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Infection and Immunity, April 2001, p. 2123-2129, Vol. 69, No. 4
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.4.2123-2129.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Preexposure of Murine Macrophages to CpG Oligonucleotide
Results in a Biphasic Tumor Necrosis Factor Alpha Response to
Subsequent Lipopolysaccharide Challenge
Traves D.
Crabtree,1
Long
Jin,1
Daniel P.
Raymond,1
Shawn J.
Pelletier,1
C. Webster
Houlgrave,1
Thomas G.
Gleason,1
Timothy L.
Pruett,1,2 and
Robert G.
Sawyer1,*
Surgical Infectious Disease Laboratory,
Department of Surgery,1 and Department
of Internal Medicine2, University of
Virginia, Charlottesville, Virginia 22908
Received 17 August 2000/Returned for modification 11 October
2000/Accepted 8 January 2001
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ABSTRACT |
Bacterial DNA and synthetic oligonucleotides containing CpG
sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-
], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF--mediated mechanism. It was
hypothesized that preexposure of macrophages to CpG-ODN would result in
an increased TNF- response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a
rapid proinflammatory cytokine response (TNF-) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of
macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF- secretion and mRNA accumulation following subsequent LPS challenge (1 µg/ml). However, prolonged preexposure to CpG-ODN (6 to
9 h) resulted in suppression of the TNF- protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during
preexposure resulted in an increase in the LPS-induced TNF- response
over that induced by CpG-ODN preexposure alone. Thus, while brief
preexposure of macrophages to CpG-ODN augments the proinflammatory
cytokine response to subsequent LPS challenge, prolonged preexposure
elicits IL-10 production, which inhibits the TNF- response. Although
the initial proinflammatory effects of CpG-DNA are well established,
the immune response to CpG-DNA may also include autocrine or paracrine
feedback mechanisms, leading to a complex interaction of
proinflammatory and inhibitory cytokines.
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INTRODUCTION |
In the past 10 years, there has been
increasing recognition of the immunostimulatory properties of bacterial
DNA and synthetic oligonucleotides containing an unmethylated cytosine
followed by guanine (CpG-DNA and CpG-ODN). CpG-DNA was initially shown to stimulate lymphocyte proliferation, gamma interferon (IFN-
) production, and natural killer (NK) cell tumoricidal activity (21, 29, 31-33). Subsequent studies focused on CpG-DNA
stimulation of proinflammatory cytokine secretion, B-cell stimulation,
and the preferential induction of a Th1-cell response. CpG-DNA and synthetic CpG-ODN stimulate the proinflammatory cytokines interleukin-6 (IL-6), IL-12, and IFN- in mixed splenocytes but fail to stimulate IL-2, IL-3, IL-4, IL-5, or IL-10 (15, 16, 34). In
addition, prolonged incubation (12 to 24 h) with CpG-DNA or
CpG-ODN stimulates tumor necrosis factor alpha TNF- secretion in
macrophage cell lines and murine peritoneal macrophages (28,
35; T. Sparwasser, T. Miethke, G. Lipford, K. Borschert, H. Hacker, K. Heeg, and H. Wagner, Letter, Nature 386:336-337,
1997). In vivo, intraperitoneal injection of CpG-ODN produces an early
(1 to 2 h) increase in serum TNF- levels while intratracheal
administration of CpG-ODN results in increased TNF- levels in lavage
fluid (25, 28). Bacterial DNA and CpG-ODN cause
significant mortality in D-galactosamine-sensitized mice
via TNF--mediated liver cell apoptosis (Sparwasser et al., Letter).
Additionally, in vivo preexposure with bacterial DNA followed 1 to
4 h later by lipopolysaccharide (LPS) injection results in a
significant increase in serum TNF- levels and mortality in mice with
respect to LPS challenge alone (5, 28; Sparwasser et al.,
Letter). On the other hand, Gao et al recently demonstrated that
preexposure of RAW 264.7 macrophages to CpG-ODN in vitro suppresses LPS
induction of nitric oxide production with respect to that induced by
LPS alone (10) and Schwartz et al demonstrated decreased
pulmonary inflammation in response to LPS after systemic exposure to
CpG-DNA (26). Thus, despite the potential utility of the
immunostimulatory properties of the CpG-DNA, e.g., vaccine adjuvants
(6, 7, 19, 27), there remains concern regarding the
potentially detrimental effects of CpG-DNA-induced alterations in
cytokine regulation.
