SEARCH

SEARCH BY CITATION

Keywords:

  • MnTBAP;
  • lipopolysaccharide;
  • ROS;
  • TNF-α;
  • p38;
  • SAPK/JNK

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Antioxidants are able to inhibit inflammatory gene expression in response to lipopolysaccharide via down-regulating generation of intracellular reactive oxygen species (ROS) as second messengers. The effect of manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a synthetic metalloporphyrin with antioxidant activity, on tumor necrosis factor (TNF)-α production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells was examined. MnTBAP prevented the generation of intracellular ROS in lipopolysaccharide-stimulated RAW 264.7 cells and further inhibited lipopolysaccharide-induced TNF-α production. MnTBAP exclusively prevented the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK/JNK) whereas it did not affect the phosphorylation and activation of nuclear factor-κB and extracellular signal regulated kinase 1/2. MnTBAP was suggested to inhibit lipopolysaccharide-induced TNF-α production by the prevention of intracellular ROS generation and subsequent inactivation of p38 MAPK and SAPK/JNK.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion (O2), and hydroxyl radical (HO), are normal metabolic byproducts and intermediates generated in many physiological processes in macrophages (Forman & Torres, 2001, 2002). Intracellular ROS function as second messengers to activate multiple signal pathways including a series of mitogen-activated protein kinase (MAPK), c-src, Ras and Akt in macrophages (Finkel, 2000; Thannickal & Fanburg, 2000). The activation of these signal cascades leads to the induction of transcription factors, such as nuclear factor (NF)-κB, activator protein (AP)-1 and activating transcription factor (ATF) (Sen & Packer, 1996; Hsu et al., 2000; Turpaev, 2002). These transcription factors control the inducible expression of genes whose products are part of the inflammatory response. Therefore, the generation of intracellular ROS may participate in inflammatory responses of macrophages (Morikawa et al., 2004).

Lipopolysaccharide, a constituent of the cell wall of gram-negative bacteria, plays an essential role in the pathogenesis of septic shock by producing inflammatory cytokines, such as tumor necrosis factor (TNF)-α. An increasing number of reports state that antioxidants, such as ascorbic acid (AA) (Victor et al., 2000), vitamin E (Pathania et al., 1999) and N-acetylcysteine (Victor et al., 2003), are potent inhibitors of inflammatory gene expression in response to lipopolysaccharide in vivo and in vitro, and suggest the involvement of intracellular ROS in the activation of signaling molecules for TNF-α production, such as NF-κB (Flohe et al., 1997; Bowie & O'Neill, 2000) and a series of MAPKs including extracellular signal regulated kinase (ERK) 1/2, p38 and stress activated protein kinase (SAPK/JNK; Torres, 2003; Torres & Forman, 2003). Therefore, modulation of the oxidation–reduction reactions may be useful for control of lipopolysaccharide-mediated inflammation. Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) is a cell-permeable superoxide dismutase mimetic and a potent inhibitor of oxidation (Faulkner et al., 1994). It was of interest to study if and how this antioxidant prevented lipopolysaccharide-mediated inflammation. In the present study, we investigated the effect of MnTBAP on TNF-α production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Here, we report that MnTBAP inhibits lipopolysaccharide-induced TNF-α production as a result of preventing intracellular ROS generation.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Reagents

Lipopolysaccharide from Escherichia coli O55:B5 was purchased from Sigma Chemicals (St Louis, MO). MnTBAP was obtained from Alexis Biochemicals (San Diego, CA). An antibody to the phosphorylated form of p65 NF-κB was purchased from Cell Signaling Technology (Beverly, MA), and antibodies to ERK 1/2, p38, SAPK/JNK and their phosphorylated forms were obtained from Promega, (Madison, WI). Antibodies to cyclooxygenase-2 (COX-2) and TNF-α were obtained from Transduction Laboratory (Lexington, KY) and Genzyme (Minneapolis, MN), respectively. SB 203580 was purchased from Calbiochem (San Diego, CA).

