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Through a Post-Transcriptional Mechanism1
*
Department of Basic Medical Sciences, University of Missouri, and
Saint Luke’s Hospital, Kansas City, MO 64111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, an important inflammatory mediator in
bacterial infection-related diseases such as septic shock. We
demonstrate here that while inducing only low levels of TNF- alone,
DNA from both Gram-negative and Gram-positive bacteria synergizes with
subthreshold concentrations of LPS (0.3 ng/ml) to induce TNF- in the
RAW 264.7 macrophage-like cell line. The bacterial DNA effects are
mimicked by synthetic CpG-containing oligodeoxynucleotides, but not
non-CpG-containing oligodeoxynucleotides. Pretreatment of macrophages
with either DNA for 2–8 h inhibits macrophage TNF- production in
responses to DNA/LPS. However, when pretreatment was extended to
24 h, DNA/LPS synergy on TNF- is further enhanced. RT-PCR
analysis indicates that mRNA levels of the TNF- gene, however, are
not synergistically induced by bacterial DNA and LPS. Analyses of the
half-life of TNF- mRNA indicate that TNF- message has a longer
half-life in bacterial DNA- and LPS-treated macrophages than that in
bacterial DNA- or LPS-treated macrophages. These findings indicate that
the temporally controlled, synergistic induction of TNF- by
bacterial DNA and LPS is not mediated at the transcriptional level.
Instead, this synergy may occur via a post-transcriptional
mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(3, 4), IL- 1 (4), IL-6
(5), IL-12 (6, 7, 8), IFN-
(9, 10), IFN-
(9), and the reactive nitrogen
intermediate, NO (11, 12). Subtle structural differences
between bacterial and eukaryotic DNA apparently account for the ability
of bacterial DNA to serve as an immune-activating agent. Specifically,
bacterial DNA is thought to activate inflammatory cells because of its
high content of short sequences with unmethylated CpG dinucleotides
(13). In mammalian DNA, CpG-containing sequences occur at
a much lower frequency than in bacterial DNA, and the cytosine present
in CpG dinucleotides of mammalian DNA is usually methylated (14, 15).
In vivo studies support the concept that bacterial DNA is an important
proinflammatory stimulus, as bacterial DNA has been shown to trigger
septic shock in D-galactosamine treated mice.
(16). Also of interest is the finding that bacterial DNA
acts synergistically with LPS to induce TNF- production in vivo,
resulting in lethal shock in mice (11, 17). The molecular
mechanism(s) by which bacterial DNA acts synergistically with LPS to
induce TNF- production remains to be determined. LPS, a constituent
of the Gram-negative bacterial cell wall, is well known as a potent
inducer of mouse macrophage activation, resulting in production of many
inflammatory mediators, including TNF-, that play a key role in the
development of septic shock (reviewed in Refs. 18 and
19). The induction of TNF- secretion from mouse
macrophages in response to LPS stimulation is controlled at
transcriptional, post-transcriptional, and translational levels
(17, 20, 21).
In this communication we explore the mechanisms by which LPS and
bacterial DNA act synergistically to activate macrophages. We present
in vitro data demonstrating that bacterial DNA acts synergistically
with substimulatory concentrations of LPS to enhance TNF- secretion
by the murine RAW 264.7 macrophage-like cell line. The observed synergy
depends upon the presence of unmethylated CpG residues in DNA and is
also dependent upon the temporal order of treatment by LPS and
bacterial DNA. Enhanced TNF- secretion by RAW 264.7 cells
simultaneously exposed to bacterial DNA and LPS is not accompanied by
enhanced transcription of the TNF- gene. Analyses of the half-life
of TNF- in differentially treated macrophages suggest that bacterial
DNA and LPS act synergistically to enhance TNF- production through a
post-transcriptional event.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified LPS from Escherichia coli O111: B4 was obtained from List Biological Laboratories (Campbell, CA). E. coli strain B genomic DNA, salmon sperm genomic DNA, and RNase-free DNase I were purchased from Sigma (St. Louis, MO). Synthetic oligodeoxynucleotides (ODNs;3 T3, 5'-AACGTT AACGTT AACGTT-3'; C3, 5'-CCATGGCCATGGCCATGG-3') were obtained from Sigma-Genosys (The Woodlands, TX). The endotoxin levels in these ODNs are <0.01 ng/µg of DNA based upon the Limulus amebocyte lysate assay.