To further characterize the macrophage cytokine response to CpG motifs,
we used a murine macrophage cell line, RAW 264.7, and elicited murine
peritoneal macrophages. We hypothesized that CpG-ODN preexposure in
vitro would result in a sensitization of the macrophage TNF-
response to LPS in a time-dependent manner. It was discovered, however,
that although short CpG-ODN preexposure led to early sensitization of
macrophages to LPS, with a resultant increase in TNF- secretion with
respect to that due to LPS alone, prolonged preexposure (6 to 9 h)
resulted in desensitization of the response to LPS, with decreased
levels of TNF- mRNA and protein secretion. This desensitization was
shown to be partially dependent on IL-10-mediated inhibition of TNF-
transcription, suggesting a complex system of cytokine responses to
CpG-DNA that include negative-feedback mechanisms following an initial
proinflammatory phase.
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MATERIALS AND METHODS |
Mice and peritoneal macrophages.
In vivo experiments
were performed using female BALB/c mice (Hilltop Labs, Scotsdale, Pa.)
weighing 20 to 25 g each. The animals were housed in a
pathogen-free environment and fed laboratory chow (Purina, St. Louis,
Mo.) and water ad libitum, in accordance with National Research Council
Standards. All procedures were approved by the University of Virginia
Animal Use Committee.
Three days prior to macrophage harvest, mice were injected
intraperitoneally with 1 ml of sterilized 3% Brewer thioglycolate medium (Difco Products, Becton-Dickinson) containing 1% (each) penicillin and streptomycin. The mice were subsequently sacrificed by
halothane anesthesia and cervical dislocation. Peritoneal macrophages were then harvested under sterile conditions, washed twice in phosphate-buffered saline, and resuspended in medium at the desired concentration. An -naphthyl acetate esterase assay (Sigma, St. Louis, Mo.) was performed on a sample of the cell suspension to confirm
the purity of macrophages within the cell population (they were >80%
pure), and viability was determined using trypan blue exclusion.
Cell culture techniques.
For most in vitro experiments, the
murine macrophage cell line RAW 264.7 (ATCC TIB 71; American Type
Culture Collection, Rockville, Md.) was used. Cells were cultured in
250-ml sterile culture flasks (Corning, Inc., Corning, N.Y.) containing
Dulbecco's modified Eagle's medium with 4 mM L-glutamine
and 4.5 g of glucose per liter, supplemented with 1.0 mM sodium
pyruvate and 10% fetal bovine serum (Gibco BRL, Life Technologies,
Inc., Grand Island, N.Y.). The cells were incubated at 37°C under 5%
CO2 and, prior to each experiment, were washed twice in
phosphate-buffered saline and resuspended in medium. The macrophages
were placed in 96-well polystyrene plates, using 1.5 × 106 macrophages/well in Dulbecco's modified Eagle's
medium for in vitro assays.
CpG- and non-CpG-containing oligonucleotides and LPS.
CpG-containing oligonucleotides 5'-ATA ATC
GAC GTT CAA GCA AG (CpG) and
non-CpG-containing sequences 5'-ATA ATA GAG CTT CAA GCA AG
(non-CpG) were synthesized on a DNase-resistant phosphorothioate backbone (Bio-Synthesis, Inc., Lewisville, Tex.) as previously described (5, 16, 25). A standard Limulus
amebocyte lysate assay (Endosafe) showed that the endotoxin content of
the synthesized oligonucleotides after reconstitution was less than 0.3 pg/µg of oligonucleotide (N = 3). LPS from E. coli strain O128:B12 (Sigma) was suspended in sterile 0.15 M NaCl
for in vivo experiments or medium for in vitro experiments. All
experiments were confirmed using a second CpG-containing
oligonucleotide, 5'-TCC ATG ACG TTC CTG ATG CT.
For all experiments, an ODN concentration of 1.5 µg/ml was
utilized, based on a prior investigation which revealed 1.5 µg/ml to
be the lowest concentration of CpG-ODN capable of eliciting consistent
TNF- secretion in RAW 264.7 cells (Fig. 1).
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FIG. 1.