Cell culture

The murine macrophage cell line RAW 264.7 was obtained from the Health Science Resource Bank (Tokyo, Japan), and maintained in RPMI 1640 medium (Sigma) containing 5% heat-inactivated fetal calf serum (Life Technologies, Grand Island, NY) and antibiotics at 37°C with 5% CO2. The cells were washed gently with saline, removed from the flasks and used for experiments.

Cell viability

RAW 264.7 cells were treated with various concentrations of MnTBAP in a 96-well plate for 24 h. Cell viability was determined by MTT activity using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Chemicon, Temecula, CA) (Hanelt et al., 1994). The OD at 540 nm was determined with a microplate reader. All measurements were corrected for the interference of MnTBAP at this wavelength.

Determination of intracellular ROS generation

Intracellular ROS generation was determined by oxidation of the fluorescent MitoSOX red reagent (Molecular Probes, Invitrogen, Carlsbad, CA) for highly selective detection of mitochondrial superoxide. Briefly, RAW 264.7 cells were treated with 0.4 mM MnTBAP for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for another 30 min. The culture medium was removed and the cells were treated with 5 μM MitoSOX red reagent in the dark for 15 min. The fluorescence intensity was determined by a laser flow cytometer (Becton Dickinson, Palo Alto, CA).

Determination of TNF-α and prostaglandin E2 (PGE2) production

The supernatant was obtained from the culture of RAW 264.7 cells stimulated with 100 ng mL−1 lipopolysaccharide for 1 h (TNF-α) and for 24 h (PGE2) with or without 30 min pretreatment of MnTBAP. The concentrations of TNF-α and PGE2 were determined by an enzyme-linked immunosorbent assay (ELISA) kit from Biosource (Camarillo, CA) and R&D systems (Minneapolis, MN), respectively. For each group, the mean of three wells with SD is expressed.

Immunoblotting

Immunoblotting was carried out as described previously (Morikawa et al., 2000). Briefly, RAW 264.7 cells were pretreated with MnTBAP for 30 min, and incubated with lipopolysaccharide (100 ng mL−1) for the indicated time. The cell lysates were extracted by a lysis buffer. The protein concentration of the samples was determined by the BCA protein assay reagent (Pierce, Rockford, IL). Each cell lysate containing 20 μg of protein was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% gradient gel under reducing conditions and transferred to the membranes. The membranes were blocked with 5% skimmed milk and treated with appropriately diluted antibodies to various signal molecules overnight. The immune complexes were detected with a 1 : 2000 dilution of horseradish peroxidase-conjugated secondary antibody and the bands were visualized with a chemiluminescent reagent (Pierce). Antibodies to COX-2 and TNF-α were also used for immunoblotting analysis.

Luciferase reporter gene assay for NF-κB, AP-1 and CREB activation

RAW 264.7 cells (3 × 105 mL−1) were cultured in a 24-well plate for 24 h. The cells were transfected with 0.3 μg mL−1 of the pNFκB-TA, pAP1(1)-TA, pCRE-TA luciferase reporter gene (BD Biosciences Clontech, Palo Alto, CA) or 0.1 μg mL−1 of control β-galactosidase expression plasmid (pCMV-β-gal) using lipofectamine 2000 transfection reagent (Gibco-BRL, Gaithersburg, MD). The transfection medium was then replaced with the growth medium containing 10% fetal calf serum and the cells were maintained for 24 h. The cells were treated with MnTBAP for 30 min and further stimulated with lipopolysaccharide (100 ng mL−1) for 6 h to determine the activity of NF-κB. The activity of AP-1 and cAMP response element binding protein (CREB) was determined 3 h after lipopolysaccharide treatment. The cells were lysed and the relative luciferase activity was calculated by the β-galactosidase activity as a normalizing factor. The relative fold induction of the luciferase activity was expressed by the ratio in comparison with control cells.

