Induction of TNF-alpha and MnSOD by endotoxin: role of membrane CD14 and Toll-like receptor-4

Min-Fu Tsan1,2, Robert N. Clark1, Sanna M. Goyert3, and Julie E. White1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Endotoxin (LPS) is a potent inducer of tumor necrosis factor-alpha (TNF-alpha ) and manganese superoxide dismutase (MnSOD). Recent evidence suggests that LPS induction of TNF-alpha 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-alpha 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-alpha or MnSOD mRNA in CD14-KO macrophages. However, LPS at 1,000 ng/ml induced TNF-alpha and MnSOD mRNAs equally in macrophages from CD14-KO and control mice. LPS (1, 10, or 1,000 ng/ml) failed to induce TNF-alpha or MnSOD mRNA and failed to activate nuclear factor-kappa B in C3H/HeJ or C57BL/10ScCr macrophages. Measurements of TNF-alpha and MnSOD enzyme activity paralleled TNF-alpha and MnSOD mRNA levels. These data demonstrate that, like TNF-alpha , induction of MnSOD by LPS is mediated by mCD14 and TLR4 in murine macrophages.

tumor necrosis factor-alpha ; manganese superoxide dismutase; endotoxin resistance; nuclear factor-kappa B; peritoneal macrophage


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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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-alpha (TNF-alpha ) and interleukin-1beta (IL-1beta ) (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-alpha 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-alpha , 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-alpha by LPS. However, at higher concentrations, e.g., >100 ng/ml, LPS can induce TNF-alpha 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-alpha , induction of MnSOD by LPS is mediated by mCD14 and TLR4 in murine macrophages.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
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-alpha 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-alpha 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-alpha activity released into the media were determined by the anti-TNF-alpha antibody-inhibitable lysis of murine L929 fibroblasts as described previously (31).

Northern blot analysis of TNF-alpha 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-alpha (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA), huMnSOD, and mubeta -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-alpha and MnSOD (1- and 4-kb bands) mRNAs was corrected for beta -actin mRNA to minimize the effect of potential unequal loading of RNA. For the purpose of comparison, levels of TNF-alpha 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-kappa 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-kappa B (NF-kappa B) consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') that had been 5'-end labeled with [gamma -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.


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Role of mCD14 in LPS induction of TNF-alpha and MnSOD. To determine the role of CD14, we studied the effect of LPS on the induction of TNF-alpha 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.

The effect of LPS on the induction of TNF-alpha is summarized in Fig. 1. Figure 1A shows the results of TNF-alpha production, Fig. 1B is a representative Northern blot of TNF-alpha mRNA, and Fig. 1C shows the results of densitometric quantification. At 3 h after treatment, LPS, at concentrations ranging from 1 to 1,000 ng/ml, markedly stimulated the production of TNF-alpha and steady-state levels of TNF-alpha mRNA in control, BALB/c peritoneal macrophages. In contrast, induction of TNF-alpha production and mRNA in CD14-deficient macrophages was markedly impaired at LPS concentrations of 1 and 10 ng/ml, but not at 1,000 ng/ml.


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Fig. 1.   Role of membrane CD14 (mCD14) in endotoxin/lipopolysaccharide (LPS) induction of tumor necrosis factor-alpha (TNF-alpha ). Adherent macrophages from control (BALB/c) or CD14-knockout mice (CD14-KO) were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h. Cellular RNAs were extracted and aliquots (1 µg/lane) were subjected to Northern blot analysis for TNF-alpha mRNA. The activity of TNF-alpha in the media was also determined. A: TNF-alpha production; n = 5; *P < 0.005 vs. control. B: a representative Northern blot of TNF-alpha mRNA. Lanes 1 and 5, LPS 0 ng/ml; lanes 2 and 6, LPS 1 ng/ml; lanes 3 and 7, LPS 10 ng/ml; lanes 4 and 8, LPS 1,000 ng/ml. C: densitometric quantification of TNF-alpha mRNA; n = 4; **P < 0.01; §P < 0.05.