Dose response of oligonucleotide stimulation of TNF-
secretion in RAW 264.7 cells. A total of 1.5 × 106 cells
were treated with different concentrations of CpG-ODN, non-CpG-ODN, or
medium alone for 24 h, and the TNF- level in supernatant was
measured. Values represent the means of at least three experiments.
Error bars represent the standard error of the mean. *, P < 0.05 versus non-CpG-ODN.
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Measurement of cytokine levels.
IL-10 secretion was measured
by an enzyme-linked immunosorbent assay (ELISA) using a mouse IL-10
Duoset kit (R&D/Genzyme, Cambridge, Mass.) containing primary and
secondary antibodies along with horseradish peroxidase-streptavidin.
TMB (3,3',5,5'-tetramethylbenzidine) was used as a substrate (Sigma),
and the plates were read at 450 nm on an ELISA plate reader. To inhibit
IL-10 bioactivity in vitro, anti-mouse IL-10 monoclonal antibody (clone
JES5-16E3; Pharmingen, San Diego, Calif.) was added to designated wells
at a concentration of 10 µg/ml. Additional controls were performed
using an isotype control antibody (rat immunoglobulin G2b [IgG2b],
clone A95-1; Pharmingen). Preliminary experiments with 20 µg of
anti-IL-10 antibody per ml showed no difference from those with 10 µg/ml in IL-10 neutralization for macrophages in our system. TNF-
was measured using a TNF- ELISA Minikit (Endogen, Woburn, Mass.) containing the primary and secondary antibodies and horseradish peroxidase-streptavidin.
Measurement of cellular cytokine mRNA levels and cell surface
markers.
TNF-, IL-6, and transforming growth factor 
(TGF-) mRNA were quantified using RNase protection assays
(Pharmingen). Following treatment of RAW 264.7 cells (2 × 106) with CpG-ODN, non-CpG-ODN, or medium alone under
various conditions, the cells were washed and total RNA was isolated
and purified using an RNA purification kit (RNeasy Minikit; Qiagen,
Inc., Valencia, Calif.). To generate the TNF- mRNA probes, the MCk-3
template set (Pharmingen) (which includes a template for multiple
murine cytokine mRNA probes including TNF-; the TNF- probe
protects a 287-base sequence) was incubated with [32P]UTP
in the presence of RNasin, GACU, dithiothreitol, RNA polymerase, and
transcription buffer (in vitro transcription kit, Pharmingen) and
incubated for 1 h. Following treatment with DNase, we added EDTA,
Tris-saturated phenol, chloroform-isoamyl alcohol (50:1), and yeast
tRNA. The aqueous phase was removed, treated with 4 M ammonium acetate
and ice-cold ethanol, and incubated for 30 min at 
70°C; the pellet
was washed with 70% ethanol, air dried, and solublized in buffer.
Using a scintillation counter, representative samples were quantified
(Cerenkov counts per microliter). The previously prepared RNA and an
aliquot of the probe set were incubated at 56°C for 12 to 16 h
in an Omnigene thermal cycler (Hybaid, Inc., Woodbridge, N.J.) and then
treated with RNase. Samples were electrophoresed on an
acrylamide-bisacrylamide (19:1) gel containing 40% acrylamide and 2%
bisacrylamide (Bio-Rad Laboratories, Hercules, Calif.). The gel was
dried and placed on film, and the film was exposed at 70°C
overnight and developed. Using the undigested probes as markers, a
standard curve was plotted to establish the identity of the
RNase-protected bands in experimental samples. The films were developed
using photodensitometry to quantify 32P activity associated
with the mRNA in each sample. Levels are reported as the ratio of
TNF- to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to control
for the amount of RNA loaded in each sample on the gel. GAPDH mRNA is
constituitively expressed in these cells.
To quantify relative changes in cell surface CD14 and MAC-1 expression
in vitro, RAW 264.7 cells were incubated for various
time intervals
with CpG-ODN or non-CpG-ODN. At each designated
time interval, the
cells were washed and labeled with fluorescein
isothiocyanate-conjugated rat anti-mouse CD11b antibody (clone
M1/70)
or rat IgG2b isotype control antibody (clone R35-38; Pharmingen)
and
incubated for 15 to 30 min at 37°C. Additional cells were
labeled
with fluorescein isothiocyanate-conjugated rat anti-mouse
CD14 antibody
(clone rmC5-3) or rat IgG1 isotype control antibody
(clone R3-34;
Pharmingen) and incubated for 15 to 30 min at 37°C.