Culture of macrophages
The murine macrophage-like cell line RAW 264.7 (American Type Culture Collection, Manassas, VA) was used in all the studies described here. Macrophages were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% heat-inactivated FBS (endotoxin content of <0.06 ng/ml; Sigma) at 37°C in a humidified, 5% CO2 environment. Before being stimulated, macrophages were seeded into culture plates and cultured overnight.
DNA manipulation
E. coli DNA and salmon sperm DNA (Sigma) were further purified by two-step CsCl ultracentrifugation. DNA from Staphylococcus aureus was extracted exactly as described by Dyer and Iandolo (22). DNA digestion was performed using RNase-free DNase I (2 U/µg of DNA) in buffer (pH 7.6) containing 20 mM Tris-HCl and 20 mM MgCl2 at 37°C for 3 h. The endotoxin levels in these DNA preparations were <0.001 ng/µg of DNA according to the Limulus amebocyte lysate assay.
TNF- analysis
After 20 h of stimulation, TNF- production in macrophage
culture supernatants was analyzed using the ELISA Duoset kit (purchased
from R&D Systems, Minneapolis, MN). The protocol from the manufacturer
was followed exactly for the assay. All data for TNF- represent the
average of duplicate samples ± SEM. Each experiment was repeated
at least twice.
RNA isolation and RT-PCR analysis
RAW 264.7 macrophages were stimulated for various periods of
time with different combinations of stimuli as described in
Results. Total RNA from macrophages was isolated using
TRIzol reagent (Life Technologies) according to the manufacturer’s
instructions. A total of 1 µg of RNA from each sample was used for
RT-PCR using the One-Step RT-PCR kit from Qiagen (Valencia, CA)
according to the manufacturer’s protocols. The sequences of the
specific primers used in these studies are: mouse TNF- sense, 5'-GGC
AGG TCT ACT TTG GAG TCA TTG C-3'; mouse TNF- antisense, 5'-ACA TTC
GAG GCT CCA GTG AAT TCG G-3'; mouse -actin sense, 5'-TGT GAT GGT GGG
AAT GGG TCA G-3'; and mouse -actin antisense, 5'-TTT GAT GTC ACG CAC
GAT TTC C-3'. PCR products were analyzed using agarose gel
electrophoresis, stained with ethidium bromide, and photographed. The
photographs were scanned using Adobe PhotoShop software (Adobe Systems,
San Jose, CA) and analyzed using a GelPro Analyzer (Meyer Instruments,
Houston, TX).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in vivo, which
contributes to septic shock in mice (3, 23). Because
macrophages are a major source of TNF- in vivo, we endeavored to
reproduce this phenomenon in vitro with cultured mouse macrophages. RAW
264.7 macrophages were exposed to various concentrations of purified
E. coli DNA with or without LPS at a subthreshold
concentration (0.3 ng/ml; Fig. 1
). At
this subthreshold concentration LPS by itself either fails to induce
TNF- secretion or induces a minimal response (data not shown). RAW
cells stimulated with E. coli DNA alone produced relatively
low levels of TNF- (<2000 pg/ml) even at the highest DNA
concentration tested (30 µg/ml) in this study (Fig. 1A,
). However, in the presence of a subthreshold LPS stimulus the
TNF- response to E. coli DNA was dramatically enhanced.
For example, TNF- production induced by 3.0 µg/ml E.
coli DNA was increased 
14-fold by adding 0.3 ng/ml LPS (Fig. 1A, ). In the presence of LPS, TNF- production was
dose dependent for E. coli DNA stimuli between 1.0 and 10
µg/ml, then subsequently declined. Importantly, TNF- secretion in
response to E. coli DNA plus a subthreshold LPS stimulus was
almost completely abrogated by treatment with DNase I, indicating that
the response is specific to DNA (Fig. 1A, 
). Salmon sperm
DNA, which contains highly methylated CpG dinucleotides, induced a weak
TNF- response by itself and failed to synergize with LPS, suggesting
that the macrophage-activating ability of DNA is dependent upon
nonmethylated CpG dinucleotides (Fig. 1A, • and
).
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(Fig. 1
B, ). However, in the presence of a subthreshold LPS
stimulus (0.3 ng/ml), S. aureus DNA induced a greatly
enhanced TNF- response that was dose dependent (Fig. 1B,
). This synergistic induction of TNF- by S. aureus DNA
plus LPS was also abolished by treatment with DNase I (Fig. 1B, ). Control salmon sperm DNA did not stimulate
macrophages to produce significant levels of TNF-, either alone or
in the presence of LPS (Fig. 1B, • and ).
