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CUTTING EDGE |
Departments of
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Microbiology, Molecular Genetics, and Immunology and
Biochemistry, University of Kansas Medical Center, Kansas City, KS 66160;
U.S. Human Health Division, Merck & Company Inc., West Point, PA 19044;
§
Department of Basic Medical Sciences, University of Missouri, Kansas City, MO 64108; and
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Saint Luke’s Hospital of Kansas City, Kansas City, MO 64111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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, IL-1, and IL-6
(9, 10, 11) as well as the secretion of NO from IFN-
-primed
mouse macrophages (12). Here we present data demonstrating that bacterial DNA can synergize with subthreshold concentrations of LPS (0.3 ng/ml) in inducing the murine RAW 264.7 macrophage-like cell line to secrete NO. The observed synergy appears to depend upon the temporal order of treatments by LPS and bacterial DNA, and upon the presence of CpG residues, and most likely results from up-regulated expression of the iNOS gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Purified LPS from Escherichia coli O111:B4 was
purchased from List Biological Laboratories (Campbell, CA).
Neutralizing rat anti-mouse IFN- mAb and control rat IgG were
obtained from PharMingen (San Diego, CA) (with endotoxin levels of
<0.01 ng/ml). Neutralizing rabbit anti-mouse IFN-ß Ab and
control rabbit IgG were gifts of Dr. William J. Murphy (University of
Kansas Medical Center; described in Ref. 13). RNase-free
DNase I was purchased from Sigma (St. Louis, MO). Synthetic
oligodeoxynucleotides (ODNs) (T3, 5'-AAC GTT AAC GTT AAC GTT-3';
C3, 5'-CCA TGG CCA TGG CCA TGG-3') were obtained from Genosys
(The Woodlands, TX). The endotoxin levels in these ODNs are <0.01
ng/µg of DNA, based upon Limulus amoebocyte lysate
assay.
Isolation and culture of macrophages
Female C57BL/6 mice from The Jackson Laboratory (Bar Harbor, ME) were used at 6–8 wk. Mice were injected i.p. with 1.5 ml of 4% Brewer thioglycollate (Difco, Detroit, MI), and peritoneal macrophages were harvested 5 days later by lavage with RPMI 1640 culture medium (Life Technologies, Grand Island, NY). Both C57BL/6 peritoneal macrophages and the murine macrophage-like cell line RAW 264.7 (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium 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.
DNA manipulation
DNA from E. coli strain B and salmon testes was purchased from Sigma and further purified by two-step CsCl ultracentrifugation. DNA from Staphylococcus aureus was purified exactly as described by Dyer and Iandolo (14). DNA digestion was performed using RNase-free DNase I (2 U/µg of DNA) at 37°C for 2–3 h in buffer (pH 7.6) containing 20 mM Tris-HCl and 20 mM MgCl2. The endotoxin levels in these DNA preparations were <0.001 ng/µg of DNA.
Nitrite assays
The NO in culture supernatants was measured as concentrations of nitrite using Griess reagent (15) and quantitated by comparison with a standard curve generated using sodium nitrite. The data presented represent averages of triplicate cultures ± SEM and are representative of at least three similar experiments.
RNA isolation and RT-PCR analysis
RAW 264.7 macrophages (2.5 x 106 cells/well) were seeded into six-well tissue culture plates and incubated for 2.0 h to allow for adherence. After simulation for 2.5 h with various stimuli as described in Results, total RNA was isolated using Trizol reagent (Life Technologies) exactly according to the manufacturer’s instructions. A total of 1 µg of total RNA from each sample was used for reverse transcription using the GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg, NJ). The manufacturer’s protocols were followed exactly for both reverse transcription and PCR. The sequences of the specific primers used in these studies are: mouse iNOS sense, 5'-TCA CTG GGA CAG CAC AGA AT-3'; mouse iNOS antisense, 5'-TGT GTC TGC AGA TGT GCT GA-3'; mouse ß-actin sense, 5'-TGT GAT GGT GGG AAT GGG TCA G-3'; mouse ß-actin antisense, 5'-TTT GAT GTC ACG CAC GAT TTC C-3'. PCR products were subjected to agarose gel electrophoresis, stained with ethidium bromide, and photographed. The photographs were scanned using Adobe PhotoShop software (Adobe Systems Incorporated, San Jose, CA) and analyzed using a GelPro Analyzer (Meyer Instruments, Houston, TX).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A). As shown by
the data in Fig. 1A, E. coli DNA, by itself, was
at best only a weak inducer of NO even at concentrations as high as 10
µg/ml (Fig. 1A, controls). In the presence of 0.3 ng/ml of
LPS, however, NO production was dramatically enhanced (Fig. 1A, 
). NO induction by E. coli DNA at a
concentration as low as 1.0 µg/ml in the presence of 0.3 ng/ml of LPS
was at least 20-fold higher compared with that induced by either 1.0
µg/ml of E. coli DNA or 0.3 ng/ml of LPS alone. Of
importance, this enhanced production of NO was completely abrogated by
pretreatment with DNase I (Fig. 1A, controls). As a control
for these studies, a eukaryotic source of DNA, specifically DNA
obtained from salmon sperm, neither induced NO by itself nor was
capable of enhancing the mouse macrophage response to LPS (Fig. 1A, controls). These observations also extend to other
microbial DNA preparations (e.g., Gram-positive DNA and synthetic
CpG-containing oligonucleotides). Similar to the results found using
E. coli DNA, S. aureus DNA by itself also did not
induce significant production of NO, even at concentrations as high as
10 µg/ml (Fig. 1A, controls). However, in the presence of
0.3 ng/ml of LPS, NO production can again be shown to be dramatically
enhanced by S. aureus DNA (Fig. 1A, 
). As
anticipated, the enhanced production of NO by the DNA was completely
abolished by treatment of DNase I (Fig. 1A, controls).