Flow cytometric
analysis was performed using a FacSTAR flow cytometer
system (Becton
Dickinson, Mountain View, Calif.). Unstained cells
were washed and
treated in a similar manner to measure the level
of autofluorescence.
Values are reported as mean channel
fluorescence.
Statistical analysis.
Values for protein concentration and
mRNA levels were compared using analysis of variance and post hoc by
Tukey's honestly significant difference test to compare the means. The
slopes of lines were compared using linear regression. P 
0.05 was considered significant. Values are reported as the mean
and standard error of the mean. All calculations were performed using
statistical software (Statistica; Statsoft, Tulsa, Okla.).
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RESULTS |
CpG-ODN stimulates delayed secretion of IL-10 in
macrophages.
Although CpG motifs augment a proinflammatory
cytokine response (e.g., TNF-, IL-12, IL-6, and IFN-), there are
conflicting data regarding the role of CpG oligonucleotides in
stimulation of inhibitory or regulatory cytokines such as IL-10. A
total of 1.5 × 106 RAW 264.7 cells were incubated
with 1.5 µg of CpG or non-CpG-ODN per ml for various periods, and the
IL-10 level in the supernatant was measured. Unlike non-CpG-ODN,
CpG-ODN stimulation of RAW 264.7 cells produced an increase in IL-10
secretion after 6 and 9 h of incubation (Fig.
2). This finding prompted evaluation for
a potential IL-10-mediated regulation of the proinflammatory response following the initial CpG stimulation of TNF-. Treatment with an
anti-IL-10 antibody (10 µg/ml) did not, however, alter the primary
TNF- response to CpG-ODN stimulation with respect to the response to
CpG-ODN alone (Fig. 3).
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FIG. 2.
CpG-ODN stimulation of IL-10 protein secretion in
supernatants of RAW 264.7 cells. A total of 1.5 × 106
cells were treated with CpG-ODN (1.5 µg/ml), non-CpG-ODN, or medium
alone for 0.5 to 9 h, and the IL-10 level in supernatant was
measured. Values represent the means of at least three experiments.
Error bars represent the standard error of the mean. *, P < 0.05 for CpG versus other groups.
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FIG. 3.
Effects of anti-IL-10 antibody on CpG-ODN stimulation of
TNF- production in RAW 264.7 cells. A total of 2.0 × 106 cells were treated with CpG-ODN (1.5 µg/ml) with or
without anti-IL-10 antibody (Ab) (10 µg/ml), non-CpG-ODN with or
without anti-IL-10 antibody, medium, or anti-IL-10 antibody alone for
0.5 to 9 h, and the TNF- level in supernatant was subsequently
measured. Values represent the means of at least three experiments.
Error bars represent the standard error of the mean. *, P < 0.01 for CpG or CpG plus anti-IL-10 antibody versus
non-CpG-ODN, non-CpG-ODN plus anti-IL-10 antibody, anti-IL-10 antibody
alone, or medium.
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CpG-ODN-stimulated IL-10 inhibits late TNF- mRNA
production.
IL-10 has previously been shown to significantly
regulate the LPS-induced production of TNF- mRNA in vitro (5,
30). Although anti-IL-10 antibody had a minimal effect on
CpG-induced TNF- protein secretion during prolonged incubations, the
role of IL-10 regulation of CpG-DNA-induced stimulation of TNF- mRNA was studied. RAW 264.7 cells (2 × 106) were incubated
for various periods with CpG or non-CpG-ODN (1.5 µg/ml) in the
presence or absence of anti-IL-10 antibody (10 µg/ml) and harvested,
and the level of TNF- mRNA was measured and quantified. During the
initial CpG-ODN augmentation of TNF- mRNA production, anti-IL-10
antibody appeared to have little effect. However, longer treatment with
anti-IL-10 antibody plus CpG-ODN resulted in persistent elevation of
TNF- mRNA levels while incubation with CpG-ODN alone resulted in a
decline of TNF- mRNA levels (Fig. 4).
Additionally, treatment under identical conditions with isotype control
antibody revealed no significant differences from treatment with
CpG-ODN alone at all time points.
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FIG. 4.