Statistical analysis

Statistical analysis was performed using Student's t-test, with P<0.05 considered to indicate a significant difference. Experimental results are expressed as the mean value of triplicates±SD in at least three independent experiments.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

MnTBAP exhibits an antioxidant action on intracellular ROS generation in lipopolysaccharide-stimulated RAW 264.7 cells

We examined the effect of MnTBAP on intracellular ROS generation in lipopolysaccharide-stimulated RAW 264.7 cells using the fluorescent MitoSOX red reagent. RAW 264.7 cells were pretreated with or without 0.4 mM MnTBAP for 30 min and then stimulated with lipopolysaccharide (100 ng mL−1) for 30 min. MnTBAP pretreatment significantly reduced the fluorescent intensity of lipopolysaccharide-stimulated RAW 264.7 cells (Fig. 1), indicating scavenging of the intracellular ROS generated.

image

Figure 1.  Effect of MnTBAP on intracellular ROS generation in lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with 0.4 mM MnTBAP for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for 30 min. The cells were treated with 5 μM MitoSOX and the fluorescence intensity was determined by a laser flow cytometer. One typical experiment of three is shown.

Download figure to PowerPoint

MnTBAP inhibits lipopolysaccharide-induced TNF-α production

Having confirmed that MnTBAP exhibited an antioxidant action in lipopolysaccharide-induced ROS generation, we examined the effect of MnTBAP on TNF-α production in lipopolysaccharide-stimulated RAW 264.7 cells in order to clarify the involvement of intracellular ROS in the production (Fig. 2). First, cells were pretreated with various concentrations of MnTBAP for 30 min and then stimulated with lipopolysaccharide (100 ng mL−1) for 1 h. The experimental result is shown in Fig. 2a. Lipopolysaccharide induced a large amount of TNF-α production whereas pretreatment with MnTBAP at 0.4 and 0.2 mM significantly inhibited it (P<0.05). In addition, MnTBAP at the concentrations tested exhibited no cytotoxic action against RAW 264.7 cells based on an MTT activity assay (data not shown), excluding the possibility that the inhibition was due to nonspecific cytotoxicity of MnTBAP. Furthermore, MnTBAP inhibited TNF-α production in RAW 264.7 cells treated with phorbol 12-myristate 13-acetate (PMA) as well as lipopolysaccharide (c. 50% inhibition).

image

Figure 2.  Effect of MnTBAP on TNF-α production in lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with various concentrations (mM) of MnTBAP for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for 60 min. Release of TNF-α was determined by ELISA (a). The experimental data are expressed as the mean±SD of three independent experiments. Expression of intracellular TNF-α was determined by immunoblotting with anti-TNF-α antibody (b). Antiactin antibody was used as a control. One typical experiment of three is shown.

Download figure to PowerPoint

Second, the effect of MnTBAP on the expression of intracellular TNF-α in lipopolysaccharide-stimulated RAW 264.7 cells was examined by immunoblotting (Fig. 2b). Lipopolysaccharide definitely induced expression of intracellular TNF-α whereas pretreatment with MnTBAP at 0.4 mM significantly inhibited it. This suggested that the inhibition of TNF-α production was dependent on transcriptional and translational levels, but not on disturbance to the secretion.

MnTBAP does not inhibit the activation of p65 NF-κB in lipopolysaccharide-stimulated RAW264.7 cells

