1 Research Service, Stratton Veterans Affairs Medical Center, and 2 Department of Medicine and Center for Cardiovascular Biology, Albany Medical College, Albany 12208; and 3 Division of Molecular Medicine, North Shore University Hospital, New York University School of Medicine, Manhasset, New York 11030
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ABSTRACT |
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Endotoxin (LPS) is a potent inducer of
tumor necrosis factor- (TNF-
) and manganese superoxide dismutase
(MnSOD). Recent evidence suggests that LPS induction of TNF-
and
MnSOD mRNAs is mediated through distinct intracellular signal
transduction pathways. Membrane CD14 (mCD14) and Toll-like receptor-4
(TLR4) mediate LPS induction of TNF-
in macrophages. In the current study, we evaluated the role of mCD14 and TLR4 in LPS induction of
MnSOD using peritoneal macrophages from CD14 knockout (CD14-KO) mice
and mice with the Tlr4 gene point mutation (C3H/HeJ) or
deletion (C57BL/10ScCr). We studied mCD14-dependent (1 and 10 ng/ml)
and mCD14-independent (1,000 ng/ml) concentrations of LPS. Compared with control (BALB/c) macrophages, LPS at 1 and 10 ng/ml failed to
induce TNF-
or MnSOD mRNA in CD14-KO macrophages. However, LPS at
1,000 ng/ml induced TNF-
and MnSOD mRNAs equally in macrophages from
CD14-KO and control mice. LPS (1, 10, or 1,000 ng/ml) failed to induce
TNF-
or MnSOD mRNA and failed to activate nuclear factor-
B in
C3H/HeJ or C57BL/10ScCr macrophages. Measurements of TNF-
and MnSOD
enzyme activity paralleled TNF-
and MnSOD mRNA levels. These data
demonstrate that, like TNF-
, induction of MnSOD by LPS is mediated
by mCD14 and TLR4 in murine macrophages.
tumor necrosis factor-; manganese superoxide dismutase; endotoxin resistance; nuclear factor-
B; peritoneal macrophage
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INTRODUCTION |
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ENDOTOXIN, A LIPOPOLYSACCHARIDE (LPS) of gram-negative bacterial cell wall, is responsible for a host of toxic effects that occur in patients infected with gram-negative bacteria, including fever, disseminated intravascular coagulation, and hemodynamic changes that may lead to multiple organ failure characteristics of the septic shock (20, 25). LPS also exhibits immunostimulatory effects (20, 25) and induces the antioxidant enzyme, manganese superoxide dismutase (MnSOD) (2, 26), effects that are beneficial to the host. However, the serious toxicity of LPS limits any potential clinical utility of its beneficial effects.
The endotoxic and immunostimulatory effects of LPS are primarily caused
indirectly through the activation of monocytes and macrophages, leading
to the release of proinflammatory cytokines such as tumor necrosis
factor- (TNF-
) and interleukin-1
(IL-1
) (4, 5, 20,
25). While much is known about the mechanism by which LPS
induces proinflammatory cytokines, little is known about the mechanism
for the induction of MnSOD by LPS. Recent studies have demonstrated
that induction of TNF-
and MnSOD by LPS is mediated through distinct
intracellular signal transduction pathways (30, 32).
Activation of protein tyrosine kinase/Ras/Raf1/MEK/mitogen-activated protein kinase (MAPK) signaling pathway is important for the induction of TNF-
, but it is not necessary for LPS induction of MnSOD
(30, 32).
Membrane CD14 (mCD14), a glycosylphosphatidylinositol-anchored
membrane glycoprotein expressed by monocytes and macrophages (9), is a membrane receptor for LPS (35),
which is part of a receptor complex containing additional molecules
required for signal transduction, including Toll-like receptor-4
(TLR4), a member of the IL-1 receptor family (14,
22-24). At low concentrations of LPS, e.g., <100 ng/ml,
both mCD14 and TLR4 are required for the induction of TNF- by LPS.