Effects of anti-IL-10 antibody on CpG-ODN stimulation of
TNF- mRNA production in RAW 264.7 cells. A total of 2.0 × 106 cells were treated with CpG-ODN (1.5 µg/ml) with or
without anti-IL-10 antibody (IL-10) or isotype control antibody (10 µg/ml), non-CpG-ODN with or without anti-IL-10 antibody, medium (M)
with or without anti-IL-10 antibody, or isotype control antibody for
0.5 to 9 h. The cells were harvested, and the TNF- mRNA level
was measured using the RNase protection assay. (a) Representative gel
from at least three experiments. Each experiment was performed under
identical conditions with similar results. Lanes: M, medium; *,
medium plus anti-IL-10 antibody;  , medium plus isotype control. (b)
Graphical depiction of TNF- mRNA level as a function of time
normalized to GAPDH [32P]mRNA expression. Error bars
represent the standard error of the mean. *, P < 0.05 for CpG or CpG plus anti IL-10 antibody (Ab) versus non-CpG-ODN
and non-CpG-ODN plus anti-IL-10 antibody; , P < 0.05
for CpG plus anti IL-10 antibody versus non-CpG-ODN and non-CpG-ODN
plus anti-IL-10 antibody.
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In vitro preexposure of macrophages to CpG-ODN results in a
biphasic TNF- response to subsequent LPS challenge.
Following
the demonstration of the direct immunomodulatory activity of CpG-ODN,
subsequent analyses were performed to characterize the effects of
CpG-ODN preexposure of macrophages on the response to a second
proinflammatory stimulus, LPS. Based on previous in vivo data (5,
28; Sparwasser et al., Letter), it was hypothesized that CpG-ODN
preexposure of macrophages in vitro would result in a time-dependent
sensitization of the TNF- response to LPS challenge. RAW 264.7 cells
(1.5 × 106) were incubated with CpG-ODN or
non-CpG-ODN (1.5 µg/ml) for various periods in medium, washed, and
incubated with 1 µg of LPS per ml for 1.5 h, and the TNF-
level in supernatant was immediately measured. In the early periods (1 to 3 h), preexposure to CpG-ODN did result in a significant
increase in LPS-induced TNF- levels in supernatant with respect to
exposure to non-CpG-ODN and LPS alone; however, preexposure to CpG-ODN
for longer periods (6 to 9 h) resulted in a decreased LPS-induced
TNF- response with respect to controls (Fig.
5a). To examine the potential regulatory
role of IL-10 in this process, RAW 264.7 cells were treated with
CpG-ODN plus 10 µg of anti-IL-10 antibody per ml for various periods
and subjected to LPS stimulation. The addition of anti-IL-10 antibody during CpG-ODN preexposure for 6 and 9 h augmented LPS-induced TNF- secretion with respect to CpG-ODN preexposure alone, suggesting that in this system local IL-10 at least partially mediates macrophage insensitivity to LPS as measured by TNF- secretion (Fig. 5b). Additional experiments revealed no difference in the LPS-induced TNF- response following preexposure to CpG-ODN alone and CpG-ODN plus isotype control antibody over all time points (data not shown). Furthermore, cell viability remained greater than 85% in the 6- and
9-h groups, thus excluding cell death as the cause of decreased TNF-
production at these time points.
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FIG. 5.
(a) Effect of CpG-ODN preexposure of RAW 264.7 cells on
TNF- secretion following subsequent LPS challenge. A total of
1.5 × 106 cells were treated with 1.5 µg of CpG-ODN
or non-CpG-ODN per ml or medium alone for 0.5 to 9 h. After each
period, the cells were washed and treated with LPS (1 µg/ml) for an
additional 1.5 h (excluding the medium-no LPS series in which
cells were treated similarly except for LPS exposure), and the TNF-
level in supernatant was measured. Values represent the means of at
least three experiments. Error bars represent the standard error of the
mean. *, P < 0.05 for CpG versus other groups; ,
P < 0.05 for CpG or medium-no LPS versus all other
groups. (b) LPS-induced TNF- secretion following preexposure of RAW
264.7 cells to CpG-ODN in the presence or absence of anti-IL-10
antibody (Ab). A total of 2.0 × 106 cells were
treated with CpG-ODN (1.5 µg/ml) with or without anti-IL-10 antibody
(10 µg/ml), non-CpG-ODN with or without anti-IL-10 antibody, medium,
or anti-IL-10 alone for 0.5 to 9 h. After each period, the cells
were washed and treated with LPS (1 µg/ml) for an additional 1.5 h. The TNF- level in supernatant was measured. Values represent the
means of at least three experiments. Error bars represent the standard
error of the mean. *, P < 0.05 for CpG versus other
groups; , P < 0.05 CpG plus anti-IL-10 antibody
versus other groups.