It is well known that NF-κB is a pivotal transcription factor for TNF-α production (Collart et al., 1990; Shakhov et al., 1990). Therefore, the effect of MnTBAP on lipopolysaccharide-induced NF-κB activation was examined (Fig. 3). The expression and phosphorylation of p65 NF-κB was analysed by immunoblotting using an antibody to the phosphorylated form of p65 NF-κB (Fig. 3a). RAW 264.7 cells were treated in the presence or absence of 0.4 mM MnTBAP for 30 min, followed by stimulation with lipopolysaccharide (100 ng mL−1) for 30 or 60 min. Whereas the cell lysate from unstimulated macrophages showed a faint band of phosphorylated p65 NF-κB (pp65), lipopolysaccharide markedly enhanced the phosphorylation of p65 30 min after the stimulation. MnTBAP did not inhibit the phosphorylation of p65 30 min after lipopolysaccharide stimulation. Further, no inhibition of the p65 phosphorylation was seen 60 min after lipopolysaccharide stimulation.

image

Figure 3.  Effect of MnTBAP on NF-κB activation in lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated concentrations (mM) of MnTBAP for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for 30 and 60 min. (a) Phosphorylation of p65 NF-κB was analysed by immunoblotting using an antibody to phosphorylated p65 (pp65). Antiactin antibody was used as a control. (b) NF-κB activation was determined by the luciferase reporter gene assay. Experimental data are expressed as the mean fold increase from three independent experiments.

Download figure to PowerPoint

We next wished to confirm no inhibition of NF-κB activation by MnTBAP via a luciferase reporter gene assay (Fig. 3b). RAW 264.7 cells were pretreated with MnTBAP at 0.1, 0.2 or 0.4 mM for 30 min and further stimulated with lipopolysaccharide (100 ng mL−1) for 6 h. MnTBAP pretreatment produced no inhibitory action on lipopolysaccharide-induced NF-κB activation in the reporter gene assay, although lipopolysaccharide resulted in an approximately three-fold increase in luciferase activity. The findings with the reporter gene assay were consistent with the immunoblotting analysis.

Expression of COX-2 is known to be exclusively dependent on NF-κB signaling in lipopolysaccharide-stimulated macrophages (D'Acquisto et al., 1997; Inoue & Tanabe, 1998). Therefore, we examined the effect of MnTBAP on the production of PGE2 and the expression of COX-2 in lipopolysaccharide-stimulated RAW 264.7 cells (Fig. 4). COX-2-dependent PGE2 production was not inhibited in MnTBAP-pretreated cells in response to lipopolysaccharide (Fig. 4a). Moreover, there was no significant difference in lipopolysaccharide-induced COX-2 expression between untreated and MnTBAP-pretreated cells in response to lipopolysaccharide (Fig. 4b). These findings excluded the involvement of NF-κB signaling in the inhibition by MnTBAP of lipopolysaccharide-induced TNF-α production.

image

Figure 4.  Effect of MnTBAP on PGE2 production and COX-2 induction in lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated concentrations (mM) of MnTBAP for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for 24 h. (a) Production of PGE2 was determined by ELISA using the supernatant. The experimental data are expressed as the mean±SD from three independent experiments. (b) Induction of COX-2 was analysed by immunoblotting using anti-COX-2 antibody. One typical experiment of three is shown.

Download figure to PowerPoint

p38 MAPK and SAPK/JNK are involved in inhibition by MnTBAP of lipopolysaccharide-induced TNF-α production

Lipopolysaccharide-induced TNF-α production is also regulated by a series of MAPKs, such as ERK 1/2 (Geppert et al., 1994; Zhu et al., 2000), p38 (Zhu et al., 2000) and SAPK/JNK (Swantek et al., 1997; Zhu et al., 2000). We examined the effect of MnTBAP on activation of a series of MAPKs in lipopolysaccharide-stimulated RAW 364.7 cells (Fig. 5). RAW 264.7 cells were pretreated with MnTBAP at 0.4 mM for 30 min and then stimulated with 100 ng mL−1 lipopolysaccharide for 30 and 60 min. The activation of MAPKs was analysed by immunoblotting with antibodies to their phosphorylated forms. Pretreatment of MnTBAP significantly inhibited the phosphorylation of p38 and SAPK/JNK. However, it did not inhibit the phosphorylation of ERK 1/2 (data not shown).

image

Figure 5.  Effect of MnTBAP on activation of p38 and SAPK/JNK in lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated concentrations (mM) of MnTBAP for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for 30 or 60 min. Activation was determined by phosphorylation of p38 and SAPK/JNK and it was detected by immunoblotting with antibodies to phosphorylated forms of p38 (pp38) and SAPK/JNK (pSAPK). Antiactin antibody was used as a control. One typical experiment of three is shown.