However, at higher concentrations, e.g., >100 ng/ml, LPS can induce
TNF-
in the presence of TLR4 alone, independent of mCD14
(10). Whether mCD14 and TLR4 also mediate LPS induction of
MnSOD is not clear. Gibbs et al. (8) reported that LPS was
capable of inducing MnSOD in peritoneal macrophages, but not
endothelial cells, derived from LPS-resistant mice C3H/HeJ, suggesting
that TLR4 may not be necessary for LPS induction of MnSOD in
macrophages. Recent evidence also suggests that LPS induction of
acute-phase proteins is CD14 independent, but requires a functional
TLR4 (11).
In the current study, we investigated the role of mCD14 and TLR4 in LPS
induction of MnSOD using peritoneal macrophages derived from CD14
knockout (CD14-KO) mice (10) and mice with the
Tlr4 gene point mutation (C3H/HeJ) and deletion
(C57BL/10ScCr) (22, 24). Our data suggest that, similar to
TNF-, induction of MnSOD by LPS is mediated by mCD14 and TLR4 in
murine macrophages.
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MATERIALS AND METHODS |
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Materials.
Protein-free LPS (from JM83 Escherichia coli K-12, rough
strain) was kindly provided by Dr. John E. Somerville of Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ. Before use,
LPS was dissolved in sterile, pyrogen-free water, sonicated in a bath
sonifier, and diluted with Hanks' balanced salt solution (HBSS).
Purified MnSOD (from E. coli) and CuZnSOD (from bovine erythrocyte) were obtained from Sigma Chemical (St. Louis, MO). Both
MnSOD and CuZnSOD had a specific activity of 4,400 U/mg protein as
determined according to the method of McCord and Fridovich (18). Polyclonal goat anti-recombinant human TNF-
antibody (affinity-purified IgG, 1 mg/ml; 50% neutralization dose
0.02-0.04 µg/ml by cell lytic assay using L929 fibroblasts) was
purchased from R&D Systems (Minneapolis, MN).
Mice. CD14-knockout mice were produced by removal of a 272-bp segment containing the CD14 initiation codon at the 3' end of exon 1, the 97-bp intron and 172 bp of the coding region in exon 2, using targeted homologous recombination as previously described by Haziot et al. (10). Male CD14-KO mice, 6-8 wk old, back-crossed 10 times to BALB/c mice, were kindly provided by the North Shore-Long Island Jewish Research Corp., Manhasset, NY. BALB/c mice (Taconic Farm, Germantown, NY), age and sex matched, were used as the control. Six-week-old male C3H/HeN, C3H/HeJ, and C57BL/10ScCr mice were obtained from Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD. Five-week-old male C57BL/10SnJ mice were purchased from the Jackson Laboratory, Bar Harbor, ME. Mice were housed in a conventional animal research facility.
Isolation of murine peritoneal macrophages. Peritoneal macrophages were isolated from mice 4 days after intraperitoneal injection of 2 ml 4% thioglycollate broth (Difco Laboratories, Detroit, MI) by peritoneal lavage using calcium/magnesium-free HBSS. Peritoneal macrophages (2 × 106/ml in RPMI 1640 plus 10 mM HEPES, 2 mM L-glutamine, and antibiotics) were allowed to adhere onto tissue culture plates for 1 h. The adherent cells, which consisted of ~100% viable peritoneal macrophages as judged by trypan blue dye exclusion, were then used for the current studies. All experiments were performed in the presence of 2% autologous serum.
Measurements of TNF- bioactivity.
Adherent macrophages were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h at 37°C. The amounts of TNF-
activity released
into the media were determined by the anti-TNF-
antibody-inhibitable lysis of murine L929 fibroblasts as described previously
(31).
Northern blot analysis of TNF- and MnSOD mRNAs.