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Figure
6 demonstrates a similar biphasic
response in murine peritoneal macrophages, although the time course of
subsequent
insensitivity to LPS measured by TNF-
release was
slightly delayed.
After 12 h of preexposure to CpG-ODN, there was
significant suppression
of LPS-induced TNF-
secretion compared to
the secretion after
preexposure to medium alone. In addition,
preexposure to CpG-ODN
plus anti-IL10 antibody for 12 h resulted
in augmentation of the
LPS-induced TNF-
response, presumably due to
the elimination
of the IL-10-mediated suppression seen in the
CpG-ODN-alone and
CpG-ODN-plus-isotype control antibody groups.
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FIG. 6.
LPS-induced TNF- secretion following preexposure of
murine peritoneal macrophages to CpG-ODN in the presence or absence of
anti-IL-10 antibody. A total of 1.5 × 106 cells were
treated with CpG-ODN (1.5 µg/ml) with or without anti-IL-10 antibody
(Ab) (10 µg/ml) or isotype control antibody, non-CpG-ODN (1.5 µg/ml) with or without anti-IL-10 antibody (10 µg/ml), or medium
alone for 1 to 12 h. After each period, the cells were washed and
treated with LPS (1 µg/ml) for an additional 1.5 h. The TNF-
level in supernatant was measured. Values represent the means of at
least three experiments. Error bars represent the standard error of the
mean. *, P < 0.05 for CpG and (CpG plus isotype
antibody) versus other groups.
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Further investigation was undertaken to investigate the relationship
between CpG-ODN preexposure dose and modulation of the
macrophage
response to subsequent LPS challenge. RAW 264.7 cells
(1.5 × 10
6) were incubated with CpG-ODN at different
concentrations for
3 and 9 h in medium washed, and incubated with 1 µg of LPS per
ml for 1.5 h. The TNF-
level in supernatant was
then immediately
measured. The CpG-ODN dose required for both
augmentation of LPS-induced
TNF-
production following 3 h of
CpG-ODN preexposure (Fig.
7a)
and
suppression of LPS-induced TNF-
production following 9 h
of
CpG-ODN preexposure (Fig.
7b) was similar to that required
for direct
CpG-ODN stimulation of TNF-
from RAW 264.7 cells (Fig.
1).
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FIG. 7.
Impact of the CpG-ODN dose on modification of
LPS-induced TNF- secretion following CpG-ODN preexposure in RAW
264.7 cells. A total of 1.5 × 106 cells were treated
with 0 to 6 µg of CpG-ODN per ml for 3 and 9 h, washed, and then
treated with LPS (1 µg/ml) for an additional 1.5 h. The TNF-
level in supernatant was measured. Values represent the means of at
least three experiments. Error bars represent the standard error of the
mean. (a) Dose-response curve for stimulation of LPS-induced TNF-
secretion in RAW 264.7 cells following a 3-h CpG-ODN preexposure. (b)
Dose-response curve for suppression of LPS-induced TNF- secretion in
RAW 264.7 cells following a 9-h CpG-ODN preexposure.
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IL-10 inhibits TNF- mRNA transcription following prolonged
CpG-ODN preexposure with subsequent LPS challenge.
To examine the
autocrine or paracrine effects of IL-10 on TNF- mRNA formation
following CpG-ODN preexposure and LPS challenge, 2 × 106 RAW 264.7 cells were incubated with CpG or non-CpG-ODN
(1.5 µg/ml) in the presence or absence of anti-IL-10 antibody (10 µg/ml) for various periods, washed, and incubated with LPS (1 µg/ml) for an additional 1.5 h. Long-term treatment (6 to 9 h)
with CpG-ODN plus anti-IL-10 antibody resulted in a return of TNF-
mRNA levels to the same level as in controls (medium or non-CpG-ODN
preexposure followed by LPS exposure), in contrast to suppression of
TNF- mRNA by CpG-ODN preexposure (Fig.