Download figure to PowerPoint

MnTBAP inhibits activation of AP-1 and CREB transcription factors in response to lipopolysaccharide

Based on the fact that p38 and SAPK/JNK activate transcription factors, such as ATF, CREB and AP-1 (Jiang et al., 1996; Whitmarsh & Davis, 1996; Deak et al., 1998), we studied the activation of AP-1 and CREB using the luciferase reporter gene assay (Fig. 6). RAW 264.7 cells transfected with luciferase reporter genes were pretreated with or without MnTBAP for 30 min and then stimulated with lipopolysaccharide (100 ng mL−1) for 3 h. The experimental result is shown in Fig. 6. Lipopolysaccharide resulted in an approximately four-fold increase in the luciferase activities of AP-1 and CRE compared with those of unstimulated cells. By contrast, pretreatment with MnTBAP abolished the increase of their luciferase activities in response to lipopolysaccharide, suggesting that MnTBAP inhibited the activation of AP-1 and CREB transcription factors in lipopolysaccharide-stimulated RAW 264.7 cells.

image

Figure 6.  Effect of MnTBAP on activation of AP-1 and CREB transcription factors in lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were transfected with pAP1-TA-luc, pCRE-TA-luc and control β-galactosidase expression plasmid. The transfected cells were pretreated with the indicated concentrations (mM) of MnTBAP for 30 min and stimulated with 100 ng mL−1 for 3 h. The relative luciferase activity in cell lysates was determined as β-galactosidase activity using a microplate reader. The experimental data are expressed as the mean±SD from three independent experiments.

Download figure to PowerPoint

SB203580 inhibits lipopolysaccharide-induced TNF-α production

The effect of SB203580, an inhibitor of p38 MAPK, on lipopolysaccharide-induced TNF-α production was studied (Fig. 7). RAW 264.7 cells were pretreated with 30 mM SB203580 for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for 1 h. SB203580 significantly inhibited TNF-α production in lipopolysaccharide-stimulated RAW 264.7 cells (P<0.05). This was consistent with the finding that MnTBAP inhibited lipopolysaccharide-induced TNF-α production by preventing activation of p38 and SAPK/JNK signaling.

image

Figure 7.  Effect of SB203580 as a p38 inhibitor on TNF-α production in lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with 30 mM SB203580 or 0.4 mM MnTBAP for 30 min and stimulated with 100 ng mL−1 lipopolysaccharide for 60 min. The experimental data are expressed as the mean±SD from three independent experiments.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The present study demonstrates that MnTBAP inhibits TNF-α production in lipopolysaccharide-stimulated RAW 264.7 cells by preventing intracellular ROS generation. Several lines of evidence suggest that MnTBAP may inhibit lipopolysaccharide-induced TNF-α production by preventing activation of p38 and SAPK/JNK signaling: first, MnTBAP attenuates lipopolysaccharide-induced intracellular ROS generation; second, MnTBAP inhibits lipopolysaccharide-induced TNF-α production; third, MnTBAP exclusively inhibits the activation of oxidative stress-dependent p38 and SAPK but did not affect the activation of NF-κB and ERK1/2; finally, a p38 inhibitor prevents lipopolysaccharide-induced TNF-α production. Therefore, MnTBAP is suggested to inhibit TNF-α production by scavenging intracellular ROS and subsequent inactivation of p38 and SAPK.