Essentially parallel results to those obtained with E. coli
and S. aureus DNA were obtained using the CpG-containing ODN
(T3). By itself, this oligonucleotide induced little production of NO
in RAW 264.7 macrophages, but in the presence of 0.3 ng/ml of LPS, a
high level of NO production was observed (Fig. 1A, 
). In
contrast, a second ODN (C3), which does not contain CpG dinucleotides,
induced little NO yield either with or without the presence of LPS
(Fig. 1A, controls).
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B, when macrophages
were treated with relatively low concentrations of LPS alone (e.g.,
0.3–10.0 ng/ml), increasing amounts of NO production were detected in
response to increasing concentrations of LPS until a plateau of
LPS-dependent NO secretion was attained at 
3.0 ng/ml of LPS.
However, in the presence of 1.0 µg/ml of E. coli DNA
(itself only weakly stimulatory), the LPS-induced NO production, as
expected, was significantly potentiated. Once again, the findings that
DNase I treatment almost completely ablated the observed enhancement of
LPS-induced NO production by E. coli DNA and that salmon
sperm DNA at equivalent concentrations did not significantly enhance
LPS-induced NO production provide support for the conclusion that the
observed effects are most likely microbial DNA-specific (Fig. 1B). Collectively, these data presented in Fig. 1 would
strongly support the conclusion that microbial DNA and LPS can
synergize in their immunostimulatory capacities to promote the release
of NO from RAW 264.7 macrophages.
Because bacterial DNA has been reported to induce both type I and type
II IFN from mouse spleen cells in vitro (22, 23), and
because IFN has been known to synergize with LPS to induce NO
production in mouse macrophages (24, 25), it was of
interest to examine whether the observed synergy between bacterial DNA
and LPS in their ability to induce NO synthesis might reflect the
ability of DNA to induce production of autocrine IFN. To test this
hypothesis, RAW 264.7 macrophages were treated with 3 µg/ml of
E. coli DNA plus 0.3 ng/ml of LPS in the presence of
neutralizing Abs against mouse IFN-ß, IFN-, or both. Neither
Abs against IFN-ß, IFN- (used at 0.3 µg/ml and 1 µg/ml,
respectively), or the combination of both significantly inhibited NO
production in response to E. coli DNA plus 0.3 ng/ml of LPS,
despite the fact that the two Abs were potent inhibitors of NO
production in LPS- and LPS plus IFN--stimulated mouse macrophages,
respectively (Fig. 2). These findings
would suggest that the observed synergy between LPS and bacterial DNA
is not mediated through the production of macrophage-derived
IFN.
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. RNA isolated from these stimulated
samples was then subjected to RT-PCR analysis. As shown by the data in
Fig. 3, treatment of macrophages for 2.5 h with E. coli
DNA (1.0 µg/ml) alone or LPS (0.3 ng/ml) alone induced only a modest
increase of iNOS mRNA, consistent with the finding
reported above for detectable NO in culture supernatants. However, when
E. coli DNA and LPS were added to macrophages together, a
readily detectable level of iNOS mRNA was observed, and
this potentiated induction of iNOS mRNA was ablated by
pretreatment of the E. coli DNA with DNase I. As controls,
culture medium alone or salmon sperm DNA in the absence or presence of
LPS induced little or no production of iNOS mRNA. In
this experiment, a parallel set of macrophages was treated and cultured
for 20 h, followed by assay of culture supernatants for nitrite
exactly as described earlier. The results obtained are fully consistent
with the iNOS mRNA levels shown in Fig. 3, in that
little NO was induced by either E. coli DNA alone or LPS
alone, but together E. coli DNA plus LPS elicited
significant induction of NO (data not shown).