Adherent macrophages were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h at 37°C. The total cellular RNA was then isolated
by the single-step method of Chomczynski and Sacchi (6)
using an RNeasy Mini Kit (Qiagen, Chatsworth, CA). For Northern blots,
denatured RNA samples (1.0 µg/lane) were electrophoresed in 1.2%
agarose gels, transferred to nylon membrane (Genescreen plus; New
England Nuclear, Boston, MA) by capillary blotting. The membrane was
then prehybridized as described previously (33). Hybridization was carried out with ULTRAhyb (Ambion, Austin, TX) and
cDNA probes that had been labeled by random hexanucleotide priming
(GIBCO/BRL, Gaithersburg, MD) to a specific activity of >109 cpm/µg DNA. The cDNA probes used included muTNF-
(DNAX Research Institute of Molecular and Cellular Biology, Palo Alto,
CA), huMnSOD, and mu
-actin (American Type Culture Collection,
Rockville, MD). After washing, autoradiographs were obtained, and
radioactive signals were quantified using a computing densitometer
(Molecular Dynamics, Sunnyvale, CA). Quantification of levels of
TNF-
and MnSOD (1- and 4-kb bands) mRNAs was corrected for
-actin
mRNA to minimize the effect of potential unequal loading of RNA. For the purpose of comparison, levels of TNF-
or MnSOD mRNA in control, LPS-nontreated cells were normalized to 1 densitometric unit.
Measurements of SOD activity. Adherent macrophages were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 or 20 h at 37°C. Cells were collected by scraping. They were then sonicated, and protein contents were determined by using bicinchoninic acid according to Smith et al. (27). Aliquots of cell extracts (75 µg/lane for MnSOD and 15 µg/ml for CuZnSOD) were then assayed for SOD activity using nondenaturing polyacrylamide gel (10%) electrophoresis (PAGE) according to the method of Beauchamp and Fridovich (3), based on the inhibitory effect of SOD on the reduction of tetrazolium by superoxide generated by photochemically reduced riboflavin, as described previously (30). The SOD activity gels were quantified using a computing densitometer. In each assay, purified E. coli MnSOD and bovine erythrocyte CuZnSOD (25-800 mU) were used to obtain standard curves from which the cell extract MnSOD and CuZnSOD activities, respectively, were derived.
Electrophoresis mobility shift assay for nuclear factor-B.
Adherent macrophages were treated with or without LPS (10 or 1,000 ng/ml) for 1 h at 37°C. Nuclear extracts were obtained according
to Osnes et al. (21). For the electrophoresis mobility shift assay, 1 µg nuclear proteins were incubated for 20 min at room
temperature with ~100,000 cpm (5 ng) of an oligonucleotide containing
nuclear factor-
B (NF-
B) consensus sequence (5'-AGT TGA GGG
GAC TTT CCC AGG C-3') that had been 5'-end labeled with [
-32P]ATP using T4 polynucleotide kinase (Promega,
Madison, WI) as described previously (32). Competition was
carried out using a 100-fold excess of the unlabeled oligonucleotide 10 min before adding the radiolabeled probe. Samples were then
electrophoresed in a 6% nondenaturing polyacrylamide gel.
Autoradiographs were obtained and radioactive signals were quantified
using a computing densitometer.
Statistical analysis. Data from two groups were compared by a two-tailed t-test, and those from more than two groups were compared by one-way ANOVA with a Bonferroni correction for multiple comparisons (17) using a commercially available statistical analysis program (SPSS, Arlington, VA). A difference is considered to be significant at P < 0.05.
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RESULTS |
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Role of mCD14 in LPS induction of TNF- and MnSOD.
To determine the role of CD14, we studied the effect of LPS on the
induction of TNF-
and MnSOD in peritoneal macrophages obtained from
CD14-KO and control (BALB/c) mice. We evaluated both the CD14-dependent
(1 and 10 ng/ml) and CD14-independent (1,000 ng/ml) concentrations of LPS.