The stimulatory activity of E. coli and S. aureus
DNA could be reproduced by a synthetic nonmethylated CpG-containing ODN
(termed T3; see Materials and Methods). Although T3 by
itself was a more potent stimulus than either E. coli or
S. aureus DNA alone, it still induced only low levels of
TNF- secretion (peaking at 4000 pg/ml) from RAW macrophages (Fig. 1C, ). However, as with E. coli and S.
aureus DNA, T3-induced TNF- secretion was markedly enhanced
(peaking at 12,000 pg/ml) in the presence of a subthreshold LPS
stimulus (Fig. 1C, ). In marked contrast to the results
with T3, treatment of macrophages with a non-CpG-containing ODN (C3)
resulted in minimal TNF- secretion (<1000 pg/ml) in either the
absence or the presence of LPS (Fig. 1C, • and
).
It has been reported that TNF- production by mouse macrophages in
response to LPS stimulation is controlled at both transcriptional and
post-transcriptional levels (17, 20, 21). However, the
mechanism by which bacterial DNA plus LPS act synergistically to induce
macrophage TNF- production remains to be determined. To assess
whether synergistic induction of macrophage TNF- by bacterial DNA
and LPS is controlled at the level of gene transcription, macrophages
were treated for 2 h with E. coli DNA or LPS alone or
with a combination of E. coli DNA plus LPS (along with
appropriate controls) as described in Fig. 2. After stimulation, total RNA was
extracted from macrophages and subjected to RT-PCR analysis for
detection of TNF- mRNA. TNF- mRNA levels in macrophages treated
with either E. coli DNA (1.0 µg/ml) or LPS (0.3 ng/ml)
were elevated compared with TNF- mRNA from control macrophages
treated with cell culture medium; recall that LPS at this concentration
fails to induce a TNF- response, and E. coli DNA induced
low levels of TNF- secretion (<2000 pg/ml). Simultaneous treatment
of macrophages with both E. coli DNA and LPS did not further
enhance TNF- mRNA expression compared with treatment with either
E. coli DNA or LPS alone despite the finding that this
combined stimulus markedly enhanced TNF- secretion. Treatment of
E. coli DNA with DNase I essentially abrogated the TNF-
mRNA response to E. coli DNA alone, but did not alter the
TNF mRNA production observed in response to simultaneous E.
coli DNA plus LPS treatment. Finally, although salmon sperm DNA by
itself failed to induce significant TNF- mRNA production, TNF-
mRNA levels induced by simultaneous treatment with salmon sperm DNA and
LPS were comparable to those induced by simultaneous E. coli
DNA and LPS treatment. Together these data all support the conclusion
that E. coli DNA and LPS do not act synergistically to
enhance TNF- mRNA production.
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mRNA expression
compared with treatment with either E. coli DNA or LPS alone
for any of the stimulation periods tested (data not shown).
The data presented above indicate that although a subthreshold LPS
stimulus (0.3 ng/ml) synergizes with bacterial DNA to enhance TNF-
secretion from RAW 264.7 macrophages, LPS and bacterial DNA do not
synergize to enhance TNF- mRNA expression. It has previously been
shown that TNF- gene expression is regulated at both transcriptional
and post-transcriptional levels when LPS alone is the stimulus
(17, 20, 21). Consequently, it is reasonable to
hypothesize that enhanced TNF- production in response to a combined
LPS and bacterial DNA stimulus results from altered
post-transcriptional controls. To test this hypothesis, we determined
the half-life of TNF- mRNA in macrophages treated with E.
coli DNA (Fig. 3A) or LPS
alone (Fig. 3B), E. coli DNA combined with LPS
(Fig. 3C), or salmon sperm DNA combined with LPS (Fig. 3D). To determine the half-life of TNF- mRNA,
transcription was inhibited by treating macrophages with actinomycin D
(5.0 µg/ml) after they had been exposed to specific stimuli for a
period of 2 h. Total RNA was isolated from macrophages at various
time points after actinomycin D treatment and subjected to RT-PCR
analysis to assay for TNF- mRNA. The half-lives of TNF- mRNA in
macrophages stimulated with bacterial DNA alone, LPS alone, bacterial
DNA plus LPS, or salmon sperm plus LPS are estimated to be 36, 45, 100,
and 45 min, respectively (Fig. 3). Thus, the half-life of TNF- mRNA
in macrophages stimulated with a combination of LPS plus bacterial DNA
is 2–3 times that of TNF- mRNA from macrophages exposed to any of
the other stimuli tested. This prolonged half-life of TNF- mRNA
could account at least in part for the synergistic production of
TNF- by macrophages exposed to a combination of bacterial DNA
and LPS.
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, the
sequence and timing of exposure to bacterial DNA and LPS were altered.