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A,
filled circles). On the other hand, pretreatment of macrophages with
LPS 2–8 h before the addition of E. coli DNA caused no
statistically significant changes in NO secretion. Interestingly,
however, when macrophages were pretreated with LPS for 24 h, NO
production was dramatically increased (Fig. 4A, ). To
test whether the differential NO production reflects expression of
iNOS mRNA, RNA isolated from LPS- or E. coli
DNA-pretreated macrophages was subjected to RT-PCR analysis, as
described in the legend to Fig. 4. Interestingly, whereas LPS
pretreatment resulted in, as anticipated, increased iNOS
mRNA levels, E. coli DNA pretreatment did not cause the
expected reduction in iNOS mRNA levels. Instead,
increases in the mRNA levels in E. coli DNA-pretreated
macrophages were found to be comparable with those in
LPS-pretreated macrophages. This occurs despite the fact that, in a
parallel set of macrophages, the secreted NO levels were enhanced with
LPS pretreatment but inhibited with E. coli DNA
pretreatment (data not shown). Nevertheless, these results support the
conclusion that the temporal relationship between additions of the
activating signals of DNA and LPS to macrophages has a profound effect
upon the ability of these cells to generate and secrete NO. The precise
molecular mechanism(s) that regulate these differential responses,
however, is clearly complex and will require additional studies.
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B and AP-1 (16, 17, 18, 19, 20, 21).
Our finding that a temporally controlled synergy rather than additivity
exists between bacterial DNA and LPS would further support the concept
that these two bacterial components do, in fact, use different
signaling pathways. Additional experimental evidence supporting this
hypothesis derives from the fact that such synergy between bacterial
DNA and LPS also exists for the induction of TNF- both in vivo and
in vitro (Refs. 9 and 27 and our manuscript
in preparation). Our finding that biologically active neutralizing Abs
against IFN did not have detectable effects upon bacterial DNA plus
LPS-induced NO would be consistent with the conclusion that the
bacterial DNA signal is most likely not mediated by an autocrine
macrophage production of IFN. Synergistic induction of NO by bacterial
DNA and LPS, nevertheless, does coincide with the increase of
iNOS mRNA levels, suggesting that NO induction involves
an enhanced expression of the iNOS gene. Recently,
Cowdery et al. (28) reported that a CpG-containing
oligonucleotide induced IL-12 p40 gene promoter activity as well as the
activation of transcription factor NF-B, suggesting that
CpG-containing oligonucleotides and/or bacterial DNA may act at the
transcriptional level to induce gene expression. However, whether or
not increased expression of the iNOS gene induced by
bacterial DNA plus LPS occurs either exclusively or primarily at the
transcriptional level remain to be clarified.
Of particular interest is the finding that prolonged pretreatment of
macrophages with E. coli DNA dramatically suppressed
E. coli DNA plus LPS-induced production of NO, although
iNOS mRNA levels in E. coli DNA-pretreated
macrophages remain comparable with those in LPS-pretreated macrophages.
This suggests that E. coli DNA pretreatment regulates NO
production via a different mechanism (e.g., posttranscriptional and/or
posttranslational) from that of LPS pretreatment. Although these
different mechanisms remain to be clarified, the suppressive effect of
E. coli DNA pretreatment on NO production does coincide with
a recent in vivo study by Schwartz et al. (29). In that
study, Schwartz et al. found that systemic pretreatment of mice with
bacterial DNA or CpG-containing ODNs suppressed the airway inflammatory
response to inhaled LPS as manifested by reduced production of TNF-
and macrophage inflammatory protein-2. However, whether or not the
suppressive effect of bacterial DNA on these two studies occurs via a
similar molecular mechanism remains to be investigated. The finding
that low concentrations of LPS can potentiate bacterial DNA on NO
induction in cultured macrophages also points out the necessity of
using highly purified bacterial DNA in both in vitro and in vivo
studies, because contamination of a DNA preparation by even a small
amount of LPS may enable bacterial DNA to exert an exaggerated
stimulatory effect on target cells compared with the effect seen for
bacterial DNA by itself.
In summary, our data indicate that a strong synergy and antagonism exists between bacterial DNA and LPS on NO production in the mouse macrophage cell line RAW 264.7. Such synergy and antagonism is temporally controlled and is not mediated by autocrine production of IFN. The synergistic induction of NO by bacterial DNA and LPS coincides with the iNOS mRNA level, whereas the antagonistic production of NO does not, suggesting that different molecular mechanisms are involved. These findings may be of considerable significance in vivo, because NO production has been known to be important in controlling bacterial infection and tumor cell growth, causing tissue damage, and mediating septic shock (1, 2), and because it is likely that both microbial constituents would be anticipated to be present within the microbial milieu of a nidus of infection. Understanding the molecular mechanism of the synergy and antagonism between bacterial DNA and LPS can be anticipated to contribute to the development of therapeutic strategies against both tumor growth and diseases related to bacterial infection.
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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, Kansas City, MO 64111. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible NO synthase; ODN, oligodeoxynucleotide.
Received for publication June 29, 1999. Accepted for publication August 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-mediated shock. Eur. J. Immunol. 27:1671.[Medline]
primes macrophage responses to bacterial DNA. J. Interferon Cytokine Res. 18:263.[Medline]
ß mediates the lipopolysaccharide-induced activation of transcription factor Statl in mouse macrophages: pivotal role of Statl in induction of the inducible nitric oxide synthase gene. J. Immunol. 161:4803. in vivo and increases the toxicity of lipopolysaccharides. J. Immunol. 156:4570.[Abstract]
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