Similar results were obtained for LPS induction of MnSOD. As shown in Fig. 2 (A is a representative Northern blot of MnSOD mRNA and B shows densitometric quantification), at 3 h after treatment, LPS at concentrations ranging from 1 to 1,000 ng/ml markedly enhanced the steady-state levels of MnSOD mRNA in control peritoneal macrophages. In contrast, induction of MnSOD mRNA in CD14-deficient macrophages was markedly impaired at LPS concentrations of 1 and 10 ng/ml, but not at 1,000 ng/ml.


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Fig. 2.   Role of mCD14 in LPS induction of manganese superoxide dismutase (MnSOD) mRNA. Adherent macrophages from control (BALB/c) or CD14-knockout mice, were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h. Cellular RNAs were extracted and aliquots (1 µg/lane) were subjected to Northern blot analysis for MnSOD mRNA. A: a representative Northern Blot. Lanes 1 and 5, LPS 0 ng/ml; lanes 2 and 6, LPS 1 ng/ml; lanes 3 and 7, LPS 10 ng/ml; lanes 4 and 8, LPS 1,000 ng/ml. B: densitometric quantification of MnSOD mRNA; n = 4; *P < 0.05 vs. control.

Previous studies have demonstrated that there is a lag in LPS-induced increase in MnSOD enzyme activity. While LPS induction of MnSOD mRNA was evident at 3 h after treatment in human monocytes, the increase in MnSOD activity was not observed until 18-20 h after LPS treatment (30, 32). Therefore, in the current study, we measured SOD activity at 3 and 20 h after LPS treatment. Consistent with previous reports, no increase in MnSOD activity was noted at 3 h after LPS treatment (data not shown). However, at 20 h after treatment, LPS at concentrations ranging from 1 to 1,000 ng/ml markedly enhanced MnSOD activity in control peritoneal macrophages (Fig. 3A is a representative SOD activity gel and Fig. 3B shows densitometric quantification). In contrast, an increase in MnSOD activity in CD14-deficient macrophages was noted only at the LPS concentration of 1,000 ng/ml, but not at 1 or 10 ng/ml. In either control or CD14-deficient macrophages, LPS had no effect in the CuZnSOD activity.


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Fig. 3.   Role of mCD14 in LPS induction of MnSOD activity. Adherent macrophages from control (BALB/c) or CD14-knockout mice were treated with or without LPS (1, 10, or 1,000 ng/ml) for 20 h. The SOD activities of the cell extract (75 µg/lane for MnSOD and 15 µg/lane for CuZnSOD) were assayed using activity gel. Purified E. coli MnSOD and bovine erythrocyte CuZnSOD (25-800 mU) were used as standards. A: a representative SOD activity gel. Lanes 1-6, standards; lanes 7 and 11, LPS, 0 ng/ml; lanes 8 and 12, LPS 1 ng/ml; lanes 9 and 13, LPS 10 ng/ml; lanes 10 and 14, LPS 1,000 ng/ml. B: densitometric quantification of SOD activity; n = 3; *P < 0.05 vs. control.

Role of TLR4 in LPS induction of TNF-alpha and MnSOD. To determine the role of TLR4 in LPS induction of TNF-alpha 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-alpha and the enhancement of steady-state levels of TNF-alpha 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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Fig. 4.   Effect of Tlr4 gene deletion on LPS induction of TNF-alpha . Adherent macrophages from control (C57BL/10SnJ) or Tlr4 gene-deleted (C57BL/1-ScCr) mice were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h. Cellular RNAs were extracted and aliquots (1 µg/lane) were subjected to Northern blot analysis for TNF-alpha mRNA. The activity of TNF-alpha in the media was also determined. A: TNF-alpha production; n = 3; *P < 0.05 vs. control. B: a representative Northern blot of TNF-alpha mRNA. Lanes 1 and 5, LPS 0 ng/ml; lanes 2 and 6, LPS 1 ng/ml; lanes 3 and 7, LPS 10 ng/ml; lanes 4 and 8, LPS 1,000 ng/ml. C: densitometric quantification of TNF-alpha mRNA; n = 3; *P < 0.05 vs. control.



