prepared human PBMC were stimulated with C5a at various
concentrations and IL-8 secreted into the supernatant was measured by
ELISA. Figure 1 shows that unstimulated
PBMC did not produce detectable IL-8, whereas cells incubated with 100 nmol/L of recombinant human C5a produced up to 1,200 pg/mL of IL-8. C5a induction of IL-8 secretion was dose-dependent, detectable with 12.5 nmol/L of C5a and peaking at an agonist concentration of 500 nmol/L.
PBMC from at least 6 different donors gave similar results (Fig 1).
Natural C5a, purified from human serum, gave nearly identical results
(data not shown). The IL-8 secreted into the culture medium increased
rapidly in the first 2 hours of C5a stimulation, reaching 14-fold above
the level of an unstimulated culture
(Fig 2). This rapid increase in
IL-8 secretion suggests a direct effect of C5a on IL-8 production. IL-8
level in unstimulated culture remained low ( 100 pg/mL) during the
time period of investigation. These results are consistent with a
previous report indicating C5a induction of IL-8 production in
monocytes.22
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| Fig 1.
C5a stimulation of immunoreactive IL-8 secretion from
PBMC. PBMC were incubated with different concentrations (from 10 to
1,000 nmol/L) of recombinant human C5a. After 2 hours, cells were
removed and immunoreactive IL-8 in the medium was determined by ELISA.
For each stimulation, 0.5 × 106 freshly prepared PBMC
were used. Data presented are the mean ± SE from 6 individual
donors.
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| Fig 2.
Time course of C5a-induced IL-8 secretion from PBMC.
Approximately 0.5 × 106 freshly prepared PBMC were
incubated in the presence ( ) or the absence ( ) of
200 nmol/L of human C5a for different lengths of time (0 to 4 hours).
The cells were then removed and the supernatants were taken for
measurement of secreted IL-8 by ELISA. Results shown are the mean ± SE from three separate measurements, each in duplicates.
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C5a stimulates transcription of the IL-8 gene.
To investigate whether the induction of IL-8 by C5a is at the
transcriptional level, PBMC were treated with various transcription and
translation inhibitors for 1 hour before C5a stimulation. After 2 hours
of incubation with C5a, the supernatants were collected and assayed for
IL-8 by ELISA. As shown in Fig 3,
C5a-stimulated IL-8 secretion was blocked almost completely by the
translation inhibitor cyclohexamide (CHX) and by the transcription
inhibitor actinomycin D (ActD). These data suggest that C5a-mediated
production of IL-8 from PBMC is the result of de novo protein synthesis
and not the release of a preformed protein product. This was further confirmed by Northern blot analysis using total RNA isolated from PBMC
after 2 hours of C5a stimulation in the absence or presence of the
above-noted inhibitors. As shown in Fig 4,
C5a stimulated cytosolic accumulation of IL-8 mRNA (lane 3 v
lane 1). Both IL-8 protein secretion and mRNA accumulation were not
only inhibited by ActD, but also reduced by pyrrolidine dithiocarbamate
(PDTC), a potent inhibitor of the transcription factor NF-B (Fig 4,
lanes 4 and 5). This result suggests the involvement of nuclear factor B (NF-B) in C5a-induced transcriptional activation of IL-8 gene.
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| Fig 3.
Effect of transcription and translation inhibitors and
pertussis toxin on C5a induced IL-8 secretion. Freshly prepared PBMC
(0.5 × 106) were preincubated with different inhibitors
for 1 hour and with pertussis toxin (PTX) for 4 hours. The cells were
then stimulated with 200 nmol/L of recombinant human C5a for 2 hours.
The supernatants were taken for measurement of secreted IL-8 by ELISA.
The final concentration of PDTC was 10 µmol/L, the transcription
inhibitor ActD was added to 5 µg/mL, and the final concentration of
CHX was 10 µg/mL. PTX was added to 1 µg/mL. TNF (40 ng/mL) was
used as a positive control. Data were from three separate experiments,
each with duplicate measurements.
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| Fig 4.