8). Further study revealed no significant
difference in TNF- mRNA levels when CpG-ODN preexposure was compared
to CpG-ODN plus isotype control antibody (10 µg/ml) preexposure. In
reference to other cytokines, additional experiments demonstrated no
difference in LPS-stimulated IL-6 and TGF- mRNA levels following a
6-h preexposure to either CpG-ODN or non-CpG-ODN (n = 3) (data not
shown).
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FIG. 8.
TNF- mRNA expression after preexposure of RAW 264.7 to CpG-ODN in the presence or absence of anti-IL-10 antibody (IL-10)
or isotype control antibody, followed by LPS stimulation. A total of
2.0 × 106 cells were treated with CpG-ODN (1.5 µg/ml), non-CpG-ODN (1.5 µg/ml), or medium (M) with or without
anti-IL-10 antibody or isotype control antibody (10 µg/ml) for 0.5 to
9 h. After each period, the cells were washed and treated with LPS
(1 µg/ml) for an additional 1.5 h, and then the TNF- mRNA
level was measured using the RNase protection assay. (a) Representative
gel from at least three experiments. Each experiment was performed
under identical conditions with similar results. Lanes: M, medium; *,
medium plus anti-IL-10 antibody; , medium plus isotype control
antibody;  , medium alone, no LPS stimulation. (B) Graphical
depiction of TNF- mRNA as a function of time, normalized to GAPDH
[32P]mRNA expression. Error bars represent the standard
error of the mean. *, P < 0.05 for CpG versus other
groups.
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To rule out the possibility that the diminished LPS response in
CpG-ODN-preexposed cells was due to a decreased number of
receptors,
both major (CD14) and minor (CD11b/CD18) LPS coreceptor
expression was
assessed after 2-, 3-, and 8-h incubations. There
was no significant
difference in CD14 expression between medium-,
CpG-ODN-, and
non-CpG-ODN-exposed cells at 2 h (CD14 mean channel
number,
12.0 ± 1.9, 13.7 ± 2.3, and 12.3 ± 2.3 respectively
[
P > 0.05 comparing all groups]) or 3 h
(11.9 ± 2.0, 11.3 ± 1.9,
and 11.3 ± 2.0 [
P > 0.05]). Interestingly, CD14 expression was
increased after 8 h of incubation with 1.5 µg of CpG-ODN per ml
compared to medium or
non-CpG-ODN incubation (15.3 ± 1.8 versus
10.0 ± 1.2 or
9.4 ± 0.9, respectively [
P < 0.05 for CpG-ODN
versus
either group]). There was no significant difference in
CD11b/CD18
expression between groups at 2, 3, and 8 h (data not
shown).
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DISCUSSION |
Although CpG-DNA is known to have a stimulatory effect on
the macrophage/monocyte system, there are few in vitro data regarding the alteration of the macrophage response to LPS following CpG-DNA preexposure. This paper demonstrates that in addition to stimulation of
TNF-, CpG-ODN stimulates significant IL-10 production in vitro, leading to a late (6 to 9 h) suppression of LPS-inducible TNF- secretion via regulation of TNF- mRNA transcription. Thus, CpG-ODN stimulation of IL-10 may serve a counterregulatory function that results in suppression of macrophage sensitivity to LPS.
Although research has focused primarily on CpG-DNA stimulation of
proinflammatory cytokines such as TNF-, IL-6, and IL-12, a small
number of conflicting reports have been published regarding induction
of the counterregulatory cytokines such as IL-10 and IL-4 by CpG-DNA.
Early studies reported a failure of CpG-ODN to stimulate IL-10
secretion in various cell populations (16, 20), while
Anitescu et al have recently demonstrated stimulation of IL-10
secretion in vitro and in vivo by CpG-ODN and have shown that this late
secretion of IL-10 is an essential component of a feedback mechanism
resulting in the inhibition of IL-12 activity (2). In
addition, Huang et al. demonstrated that CpG-DNA stimulation of IL-10
in vitro downregulated the Th1-like cytokine response to heat-killed
bacteria or LPS (14). These findings are consistent with
our demonstration of a delayed CpG-ODN stimulation of IL-10 acting as
an inhibitor of TNF- secretion following subsequent LPS challenge.