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Fig. 5.   Effect of Tlr4 gene deletion on LPS induction of MnSOD mRNA. Adherent macrophages from control (C57BL/10SnJ) and Toll-like receptor-4 (TLR4) gene-deleted (C57BL/10ScCr) mice were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h. Cellular RNAs were extracted, and aliquots (1 µg/lane) were subjected to Northern blot analysis for MnSOD mRNA. A: a representative Northern blot. Lanes 1 and 5, LPS 0 ng/ml; lanes 2 and 6, LPS 1 ng/ml; lanes 3 and 7, LPS 10 ng/ml; lanes 4 and 8, LPS 1,000 ng/ml. B: densitometric quantification of MnSOD mRNA; n = 3; *P < 0.05, **P < 0.01, §P < 0.02, vs. control.

The above results suggest that TLR4 is required for the induction of TNF-alpha and MnSOD by both the mCD14-dependent (1 and 10 ng/ml) and mCD14-independent (1,000 ng/ml) concentrations of LPS, consistent with the signal transducer role of TLR4 for LPS. A previous study by Gibbs et al. (8), however, demonstrated that LPS was able to induce MnSOD in peritoneal macrophages from LPS-resistant mutant (C3H/HeJ) mice. C3H/HeJ mice have been shown to carry a point, missense mutation of the Tlr4 gene at position 712 (proline right-arrow histidine) of the polypeptide chain (22, 24). This raises the possibility that this point mutation may result in a mutant TLR4 enabling the transduction of LPS signal leading to the induction of MnSOD, but not TNF-alpha . For this reason, we studied the induction of TNF-alpha and MnSOD by LPS in C3H/HeJ macrophages.

As shown in Fig. 6, compared with control (C3H/HeN) macrophages, at 3 h after treatment, LPS at concentrations ranging from 1 to 1,000 ng/ml failed to induce the production of TNF-alpha and the enhancement of steady-state levels of TNF-alpha mRNA in C3H/HeJ TLR4-mutant macrophages. Likewise, LPS at 1-1,000 ng/ml failed to induce MnSOD mRNA in C3H/HeJ TLR4-mutant macrophages (Fig. 7).


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Fig. 6.   Effect of Tlr4 gene point mutation on LPS induction of TNF-alpha . Adherent macrophages from control (C3H/HeN) or Tlr4 gene point-mutated (C3H/HeJ) mice were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h. Cellular RNAs were extracted and aliquots were subjected to Northern blot analysis for TNF-alpha mRNA. The activity of TNF-alpha in the media was also determined. A: TNF-alpha production; n = 3; *P < 0.05 vs. control. B: a representative Northern blot. Lanes 1 and 5, LPS 0 ng/ml; lanes 2 and 6, LPS 1 ng/ml; lanes 3 and 7, LPS 10 ng/ml; lanes 4 and 8, LPS 1,000 ng/ml. C: densitometric quantification of TNF-alpha mRNA; n = 3; *P < 0.05 vs. control.



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Fig. 7.   Effects of Tlr4 gene point mutation on LPS induction of MnSOD mRNA. Adherent macrophages from control (C3H/HeN) or Tlr4 gene point-mutated (C3H/HeJ) mice were treated with or without LPS (1, 10, or 1,000 ng/ml) for 3 h. Cellular RNAs were extracted, and aliquots (1 µg/lane) were subjected to Northern blot analysis for MnSOD mRNA. A: a representative Northern blot. Lanes 1 and 5, LPS 0 ng/ml; lanes 2 and 5, LPS 1 ng/ml; lanes 3 and 7, LPS 10 ng/ml; lanes 4 and 8, LPS 1,000 ng/ml. B: densitometric quantification of MnSOD mRNA; n = 3; *P < 0.05 vs. control.