C5a-induced increase in cytosolic IL-8 mRNA. Freshly
prepared PBMC (0.5 × 106) were preincubated with either
the transcription inhibitor actinomycin D, the NF-B inhibitor PDTC,
or PTX for 1 hour (lanes 4, 5, and 6), as specified on top of each
lane. The concentrations of the inhibitors used in this study were the
same as indicated in Fig 3. The cells were then stimulated with 200 nmol/L of human C5a for 2 hours or with 40 ng/mL of TNF (lane 2) as
a positive control. Total RNA was extracted from the cell pellets,
separated in formaldehyde/agarose gel (10 µg/lane) and subjected to
Northern blot analysis using a 32P-labeled IL-8 cDNA
fragment as probe. The autoradiograph shows specific bands of IL-8
transcript. The same blot was stripped and reprobed with
32P-labeled -actin for equal loading control.
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C5a-induced nuclear expression of a B DNA binding
activity is primarily responsible for the early stage transcriptional
activation of IL-8 gene.
The IL-8 gene consists of 4 exons and 3 introns with a single TATA box
and a CAT box. The 5'-flanking region contains potential binding
sites for known transcription factors AP-1, AP-2, NF-IL-6, and
NF-B.27 The sequence from 94 to 81 on IL-8
gene promoter shows similarity with potential binding site for NF-IL-6
(CAGTTGCAAATCGT), and the sequence from 82 to 70
(GTGGAATTTCCTC) is homologous to the consensus binding element for
NF-B, as in the genes for Ig light chain, SV40 and HIV
(GGGGACTTTCC).27,28 NF-B has been shown to play an
important role in the transcription of genes for many inflammatory
factors,29 including the IL-8 gene.28 It was
therefore important to determine whether C5a is capable of activating
this transcription factor as well as other factors known to cooperate
with NF-B in IL-8 gene expression, including AP-1 and NF-IL-6.
PBMC were stimulated with C5a or with the control agonists TNF and
LPS, and nuclear extracts were prepared for gel mobility shift assays
using 32P-labeled probes for NF-B, AP-1, and NF-IL-6. As
shown in Fig 5, exposure of PBMC to C5a
resulted in a marked increase in both B and AP-1 binding activities.
However, the expression of these binding activities followed different
time courses. The B binding activity was strongly expressed within
30 minutes after C5a stimulation and reached its peak in 60 minutes.
However, the AP-1 binding activity was apparent 4 hours after C5a
stimulation (Fig 5, middle panel). C5a did not stimulate NF-IL-6
binding activity for up to 4 hours, whereas LPS induced a strong
NF-IL-6 binding activity in these cells (Fig 5, lower panel). Because
of the rapid increase in both IL-8 mRNA and protein levels in the first
2 hours after C5a stimulation (Fig 2), the above-noted results suggest
that C5a-induced NF-B is most relevant to the early stage IL-8 gene expression in PBMC.
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| Fig 5.
C5a-induced transcription factor activation in PBMC. For
each sample, 5 × 106 freshly prepared PBMC were incubated
with 200 nmol/L of human C5a at 37°C for different lengths of time
(0 to 4 hours). The cells were then harvested and nuclear extracts were
prepared as described in Materials and Methods. Nuclear extract (2.5 µg) from each time point was incubated with one of the three
different probes: 32P-labeled, double-stranded
oligonucleotides containing binding sites for B, AP-1, and NF-IL-6.
The binding mixture was subjected to 5% acrylamide gel
electrophoresis. The binding activities for NF-B (top panel), AP-1
(middle panel), or NF-IL-6 (lower panel) were measured by EMSA. The
DNA-protein complexes are marked with brackets. TNF (40 ng/mL)-stimulated cells were used as positive controls for NF-B and
AP-1 activation, and LPS (5 µg/mL; lane 6) was used as a positive
control for NF-IL-6 binding activity.