The promoter region of the TNF-α gene is reported to have the binding site of NF-κB, AP-1, ATF and CREB (Yao et al., 1997; Becker et al., 1999). NF-κB and CREB/AP-1 are important regulators of inducible TNF promoter activity (Becker et al., 1999). Maximal lipopolysaccharide induction of the TNF-α promoter is mediated by a cooperative interaction between c-Jun complexes and NF-κB (Yao et al., 1997). These transcription factors can be activated by p38 and SAPK/JNK (Jiang et al., 1996; Whitmarsh & Davis, 1996; Deak et al., 1998). Lipopolysaccharide causes generation of intracellular ROS, which then ROS activate p38 and SAPK/JNK (Guha et al., 2000; Ueda et al., 2002; Torres & Forman, 2003). Activated p38 and SAPK/JNK may augment the expression of CREB, AP-1 and ATF, and these transcription factors further augment TNF-α production in response to lipopolysaccharide. Therefore, the scavenging of intracellular ROS by MnTBAP results in the inhibition of lipopolysaccharide-induced TNF-α production via inactivation of p38 and SAPK. This is a putative mechanism for the inhibitory action of MnTBAP on lipopolysaccharide-induced TNF-α production. In fact, an ROS-dependent TRAF6–ASK1–p38 axis (Matsuzawa et al., 2005) is reported to be crucial for the production of TNF-α following lipopolysaccharide challenge in RAW 264.7 macrophages.

MnTBAP did not inhibit lipopolysaccharide-induced NF-κB activation, suggesting that intracellular ROS generation is not involved in lipopolysaccharide-induced NF-κB activation. This is consistent with the report of Chandel et al. (2000) that lipopolysaccharide activates NF-κB and TNF-α gene transcription through an ROS-independent mechanism. It was also confirmed by our finding that MnTBAP does not inhibit lipopolysaccharide-induced COX-2 expression, which is exclusively dependent on NF-κB signaling. By contrast, Ndengele et al. (2005) have reported that the removal of superoxide by Mn-containing superoxide dismutase mimetic inhibits lipopolysaccharide-induced production of TNF-α in alveolar macrophages through prevention of NF-κB activation. The reason for this difference remains unclear, although it might be due to the cell type. The relationship between intracellular ROS generation and NF-κB activation must await further characterization.

MnTBAP is a metalloporphyrin and cell-permeable superoxide dismutase (SOD) mimetic (Day et al., 1995). It is reported to scavenge intracellular ROS, such as superoxide and peroxynitrite, but not nitric oxide (Szabo et al., 1996). Furthermore, the potencies of MnTBAP seem to be related not only to SOD activities but also to the ability to act as electron acceptors (Day et al., 1999), suggesting that MnTBAP possesses a broad array of antioxidant activities. In the present study we demonstrate that MnTBAP prevents the generation of intracellular ROS in lipopolysaccharide-stimulated RAW 264.7 cells and exhibits an inhibitory action on release of proinflammatory mediators. Furthermore, MnTBAP inhibited TNF-α production induced by PMA as well as lipopolysaccharide. Therefore, MnTBAP might be a useful agent in preventing cell damage and tissue injury not only caused directly by ROS generation but also by subsequent release of ROS-dependent proinflammatory mediators.