Role of TLR4 in LPS activation of NF-kappa B. A previous study by Ding et al. (7) revealed that LPS was able to activate NF-kappa B in peritoneal macrophages derived from C3H/HeJ mice, suggesting that defective induction of TNF-alpha by LPS in these mice was not mediated by the defective activation of NF-kappa B. However, recent evidence suggests that TLR4 mediates the activation of NF-kappa B by LPS (13, 36). Therefore, we studied the role of TLR4 in the LPS activation of NF-kappa B using both Tlr4 gene-deleted (C57BL/10ScCr) and point-mutated (C3H/HeJ) mice.

As shown in Fig. 8, at 1 h after treatment, LPS (10 or 1,000 ng/ml) markedly enhanced nuclear translocation of NF-kappa B in control macrophages (Fig. 8, A and B, C57BL/10SnJ; C and D, C3H/HeN). The specificity of NF-kappa B activation was confirmed using competition by a 100-fold excess of unlabeled oligonucleotide (data not shown). In contrast, under similar conditions, LPS failed to activate NF-kappa B in TLR4-mutant macrophages (Fig. 8, A and B, C57BL/10ScCr; C and D, C3H/HeJ).


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Fig. 8.   Role of TLR4 in LPS activation of nuclear factor-kappa B (NF-kappa B). Adherent macrophages from control (C57BL/10SnJ and C3H/HeN), Tlr4 gene-deleted (C57BL/10ScCr), or Tlr4 gene point-mutated (C3H/HeN) mice were treated with or without LPS (10 and 1,000 ng/ml) for 1 h. Nuclear extracts were obtained and aliquots (1 µg/lane) were probed for NF-kappa B using electrophoresis mobility shift assay (EMSA). A: a representative EMSA for control C57BL/10SnJ (lanes 1-3) and C57BL/10ScCr (lanes 4-6) macrophages. Lanes 1 and 4, 0 ng/ml LPS; lanes 2 and 5, 10 ng/ml LPS; lanes 3 and 6, 1,000 ng/ml LPS. B: densitometric quantification of NF-kappa B; n = 4; *P < 0.005 and **P < 0.05 vs. control. C: a representative EMSA for control C3H/HeN (lanes 1-3) and C3H/HeJ (lanes 4-6) macrophages. Lanes 1 and 4, 0 ng/ml LPS; lanes 2 and 5, 10 ng/ml LPS; lanes 3 and 6, 1,000 ng/ml LPS. D: densitometric quantification of NF-kappa B; n = 4; §P < 0.01 vs. control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

The results presented in this report demonstrate that mCD14 and TLR4 mediate LPS induction of TNF-alpha 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-alpha 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-alpha 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-alpha (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-alpha production by human monocytes; it retains the capacity to induce MnSOD (30). On the other hand, induction of both TNF-alpha and MnSOD by LPS requires activation of NF-kappa B (32). In the current study, we demonstrated that LPS induction of TNF-alpha 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-alpha and MnSOD is not clear. Further studies are necessary to define the exact intracellular signaling pathways for the induction of TNF-alpha 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-alpha 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-alpha as well as defective induction of TNF-alpha and MnSOD mRNAs. The role of TLR4 in LPS induction of TNF-alpha 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-kappa 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-kappa 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-kappa 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-kappa 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-alpha , induction of MnSOD by LPS is mediated by mCD14 and TLR4. Furthermore, TLR4 also mediates LPS activation of NF-kappa B, which is necessary for the induction of TNF-alpha and MnSOD.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aliprantis, AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P, and Zychlinsky A. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285: 736-739, 1999[Abstract/Free Full Text].

2.   Asayama, K, Janco RL, and Burr IM. Selective induction of manganous superoxide dismutase in human monocytes. Am J Physiol Cell Physiol 249: C393-C397, 1985[Abstract].