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To test the specificity of C5a-induced B binding activity, an excess
amount of unlabeled B oligonucleotide was used in gel mobility shift
assays. Unlabeled B oligonucleotide, at 100-fold molar excess,
successfully competed with the labeled probe, whereas a 100-fold molar
excess of the AP-1 oligonucleotide had no effect on B binding
activity (Fig 6, lanes 4 and 5). The two
protein components of the prototypic NF-B, p65 (RelA) and p50
(NFKB1), were found to be the constituents in supershift assays by
inclusion of anti-p50 and anti-p65 polyclonal antibodies (Fig 6, lanes
6 through 8). Antibody-induced supershift was blocked by incubation with the p50 and p65 peptides, to which the antibodies were generated, indicating specificity of the antibodies for p50 and p65 (lane 9).
These results demonstrated that C5a induces activation of NF-B
containing the p50 and p65 subunits.
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| Fig 6.
Specificity of C5a-induced B binding activity. Nuclear
extracts prepared from unstimulated PBMC (basal; lane 1), PBMC
stimulated with TNF (40 ng/mL for 2 hours; lane 2), and C5a (200 nmol/L for 2 hours; lanes 3 through 9) were used for analysis of B
binding activity by EMSA, in the absence (lane 3) or presence (lanes 4 and 5) of competitive unlabeled (cold) oligonucleotide probes. Lane 4 contains an oligonucleotide with the B consensus sequence at
100-fold molar excess; lane 5 has an olinonucleotide with AP-1
consensus sequence at 100-fold molar excess. The B DNA-protein
complexes are marked with brackets. Lanes 6 through 9, nuclear extracts
from cells stimulated with C5a were incubated in the presence of Ab (2 µg/sample) against NF-B/Rel proteins: lane 6, anti-p50; lane 7, anti-p65; and lane 8, anti-p50 plus anti-p65. Lane 9 contains anti-p50
and anti-p65 plus the neutralizing peptides used for antibody
production. Samples were analyzed by EMSA, using 6% acrylamide gel and
32P-labeled double-stranded oligonucleotide containing the
NF-B binding site consensus sequence. The gel supershift bands
induced by the anti-p50 and anti-p65 antibodies were indicated with
arrows.
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C5a induction of NF-B activation and IL-8 gene
transcription in transfected cells.
To establish a correlation between C5a-induced B binding activity
and IL-8 gene transcription, B-CAT (chloramphenical
acetyltransferase) reporter gene constructs were transiently
transfected into the murine macrophage-like cell line RAW264.7, which
expresses C5a receptor. The CAT activities were determined 48 hours
after transfection. As shown in Fig 7, C5a
induced a sixfold increase in CAT activity in the cells transfected
with the wild-type IB promoter-CAT construct, p0.2kb(WT)CAT (lane
3 v lane 2), which contains one copy of a B site
(GGAAATTCCC).30 There was no increase in CAT activity in
cells transfected with a mutant IB construct [p0.2kb(M)CAT; CGAAATTAAT; Fig 7, lane 4].
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| Fig 7.
Correlation between C5a-induced B binding activity and
IL-8 gene transcription. The murine RAW264.7 macrophage cells were
transfected with 8 µg plasmid DNA for either the IB
promoter-CAT reporters (lanes 1 through 4) or the p-272CAT reporters
(lanes 5 through 8), together with 2 µg of the control plasmid
pCMV DNA to monitor transfection efficiency. Relative CAT activities
were determined in these cells after 2 hours of stimulation with C5a by
normalizing the CAT activities to the -galactosidase activities. The
acetylated forms of CAT (AcCM) are marked with brackets. The net fold
induction of relative CAT activities by C5a is shown at the bottom.
Lane 1, no reporter plasmid was transfected. The p0.2kb(WT)CAT plasmid
was used in the absence (lane 2) or presence (lane 3) of human C5a.
Lane 4 was a sample with p0.2kb(M)CAT, which contains a mutated B
site in the IB promoter. In lanes 5 through 8, CAT reporter gene
plasmids driven by the IL-8 promoter were used in the absence (lanes 5 and 7) or the presence (lanes 6 and 8) of C5a. Lanes 5 and 6 contain
the p-272CAT construct. A reporter with the B binding sequence
removed p-94( 78-71) CAT was used in lanes 7 and 8. The thin-layer
chromatography data shown here is representative of three CAT assays
with similar results.