Pretreatment of macrophages with E. coli DNA for 2–8 h
before adding E. coli DNA plus LPS (0.3 ng/ml) resulted in a
time-dependent reduction of TNF- secretion (Fig. 4, •). The
maximum observed effect in this experiment was when macrophages were
pretreated with E. coli DNA for 8 h before adding LPS;
TNF- secretion was reduced more than 3-fold compared with
macrophages that were not pretreated. Interestingly, when pretreatment
with E. coli DNA was extended to 24 h, TNF-
secretion was comparable to levels attained in macrophages that were
not pretreated (Fig. 4, compare 24 and 0 h points). Macrophages
pretreated with LPS for 2–8 h before adding E. coli DNA
plus LPS also displayed a time-dependent reduction in TNF- secretion
compared with macrophages that were not pretreated (Fig. 4, ).
However, the reduction in TNF- secretion was not as extensive as
that observed with E. coli DNA pretreatment (Fig. 4, compare
and •). As with DNA pretreatment, when pretreatment with LPS was
extended to 24 h, TNF- secretion was comparable to levels
attained with macrophages that were not pretreated (Fig. 4, compare 24
and 0 h points).
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mRNA
transcription, macrophages were pretreated with E. coli DNA,
LPS, or medium (control) for 8 h before stimulation with E.
coli DNA plus LPS for 2 h. After stimulation, total RNA was
extracted from macrophages and subjected to RT-PCR analysis for
detection of TNF- specific mRNA. TNF- mRNA levels in macrophages
pretreated with either LPS or E. coli DNA before stimulation
with E. coli DNA plus LPS were comparable to TNF- mRNA
levels in macrophages pretreated with medium (Fig. 5), results comparable to those observed
earlier regarding the relative quantity of TNF- secreted by
macrophages and the quantity of mRNA produced by macrophages.
Pretreatment of macrophages with either LPS or E. coli DNA
reduces TNF- secretion in response to a subsequent LPS plus E.
coli DNA stimulus, but appears not to alter transcription of
TNF- mRNA. These data lend additional support to the conclusion that
post-transcriptional events account for the synergistic interactions
among bacterial DNA, LPS, and macrophages that lead to altered patterns
of TNF- secretion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein and enhanced expression of TNF- mRNA (Figs. 1 and 2). When macrophages were simultaneously treated with bacterial DNA
plus LPS, secretion of TNF- protein was dramatically enhanced, while
levels of TNF- mRNA remained comparable to those attained with
either bacterial DNA or LPS stimulation alone (Figs. 1 and 2). However,
the half-life of TNF- mRNA in macrophages treated simultaneously
with bacterial DNA plus LPS was substantially longer than that in
macrophages treated with bacterial DNA or LPS alone. Additionally,
pretreatment of macrophages with either bacterial DNA or LPS alone
caused a time-dependent suppression of TNF- secretion induced by
subsequent stimulation with bacterial DNA plus LPS. This suppression of
TNF- secretion was not accompanied by reduced expression of TNF-
mRNA. These findings suggest that synergistic interactions among
bacterial DNA, LPS, and macrophages leading to enhanced TNF-
secretion are controlled by post-transcriptional events. A synthetic
oligonucleotide rich in nonmethylated CpG residues also acted
synergistically with LPS, whereas both salmon sperm DNA, which contains
highly methylated CpG residues, and a synthetic non-CpG-containing
oligonucleotide, failed to act synergistically with LPS to enhance
TNF- secretion. These data support the conclusion that nonmethylated
CpG residues are critical to bacterial DNA’s capacity to act
synergistically with LPS to enhance TNF- secretion by
macrophages.
The data we present are consistent with the results of a recent in vivo
study by Schwartz et al. (24). Schwartz et al. reported
that i.v. pretreatment of mice with CpG-containing oligonucleotides
suppressed production of TNF- and macrophage inflammatory protein-2
in lung lavage fluid in response to inhaled LPS. However, analysis of
total lung mRNA indicated that TNF- and macrophage inflammatory
protein-2 mRNA levels were not reduced by pretreatment with CpG
oligonucleotides. Thus, this in vivo study shows discrepancies between
TNF- production and TNF- mRNA transcription following
interactions between LPS, CpG DNA, and macrophages. This suggests that
modifications in TNF- production attributable to these interactions
in vivo are controlled by post-transcriptional events.