3.   Beauchamp, C, and Fridovich I. Superoxide dismutase: improved assay and an assay application to acrylamide gels. Anal Biochem 44: 276-282, 1971[ISI][Medline].

4.   Beutler, B, and Kruys V. Lipopolysaccharide signal transduction, regulation of tumor necrosis factor biosynthesis, and signaling by tumor necrosis factor itself. J Cardiovasc Pharmacol 25: S1-S8, 1995[ISI][Medline].

5.   Beutler, B, Milsark IW, and Cerami A. Passive immunization against cachetin/tumor necrosis factor (TNF) protects mice from the lethal effect of endotoxin. Science 229: 869-871, 1985[ISI][Medline].

6.   Chomczynski, P, and Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

7.   Ding, A, Hwang S, Lander HM, and Xie QW. Macrophages derived from C3H/HeJ (LPSd) mice respond to bacterial lipopolysaccharide by activating NF-kappa B. J Leukoc Biol 57: 174-179, 1995[Abstract].

8.   Gibbs, LS, Del Vecchio P, and Shaffer JB. Mn and Cu/Zn SOD expression in cells from LPS-sensitive and LPS-resistant mice. Free Radic Biol Med 12: 107-111, 1992[ISI][Medline].

9.   Haziot, A, Chen S, Ferrero E, Low MG, Silber R, and Goyert SM. The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol 141: 547-552, 1988[Abstract/Free Full Text].

10.   Haziot, A, Ferrero E, Kontgen F, Hijiya N, Yamamoto S, Silver J, Stewart CL, and Goyert SM. Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice. Immunity 4: 407-414, 1996[ISI][Medline].

11.   Haziot, A, Lin XY, Zhang F, and Goyert SM. The induction of acute phase proteins by lipopolysaccharide uses a novel pathway that is CD14-independent. J Immunol 160: 2570-2572, 1998[Abstract/Free Full Text].

12.   Hirschfeld, M, Kirchning CJ, Schwandner R, Wesche H, Weis JH, Wooten RM, and Weis JJ. Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by Toll-like receptor 2. J Immunol 163: 2382-2386, 1999[Abstract/Free Full Text].

13.   Hirschfeld, M, Ma Y, Weis JH, Vogel SN, and Weis JJ. Repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J Immunol 165: 618-622, 2000[Abstract/Free Full Text].

14.   Hoshino, K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, and Akira S. Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162: 3749-3752, 1999[Abstract/Free Full Text].

15.   Kirschning, CJ, Wesche H, Ayers TM, and Rothe M. Human Toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide. J Exp Med 188: 2091-2097, 1998[Abstract/Free Full Text].

16.   Lien, E, Sellati TJ, Yoshmura A, Flo TH, Rawadi G, Finberg RW, Carroll JD, Espevik T, Ingalls RR, Radolf JD, and Golenbock DT. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 274: 33419-33425, 1999[Abstract/Free Full Text].

17.   Mathews, DE, and Farewell VT. Data analysis. In: Using and Understanding Medical Statistics (2nd ed.), edited by Mathew DE, and Farewell VT.. Basel: Karger, 1988, p. 178-181.

18.   McCord, JM, and Fridovich I. Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 244: 6049-6055, 1969[Abstract/Free Full Text].

19.   Morrison, DC, Betz SJ, and Jacobs DM. Isolation of a lipid A bound polypeptide responsible for "LPS-initiated" mitogenesis of C3H/HeJ spleen cells. J Exp Med 144: 840-846, 1976[Abstract/Free Full Text].

20.   Morrison, DC, and Ryan JL. Bacterial endotoxins and host immune responses. Adv Immunol 28: 293-450, 1979[Medline].

21.   Osnes, LTN, Foss KB, Joo GB, Okkenhaug C, Wastvik A-B, Ovstebo R, and Kierulf P. Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kappa B/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and tumor necrosis factor-alpha (TNFalpha ) in human monocytes. Thromb Haemost 76: 970-976, 1996[ISI][Medline].