The inhibitory role of IL-10 in other systems is well established.
IL-10 is known to work in an autocrine fashion to inhibit cytokines
associated with a Th1-like response (IL-12 and IL-2) in favor of a
Th2-like response to antigen challenge (9). In addition,
IL-10 inhibits monocyte/macrophage TNF- secretion in vitro (8,
24, 30) and antagonizes LPS-induced TNF- release in vivo
(11). These findings are consistent with our demonstration of inhibition of LPS-induced TNF- by CpG-ODN-stimulated IL-10. There
are, however, several unanswered questions regarding the suppression of
TNF- in response to LPS following prolonged preexposure with
CpG-ODN. While the addition of anti-IL-10 antibody during preexposure
of cells to CpG-ODN was able to augment TNF- mRNA back to control
levels compared to preexposure to CpG-ODN alone, TNF- protein
secretion was only partially restored at the later times. This suggests
that there are other factors activated by CpG-ODN that also play a role
in the desensitization of the macrophage to LPS. Such factors could
potentially include other anti-inflammatory mediators such as TGF-
or TNF--inhibiting factor, both of which are important in other
models of monocyte hyporesponsiveness to LPS challenge (4,
24), and unrelated changes in later steps of TNF- cleavage
and secretion. Further studies are also necessary to identify the role
of delayed CpG-DNA stimulation of IL-10 in vivo.
There are many similarities between our in vitro results and classical
endotoxin tolerance. It is well established that under the proper
conditions, preexposure of macrophages in vitro to LPS can induce
suppression of the proinflammatory cytokine response to subsequent LPS
challenge. Potential cellular mechanisms of endotoxin tolerance include
alterations in nuclear factor 
B (NF-B) activity, alterations in
mitogen-activated protein kinases and p38 kinase, and alteration in
Toll-like receptor 4 (Tlr4) expression (1, 3, 17, 18, 22,
23). We are currently examining these mechanisms to evaluate
their importance to the LPS insensitivity induced by CpG-ODN in our system.
Interestingly, the in vivo correlate of endotoxin tolerance is
characterized by decreased LPS-induced mortality following in vivo
preexposure to LPS; this is in contrast to the increase in LPS-induced
mortality seen following in vivo preexposure to CpG-DNA
(28). Thus, while there is much overlap between endotoxin tolerance and the effects of CpG-DNA preexposure, there are still significant differences in these systems that preclude firm conclusions regarding their exact relationship.
We demonstrated the biphasic LPS-induced TNF- response to CpG
preexposure in both RAW 264.7 cells and murine peritoneal macrophages, with similar late augmentation of TNF- on addition of an anti-IL-10 antibody. Despite the similar responses seen between murine cell lines
and primary murine macrophages, there remains the potential for
important discrepancies between murine and human models of endotoxin
responsiveness, which are beyond the scope of this paper. It is now
clear, however, that human monocytes respond to CpG-DNA in vitro by
secreting TNF- via a mechanism that is at least temporally distinct
from that of LPS-induced TNF- secretion (12, 13).
CpG-DNA produces a rapid stimulatory effect on macrophages and
monocytes, resulting in the release of several proinflammatory cytokines. The relationship between CpG-DNA, bacterial pathogenesis, and the innate immune response, however, remains unclear. Although there is room for optimism regarding the potential clinical benefit of
using these oligonucleotides as immunomodulators, particularly for
vaccines, caution is necessary to avoid perhaps unforseen, delayed
effects on the immune response to subsequent infections. On the other
hand, the well-timed addition or deletion of CpG-DNA activity in the
actively infected patient might have a broad enough effect to be useful
as a new therapeutic modality in the treatment of human sepsis and
septic shock.
| |
ACKNOWLEDGMENTS |
This work was supported by a National Research Service Award
(1F32GM19423-01) from the National Institutes of Health (Traves D. Crabtree) and a Surgical Infection Society Junior Faculty Research Award (Robert G. Sawyer).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Surgery, UVA Health Systems, P.O. Box 800709, Charlottesville, VA
22908-0709. Phone: (804) 982-1632. Fax: (804) 924-5539. E-mail:
rws2k{at}virginia.edu.
Editor:
R. N. Moore
| |
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Infection and Immunity, April 2001, p. 2123-2129, Vol. 69, No. 4
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.4.2123-2129.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