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Role of TLR4 in LPS induction of TNF- and MnSOD.
To determine the role of TLR4 in LPS induction of TNF-
and MnSOD, we
first studied the natural mutant mice (C57BL/10ScCr) with the
Tlr4 gene deletion. As shown in Fig.
4, compared with control (C57BL/10SnJ)
macrophages, at 3 h after treatment, LPS at concentrations ranging
from 1 to 1,000 ng/ml failed to induce the production of TNF-
and
the enhancement of steady-state levels of TNF-
mRNA in C57BL/10ScCr
TLR4-deficient macrophages. Likewise, LPS at 1-1,000 ng/ml failed
to induce MnSOD mRNA in TLR4-deficient macrophages (Fig.
5).
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Role of TLR4 in LPS activation of NF-B.
A previous study by Ding et al. (7) revealed that LPS was
able to activate NF-
B in peritoneal macrophages derived from C3H/HeJ
mice, suggesting that defective induction of TNF-
by LPS in these
mice was not mediated by the defective activation of NF-
B. However,
recent evidence suggests that TLR4 mediates the activation of NF-
B
by LPS (13, 36). Therefore, we studied the role of TLR4 in
the LPS activation of NF-
B using both Tlr4 gene-deleted
(C57BL/10ScCr) and point-mutated (C3H/HeJ) mice.
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DISCUSSION |
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The results presented in this report demonstrate that mCD14 and
TLR4 mediate LPS induction of TNF- as well as MnSOD in murine peritoneal macrophages. mCD14 is required only at low concentrations of
LPS, e.g., 1 and 10 ng/ml. On the other hand, a functional TLR4 is
required for the effects of both the low and high concentrations of
LPS, even in the presence of an intact mCD14. These findings are
consistent with the current concept that mCD14 and TLR4 are part of a
receptor complex that mediates the divergent effects of LPS (14,
22-24, 35). At least one of the functions of mCD14 is to
facilitate the intracellular signal transduction via TLR4 at low
concentrations of LPS. On the other hand, at higher concentrations of
LPS, LPS signal transduction occurs in the presence of a functional TLR4 without the involvement of mCD14.
We have recently demonstrated that induction of TNF- and MnSOD by
LPS are mediated by distinct intracellular signal transduction pathways. While protein tyrosine kinase/Ras/Raf1/MEK/MAPK signaling pathway plays an important role in the induction of TNF-
by LPS, it
is not necessary for LPS induction of MnSOD (30, 32). In contrast, reactive oxygen species are involved in the LPS induction of
MnSOD but not TNF-
(34). Likewise, a mutant E. coli LPS, lacking myristoyl fatty acid at the 3'
R-3-hydroxymyristate position of the lipid A moiety
(nonmyristoyl LPS) (28), fails to stimulate TNF-
production by human monocytes; it retains the capacity to induce MnSOD
(30). On the other hand, induction of both TNF-
and
MnSOD by LPS requires activation of NF-
B (32). In the
current study, we demonstrated that LPS induction of TNF-
and MnSOD
is mediated by similar membrane receptor, mCD14, and signal transducer, TLR4. Exactly how the intracellular signal pathways diverge after LPS
activation of TLR4 leading to the induction of TNF-
and MnSOD is not
clear. Further studies are necessary to define the exact intracellular
signaling pathways for the induction of TNF-
and MnSOD and how these
signal pathways are activated by TLR4.
Our observation that C3H/HeJ macrophages with a point, missense
mutation of the Tlr4 gene are unresponsive to LPS in
inducing TNF- and MnSOD mRNAs differs from the report of Gibbs
et al. (8). These investigators reported that, at 24 h after treatment, LPS induced MnSOD mRNA in peritoneal macrophages but
not in lung endothelial cells derived from C3H/HeJ mice. The reason for
the discrepancy between our study and that of Gibbs et al. is not clear. Gibbs et al. (8) used resident peritoneal
macrophages treated with LPS for 24 h. Induction of MnSOD mRNA was
noted only at the LPS concentration of 500 ng/ml, but not at 10 ng/ml.