22.   Poltorak, A, He X, Sminova I, Lui MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, and Beutler B. Defective signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088, 1998[Abstract/Free Full Text].

23.   Poltorak, A, Sminova I, He X, Lui MY, Van Huffel C, McNally O, Birdwell D, Alejos E, Silva M, Du X, Thompson P, Chen EK, Ledesma J, Roe B, Clifton S, Vogel SN, and Beutler B. Genetic and physical mapping of the Lps locus: identification of the Toll-4 receptor as a candidate gene in the critical region. Blood Cells Mol Dis 24: 340-355, 1998[ISI][Medline].

24.   Qureshi, ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, and Malo D. Endotoxin-tolerant mice have mutations in Toll-like receptor-4 (Tlr4). J Exp Med 189: 615-625, 1999[Abstract/Free Full Text].

25.   Rietschel, TE, and Brade H. Bacterial endotoxins. Sci Am 267: 54-61, 1992[ISI][Medline].

26.   Shiki, Y, Meyerick BO, Brigham KL, and Burr IM. Endotoxin increases superoxide dismutase in cultured bovine pulmonary endothelial cells. Am J Physiol Cell Physiol 252: C436-C440, 1987[Abstract/Free Full Text].

27.   Smith, PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC. Measurement of protein using bicinchoninic acids. Anal Biochem 150: 76-85, 1985[ISI][Medline].

28.   Somerville, JE, Jr, Cassiano L, Bainbridge B, Cunningham MD, and Darveau RP. A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide. J Clin Invest 97: 359-365, 1996[Abstract/Free Full Text].

29.   Sultzer, BM, and Goodman GW. Endotoxin protein: a B-cell mitogen and polyclonal activator of C3H/HeJ lymphocytes. J Exp Med 144: 821-827, 1976[Abstract].

30.   Tian, L, White JE, Lin HY, Haran VS, Sacco J, Chikkappa G, Davis FB, Davis PJ, and Tsan MF. Induction of MnSOD in human monocytes without inflammatory cytokine production by a mutant endotoxin. Am J Physiol Cell Physiol 275: C740-C747, 1998[Abstract].

31.   Tsan, MF, Lawrence D, and White JE. Erythrocyte insufflation-induced protection against oxygen toxicity: role of cytokines. J Appl Physiol 71: 1751-1757, 1991[Abstract/Free Full Text].

32.   White, JE, Lin HY, Davis FB, Davis PJ, and Tsan MF. Differential induction of tumor necrosis factor alpha  and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase and nuclear factor kappa B. J Cell Physiol 182: 381-389, 2000[ISI][Medline].

33.   White, JE, and Tsan MF. Induction of pulmonary Mn superoxide dismutase mRNA by interleukin-1. Am J Physiol Lung Cell Mol Physiol 266: L664-L671, 1994[Abstract/Free Full Text].

34.   White, JE, and Tsan MF. Differential induction of TNFalpha and MnSOD by endotoxin: role of reactive oxygen species and NADPH oxidase. Am J Respir Cell Mol Biol 24: 164-169, 2001[Abstract/Free Full Text].

35.   Wright, SD, Ramos RA, Tobias PS, Ulevich RJ, and Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249: 1431-1433, 1990[ISI][Medline].

36.   Yang, H, Young DW, Gusovsky F, and Chow JC. Cellular events mediated by lipopolysaccharide-stimulated Toll-like receptor 4. MD-2 is required for activation of mitogen-activated protein kinases and ELK-1. J Biol Chem 275: 20861-20866, 2000[Abstract/Free Full Text].

37.   Yang, RB, Mark MR, Gray A, Huang A, Xie MH, Zhang M, Goddard A, Wood WI, Gurney AL, and Godowski PJ. Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature 395: 284-288, 1998[ISI][Medline].


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