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The IL-8 promoter-driven CAT constructs were next used to examine the
direct effect of C5a on IL-8 gene expression. C5a induced a 14-fold
increase in CAT activity in cells transfected with an IL-8 promoter
reporter p-272CAT, which contains 272 bp upstream from the
transcription initiation site (Fig 7, lane 6 v lane 5). The
reporter construct harbors NF-B, NF-IL-6, and AP-1
sites.28 To determine whether NF-B plays a crucial
function in C5a-induced IL-8 gene expression, another reporter
construct, p-94(78-71)CAT was used. In this construct, a critical
part of the B binding sequence (underlined in
GTGGAATTTCCTC) was removed, resulting in a plasmid containing
all other DNA binding sites except a functional B site. Cells
transfected with this construct did not show C5a inducible CAT activity
(Fig 7, lane 8 v lane 7). Therefore, the B site in the
5' flanking region of IL-8 gene is indispensable for
C5a-stimulated IL-8 gene transcription.
NF-B activation results from phosphorylation and degradation of the
IB proteins.31-33 Prevention of IB phosphorylation leads to inhibition of NF-B activation.34 To establish a
function of C5a-induced NF-B activation in IL-8 transcription,
RAW264.7 cells were cotransfected with the p-272CAT reporter and a
dominant negative form of IB (IBM), in which
Ser32 and Ser36 were mutated to block
agonist-induced IB phosphorylation and subsequent NF-B activation.34 Coexpression of IBM completely blocked
C5a-induced expression of the p-272CAT gene
(Fig 8, lane 4), indicating involvement of
IB phosphorylation and NF-B activation in C5a-stimulated IL-8
gene expression. Additional evidence supporting a critical role of
NF-B activation in C5a-induced IL-8 gene transcription was obtained
by using the NF-B inhibitor PDTC, which effectively blocked
C5a-stimulated CAT activity using the p-272CAT construct (Fig 9, lane 5). Similarly, the
transcription inhibitor ActD and the translation inhibitor CHX also
abolished C5a induction of CAT activity (Fig 9, lanes 3 and 4).
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| Fig 8.
Inhibition of C5a-induced CAT activity by IBM.
RAW264.7 cells were cotransfected with the dominant negative IBM
plasmid (4 µg; lane 4) and the p-272CAT reporter plasmid, using
transfection conditions described above and in Materials and Methods.
After serum starvation, the cells were either unstimulated (lane 1) or
stimulated with TNF (40 ng/mL, lane 1) or C5a (100 nmol/L, lanes 3 and 4) for 4 hours before the assay.
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| Fig 9.
Effect of inhibitors and pertussis toxin on C5a-induced
IL-8 promoter-directed CAT activities. RAW264.7 cells were transfected
with an IL-8 promoter-CAT construct, p-272CAT, and stimulated with 200 nmol/L of C5a after 1 hour of treatment with the transcription
inhibitor actinomycin D (ActD) or the translation inhibitor
cycloheximide (CHX). PTX (1 µg/mL) was added 2 hours before C5a
stimulation and was present during the course of C5a stimulation. The
inhibitors and PTX and relative net fold induction of CAT activities by
C5a are specified at the bottom of the figure.
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Involvement of pertussis toxin-sensitive and insensitive pathways in
C5a stimulation of IL-8 gene expression.
The chemotactic function of C5a, like that of other chemoattractants,
is known to be mediated through coupling of its 7-transmembrane domain
receptor with the Gi class of G protein -subunits that are sensitive to pertussis toxin (PTX) treatment.35 In
addition to Gi proteins, C5a receptor can also couple to
PTX-insensitive G protein -subunits including G 16 in
certain types of cells.36-38 The involvement of
Gi proteins in C5a-induced IL-8 gene expression was
examined in cells treated with PTX, which ADP-ribosylates and inhibits
the activities of the Gi and Go proteins.
Results from both ELISA and Northern blotting indicated only a partial blockade (~20%) of IL-8 secretion and IL-8 mRNA accumulation in the
toxin-treated cells (Figs 3 and 4). In control experiments, the same
pertussis toxin treatment was sufficient to block C5a-induced calcium
mobilization and MAP kinase activation in neutrophils (data not shown).