On the other hand, we used thioglycolate-elicited peritoneal
macrophages from C3H/HeJ mice. At 3 h after LPS treatment, we
consistently observed defective production of TNF-
as well as
defective induction of TNF-
and MnSOD mRNAs. The role of TLR4 in LPS
induction of TNF-
and MnSOD was further confirmed using peritoneal
macrophages derived from C57BL/10ScCr mice with a Tlr4 gene
deletion. In addition, we used a highly purified, protein-free LPS
preparation, while Gibbs et al. (8) used a commercially
available LPS preparation (from Sigma Chemical) without further purification.
In the current study, using macrophages from two different TLR4
mutants, C3H/HeJ and C56BL/10ScCr mice, we also demonstrated that a
functional TLR4 is necessary for the activation of NF-B by LPS. This
observation is in variance with the study of Ding et al.
(7) who reported that LPS was able to activate NF-
B in
C3H/HeJ macrophages, although to a lesser extent than in the control,
C3H/HeN macrophages. The experimental conditions used by Ding et al.
(7) were similar to those of ours. However, the LPS
preparations used in these two studies were different. We used a highly
purified, protein-free LPS preparation, while Ding et al.
(7) used a commercially available LPS preparation (from
List Biological Laboratories, Campbell, CA).
Many commercial preparations of LPS contain low concentrations of
highly bioactive contaminants previously described as "endotoxin protein" (13, 19, 29). These LPS preparations with
endotoxin protein contamination are capable of inducing IL-6 production from C3H/HeJ macrophages and NF-B activation in human embryonic kidney epithelial cell line 293 transfected with TLR2
(13). After repurification to eliminate endotoxin protein,
the noncontaminated LPS was no longer capable of activating C3H/HeJ
macrophages or the 293 cell line transfected with TLR2. However, it
retained the capacity to activate the 293 cell line transfected with
TLR4 (13). These results clearly indicate that the
contaminating endotoxin protein was responsible for the activation of
C3H/HeJ macrophages possibly through TLR2. They also support the
hypothesis that TLR4 (13, 14, 22-24), but not TLR2
(15, 37), is the sole LPS signal transducer in human and
murine macrophages.
Based on the foregoing discussion, we believe that endotoxin protein
contamination in the commercial LPS preparations used in the studies of
Gibbs et at. (8) and Ding et al. (7) can best
explain the observed discrepancies between their studies and our
current study. The biochemical nature of the contaminants in these
commercial LPS preparations responsible for the induction of MnSOD mRNA
and the activation of NF-B as observed by Gibbs et al.
(8) and Ding et al. (7), respectively, is not
clear. However, it is likely that bacterial lipoproteins could be at least in part responsible, since recent evidence suggests that these
lipoproteins possess potent bioactivity via the TLR2 signaling pathway
(1, 12, 16). Further studies are required to clarify this point.
In summary, using peritoneal macrophages from CD14-KO mice and mice
with two different natural mutations of the Tlr4 gene, we
demonstrated that, similar to TNF-, induction of MnSOD by LPS is
mediated by mCD14 and TLR4. Furthermore, TLR4 also mediates LPS
activation of NF-
B, which is necessary for the induction of TNF-
and MnSOD.
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ACKNOWLEDGEMENTS |
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This work was supported by the Medical Research Service, Office of Research and Development, Department of Veterans Affairs, and by National Institute of Allergy and Infectious Diseases Grant AI-23859.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M.-F. Tsan, Regional Office (10R), Office of Research Compliance and Assurance, VA Medical Center, 50 Irving St. NW, Washington, DC 20422 (E-mail: min-fu.tsan2{at}med.va.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 October 2000; accepted in final form 4 January 2001.
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