MnTBAP is known to inhibit lipopolysaccharide-mediated inflammatory responses (Szabo et al., 1996; Zingarelli et al., 1997). MnTBAP causes a dose-dependent inhibition of NO production in cells stimulated with lipopolysaccharide (Szabo et al., 1996). It also inhibits the inflammatory activation of human blood vessels by stimulation of low-level endotoxin (Zingarelli et al., 1997). The present study demonstrates that MnTBAP inhibits TNF-α production in lipopolysaccharide-stimulated macrophages. On the basis of these finding, we investigated the in vivo effect of MnTBAP on lethal endotoxic shock induced in mice by lipopolysaccharide and d-galactosamine. Pretreatment with MnTBAP did not protect mice from the lethal action of d-galactosamine and lipopolysaccharide (data not shown). Zingarelli et al. (1997) also reported that MnTBAP did not alter survival rates in mice challenged with a high dose of endotoxin. The anti-inflammatory action of MnTBAP may not be powerful enough to prevent in vivo lethal endotoxic shock. Prevention of lethal endotoxin shock might require inactivation of NF-κB signaling as well as p38 and SAPK/JNK signaling, thereby leading to stronger inhibition of proinflammatory mediators.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported in part by a Grant-in-Aid for Scientific Research (Kakenhi) from the Ministry of Education, Culture, Science, Sports, Science and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Becker C, Barbulescu K, Wirtz S, Meyer zum Buschenfelde KH, Pettersson S & Neurath MF (1999) Constitutive and inducible in vivo protein-DNA interactions at the tumor necrosis factor-alpha promoter in primary human T lymphocytes. Gene Expr 8: 115127.
  • Bowie A & O'Neill LA (2000) Oxidative stress and nuclear factor-κB activation. Biochem Pharmacol 59: 1323.
  • Chandel NS, Trzyna WC, McClintock DS & Schumacker PT (2000) Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin. J Immunol 165: 10131021.
  • Collart MA, Baeuerle P & Vassalli P (1990) Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Mol Cell Biol 10: 14981506.
  • D'Acquisto F, Iuvone T, Rombola L, Sautebin L, Di Rosa M & Carnuccio R (1997) Involvement of NF-kappaB in the regulation of cyclooxygenase-2 protein expression in lipopolysaccharide-stimulated J774 macrophages. FEBS Lett 418: 175178.
  • Day BJ, Shawen S, Liochev SI & Crapo JD (1995) A metalloporphyrin superoxide dismutase mimetic protects against paraquat-induced endothelial cell injury, in vitro. J Pharmacol Exp Ther 275: 12271232.
  • Day BJ, Batinic-Haberle I & Crapo JD (1999) Metalloporphyrins are potent inhibitors of lipid peroxidation. Free Radic Biol Med 26: 730736.
  • Deak M, Clifton AD, Lucocq LM & Alessi DR (1998) Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J 17: 44264441.
  • Faulkner KM, Liochev SI & Fridovich I (1994) Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo. J Biol Chem 269: 2347123476.
  • Finkel T (2000) Redox-dependent signal transduction. FEBS Lett 476: 5254.
  • Flohe L, Brigelius-Flohe R, Saliou C, Traber MG & Packer L (1997) Redox regulation of NF-κB activation. Free Radic Biol Med 22: 11151126.
  • Forman HJ & Torres M (2001) Redox signaling in macrophages. Mol Aspects Med 22: 189216.
  • Forman HJ & Torres M (2002) Reactive oxygen species and cell signaling. Am J Respir Crit Care Med 166: S4S8.
  • Geppert TD, Whitehurst CE, Thompson P & Beutler B (1994) Lipopolysaccharide signals activation of tumor necrosis factor biosynthesis through the ras/raf-1/MEK/MAPK pathway. Mol Med 1: 93103.
  • Guha M, Bai W, Nadler JL & Natarajan R (2000) Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways. J Biol Chem 275: 1772817739.
  • Hanelt M, Gareis M & Kollarczik B (1994) Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay. Mycopathologia 128: 167174.
  • Hsu TC, Young MR, Cmarik J & Colburn NH (2000) Activator protein 1 (AP-1)- and nuclear factor kappaB (NF-kappaB)-dependent transcriptional events in carcinogenesis. Free Radic Biol Med 28: 13381348.