Consistent with the results from ELISA and Northern blot experiments
shown above, PTX had a very small inhibitory effect on C5a-induced CAT
activity (Fig 9, lane 6 compared with lane 2).
These findings suggest that C5a-induced IL-8 gene expression is
mediated primarily through G proteins insensitive to PTX
treatment. Alternatively, a pathway independent of G protein activation
may be involved in activating NF-B. To examine these possibilities,
calcium mobilization assay was adopted, because C5a-induced increase of
intracellular Ca2+ level is known to follow G protein and
phospholipase C activation. Figure 10
shows the results of experiments using three different types of cells.
In RAW264.7 cells, 16 hours of PTX treatment at doses up to 1,000 ng/mL
produced no inhibitory effect on C5a-induced Ca2+ flux. In
the rat basophilic leukemia cell line RBL-2H3 stably transfected to
overexpress the human C5a receptor, PTX completely blocked
agonist-induced Ca2+ flux. Similar PTX-sensitive data were
obtained using mouse L-cell fibroblasts expressing the human
fMet-Leu-Phe receptor, FPR, which is known for its coupling to
PTX-sensitive G proteins for Ca2+
mobilization.39 These results support the notion that C5a
receptor can couple to a G protein other than Gi for the
activation of signaling pathways leading to IL-8 gene expression. C5a
receptor may also selectively couple to either PTX-sensitive or
PTX-insensitive G proteins depending on cell types.
View larger version (18K):
[in this window]
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| Fig 10.
Effects of pertussis toxin (PTX) on calcium mobilization
in three different cells. The cells were treated for 16 hours with PTX
at the concentrations indicated next to each tracing (in nanograms per
milliliter). Agonist-induced calcium mobilization was then measured
using indo-1 AM as a fluorescent calcium indicator. Relative
intracellular calcium levels are shown. The arrows mark the time points
when agonist was applied. Each horizontal bar represents approximately
50 seconds.
|
|
| |
DISCUSSION |
The anaphylatoxin C5a has been previously shown to induce the synthesis
and secretion of a number of proinflammatory cytokines including
IL-1 and IL-8.16,18,22 However, how C5a regulates the
biosynthesis of these cytokines remains unclear. Results from this work
provide direct evidence for C5a activation of NF-B in PBMC and in a
transfected macrophage cell line. Thus, NF-B activation may be an
important mediator of C5a-induced transcriptional activation of these
cytokines genes, which all contain B-binding sequence in their
promoter regions.29 This notion is supported by the finding
that C5a stimulates IL-8 secretion, which peaks at 2 hours after
agonist stimulation and follows NF-B activation, which reaches
maximum within 1 hour (Figs 2 and 5). Further attesting to the role of
NF-B in IL-8 gene expression, the antioxidant inhibitor of NF-B,
PDTC, markedly reduces C5a-induced IL-8 secretion and IL-8 message
accumulation. The effective inhibition of NF-B activation by a
dominant negative IB mutant suggests a direct role of C5a
stimulation of IB phosphorylation, which leads to NF-B
activation and IL-8 expression. Although C5a also stimulates AP-1
binding activity, the time course of AP-1 activation differs significantly from that of NF-B and cannot account for the early phase (within 2 hours) upregulation of IL-8 at the protein and mRNA
levels. The current work does not rule out the possibility that AP-1
activation contributes to IL-8 gene expression in the late phase, as
was reported in several previous studies.40-42 The observed
14-fold increase in CAT activity with the p-272CAT construct compared
with a sixfold increase using the 0.2-kb (WT)CAT construct supports a
synergistic function of AP-1 in IL-8 gene expression with prolonged C5a
stimulation, as was the condition used in CAT assays. Previously
published studies demonstrate that NF-IL-6 can act synergistically with
NF-B to enhance IL-8 transcription in a cell type-dependent manner,
but NF-IL6 alone is insufficient for IL-8 gene
expression.40 However, in this study, C5a did not induce
detectable NF-IL6 activation; therefore, the function of NF-IL-6 in
IL-8 expression may be negligible. Our results suggest a critical role
of NF-B activation in C5a-stimulated IL-8 gene expression, because a
reporter construct with a defective B site did not respond to C5a
stimulation despite the presence of the AP-1 and NF-IL-6 sites (Fig 7).