We have previously reported that bacterial DNA and LPS interact
synergistically with macrophages to enhance production of NO, another
important inflammatory mediator (12). Although the
combination of bacterial DNA plus LPS dramatically enhanced the
production of both NO and TNF-, the mechanisms responsible for the
synergistic induction of these two inflammatory mediators appear to
differ. The synergistic induction of NO is regulated primarily at the
transcriptional level, because a combination of bacterial DNA plus LPS
enhanced the production of mRNA for the inducible NO synthase gene,
which accounts for the synthesis of NO in mouse macrophages
(25). In contrast, levels of TNF- mRNA in macrophages
treated with a combination of bacterial DNA plus LPS were similar to
those in macrophages treated with either bacterial DNA or LPS alone,
suggesting that transcriptional controls do not account for enhanced
TNF- secretion under these conditions. However, the half-life of
TNF- mRNA in macrophages treated with a combination of bacterial DNA
plus LPS is 2–3 times that of TNF- mRNA in macrophages stimulated
with either bacterial DNA or LPS alone. This suggests that
post-transcriptional controls are responsible for the synergistic
induction of TNF- by bacterial DNA plus LPS.
Expression of the TNF- gene in mouse macrophages is regulated at
multiple levels. Transcriptional (20, 21),
post-transcriptional (17, 20), and translational
(17, 26, 27, 28) controls have all been reported to contribute
to regulation of TNF- gene expression. Stimulation of RAW 264.7
macrophages with LPS resulted in increased transcription and
translation of the TNF- gene, although no apparent change in TNF-
mRNA stability has been observed (20, 28). Transcriptional
regulation of the TNF- gene is mediated primarily by binding of
transcription factors (e.g., NF-
B) to the promoter region of the
TNF- gene (29, 30), whereas post-transcriptional and
translational regulation is related to the 3' untranslated region (UTR)
of TNF- mRNA. It has been reported that the UA-rich 3' UTR of
TNF- mRNA contributed to instability as well as translation
repression of TNF- mRNA (17, 26, 27, 28). Whether the
prolonged half-life of TNF- mRNA in macrophages treated with a
combination of bacterial DNA plus LPS is mediated by the 3' UTR of
TNF- mRNA and whether the 3' UTR-mediated translational mechanism
also plays a role in the synergistic induction of TNF- remain to be
investigated.
The available experimental evidence suggests that bacterial DNA and LPS
may use different signaling transduction pathways, even though both
activate common transcription factors such as NF-B and AP-1
(31, 32, 33, 34, 35). Although LPS uses TLR4 as its receptor
(36), bacterial DNA uses TLR9 as its receptor that might
be dependent on the adaptor protein MyD88 (37, 38, 39). Our
finding that a temporally controlled synergy exists between bacterial
DNA and LPS lends further support to the idea that these two microbial
components use different signaling pathways. Whereas LPS alone induces
macrophages to produce TNF- primarily through transcriptional and
translational signaling (20, 28), a combination of
bacterial DNA plus LPS may activate a signal(s) directed toward
post-transcriptional controls, which might contribute to the
synergistic production of TNF-.
Collectively, the data presented in this communication indicate that
nonmethylated, CpG-rich, bacterial DNA synergizes with LPS to enhance
TNF- secretion from the RAW 264.7 macrophage cell line cultured in
vitro. The synergistic production of TNF- induced by a combination
of bacterial DNA plus LPS appears to be controlled at
post-transcriptional levels, as these conditions enhance the half-life
of TNF- mRNA, but do not enhance the transcription of TNF- mRNA.
Our data also showed that pre-exposure of macrophages to either
bacterial DNA or LPS resulted in a time-dependent reduction in TNF-
secretion in response to a combined bacterial DNA plus LPS stimulus.
This inhibition also appears to be controlled at the
post-transcriptional level, as pretreatment with bacterial DNA or LPS
reduced TNF- secretion without impacting TNF- mRNA levels. These
findings provide a mechanistic explanation for previous reports
indicating that bacterial DNA and LPS acted synergistically to enhance
TNF- production in vivo, leading to lethal shock in mice (3, 23).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David C. Morrison, Office of Research Administration, Room 3112, Main Hospital, Saint Luke’s Hospital of Kansas City, 4401 Wornall Road, Kansas City, MO 64111. E-mail address: dmorrison{at}saint-lukes.org
3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; UTR, untranslated region.
Received for publication February 2, 2001. Accepted for publication March 21, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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regulation. J. Biol. Chem. 272:21120. by lipopolysaccharide. J. Biol. Chem. 273:974.B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor gene in primary macrophages. J. Exp. Med. 171:35. promoter in primary macrophages. J. Immunol. 147:1694.[Abstract]
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J. J. Gao, V. Diesl, T. |