  • Inoue H & Tanabe T (1998) Transcriptional role of the nuclear factor kappa B site in the induction by lipopolysaccharide and suppression by dexamethasone of cyclooxygenase-2 in U937 cells. Biochem Biophys Res Commun 244: 143148.
  • Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S & Han J (1996) Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta). J Biol Chem 271: 1792017926.
  • Matsuzawa A, Saegusa K, Noguchi T, Sadamitsu C, Nishitoh H, Nagai S, Koyasu S, Matsumoto K, Takeda K & Ichijo H (2005) ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat Immunol 6: 587592.
  • Morikawa A, Koide N, Kato Y, Sugiyama T, Chakravortty D, Yoshida T & Yokochi T (2000) Augmentation of nitric oxide production by gamma interferon in a mouse vascular endothelial cell line and its modulation by tumor necrosis factor alpha and lipopolysaccharide. Infect Immunol 68: 62096214.
  • Morikawa A, Koide N, Sugiyama T, Mu MM, Hassan F, Islam S, Ito H, Mori I, Yoshida T & Yokochi T (2004) The enhancing action of d-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells. FEMS Immunol Med Microbiol 41: 211218.
  • Ndengele MM, Muscolim C, Wang ZQ, Doyle TM, Matuschak GM & Salvemini D (2005) Superoxide potentiates NF-kappaB activation and modulates endotoxin-induced cytokine production in alveolar macrophages. Shock 23: 186193.
  • Pathania V, Syal N, Pathak CM & Khanduja KL (1999) Vitamin E suppresses the induction of reactive oxygen species release by lipopolysaccharide, IL-1 beta and TNF-alpha in rat alveolar macrophages. J Nutr Sci Vitaminol 45: 675686.
  • Sen CK & Packer L (1996) Antioxidant and redox regulation of gene transcription. FASEB J 10: 709720.
  • Shakhov AN, Collart MA, Vassalli P, Nedospasov SA & Jongeneel CV (1990) Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages. J Exp Med 171: 3547.
  • Swantek JL, Cobb MH & Geppert TD (1997) Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alpha (TNF-alpha) translation: glucocorticoids inhibit TNF-alpha translation by blocking JNK/SAPK. Mol Cell Biol 17: 62746282.
  • Szabo C, Day BJ & Salzman AL (1996) Evaluation of the relative contribution of nitric oxide and peroxynitrite to the suppression of mitochondrial respiration in immunostimulated macrophages using a manganese mesoporphyrin superoxide dismutase mimetic and peroxynitrite scavenger. FEBS Lett 381: 8286.
  • Thannickal VJ & Fanburg BL (2000) Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279: L1005L1028.
  • Torres M (2003) Mitogen-activated protein kinase pathways in redox signaling. Front Biosci 8: d369d391.
  • Torres M & Forman HJ (2003) Redox signaling and the MAP kinase pathways. Biofactors 7: 287296.
  • Turpaev KT (2002) Reactive oxygen species and regulation of gene expression. Biochemistry (Mosc) 67: 281292.
  • Ueda S, Masutani H, Nakamura H, Tanaka T, Ueno M & Yodoi J (2002) Redox control of cell death. Antioxid Redox Signal 4: 405414.
  • Victor VV, Guayerbas N & De La Fuente M (2000) Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock. Immunopharmacology 46: 89101.
  • Victor VV, Rocha M & De La Fuente M (2003) Regulation of macrophage function by the antioxidant N-acetylcysteine in mouse-oxidative stress by endotoxin. Int Immunopharmacol 3: 97106.
  • Whitmarsh AJ & Davis RJ (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74: 589607.
  • Yao J, Mackman N, Edgington TS & Fan ST (1997) Lipopolysaccharide induction of the tumor necrosis factor-alpha promoter in human monocytic cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors. J Biol Chem 272: 1779517801.
  • Zhu W, Downey JS, Gu J, Di Padova F, Gram H & Han J (2000) Regulation of TNF expression by multiple mitogen-activated protein kinase pathways. J Immunol 164: 63496358.
  • Zingarelli B, Day BJ, Crapo JD, Salzman AL & Szabo C (1997) The potential role of peroxynitrite in the vascular contractile and cellular energetic failure in endotoxic shock. Br J Pharmacol 120: 259267.