The actions of C5a are mediated by the G-protein-coupled C5a receptor
expressed in granulocytes, monocytes, and several other types of cells.
The C5a receptor is known to couple to Gi proteins that are
sensitive to pertussis toxin-mediated ADP-ribosylation and G protein
inactivation. In reported studies, PTX treatment abolishes C5a mediated
functions such as chemotaxis, degranulation, and superoxide generation
(reviewed in Gerard and Gerard5). A surprising finding from
this study is the lack of complete inhibition by PTX of C5a-stimulated
NF-B activation and IL-8 secretion. Using calcium mobilization as an
assay for G-protein-coupled response, we demonstrated that C5a
receptor can couple to either PTX-sensitive or PTX-insensitive G
proteins depending on cell types. The two likely candidates for
PTX-insensitive G proteins are Gq and G16, being both present in mononuclear cells and
insensitive to PTX treatment. G16 has been shown in a
previous report to couple to C5a receptor in transiently transfected
COS-7 and 293 cells,37,38 making it a more likely candidate
for the G protein that transduces C5a-induced signaling leading to
transcription activation. This study provides an example of coupling of
a chemoattractant receptor to PTX-insensitive G protein -subunit(s)
that effectively mediates a cellular response. Based on our data, we
cannot rule out the possibility that C5a induces IL-8 gene expression
without activating G proteins in the RAW264.7 cells. It will be
important to further investigate the possible presence of a
G-protein-independent pathway and to understand the structural basis
for C5a receptor coupling to multiple G proteins that
leads to different cellular responses.
Activation of NF-B and other transcription factors for
immediate-early genes provides an effective means for chemoattractant stimulated upregulation of proinflammatory cytokines. Results from the
current study suggest that, in addition to a synergistic role of C5a in
LPS-induced cytokine production,16,17 this peptide agonist
can directly induce NF-B activation that leads to biosynthesis of
IL-8. A rapid induction of IL-8 biosynthesis after C5a stimulation supports a direct action of this chemoattractant on IL-8 gene expression. Because chemoattractants are molecules that leukocytes encounter at an early stage of inflammation, their effect on cytokine gene expression may influence the course of inflammation. By induction of IL-8 expression, a chemoattractant such as C5a and fMet-Leu-Phe can
stimulate the generation of a potent chemotactic cytokine that, in
turn, may attract additional phagocytes and lymphocytes to the site of
inflammation. Recent studies conducted in our laboratory demonstrate
that IL-8 produced by mononuclear cells may also regulate the secretion
of other cytokines, including IL-8 itself. IL-8 binding to the IL-8
receptors can lead to transcription activation and an autocrine
regulation of IL-8 biosynthesis (unpublished result).
Additional efforts will be required to understand the mechanisms by
which chemoattractants induce transcription activation, especially the
signaling pathways leading from receptor stimulation to IB kinase
activation and nuclear translocation of the NF-B proteins.
| |
ACKNOWLEDGMENT |
The authors thank Drs Dan Van Antwerp and Inder Verma for providing the
IBM plasmid. The blood drawing service was provided by Scripps'
General Clinical Research Center, supported by National Institutes of
Health Grant No. MO1RR00833. This is manuscript 11564-IMM from The
Scripps Research Institute.
| |
FOOTNOTES |
Submitted May 11, 1998; accepted January 20, 1999.
Supported by US Public Health Services Grant No. AI40176 and an
Arthritis Foundation Biomedical Science Grant (to R.D.Y.). M.H.H. and
D.D.B. are recipients of Arthritis Foundation Postdoctoral Fellowships.
This work was performed during the tenure of an Established Investigatorship (to R.D.Y.) from the American Heart Association.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Richard D. Ye, MD, PhD, Department of
Pharmacology, MC868, University of Illinois at Chicago, 835 S Wolcott
Ave, Chicago, IL 60612; e-mail: yer{at}uic.edu.
| |
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TOP
ABSTRACT
INTRODUCTION