MnTBAP, a synthetic metalloporphyrin, inhibits production of tumor necrosis factor-α in lipopolysaccharide-stimulated RAW 264.7 macrophages cells via inhibiting oxidative stress-mediating p38 and SAPK/JNK signaling
Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Correspondence: Takashi Yokochi, Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan. Tel.: +81 561 62 3311 ext. 2169; fax: +81 561 63 9187; e-mail: firstname.lastname@example.org
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.
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
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).
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.
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 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 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.
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.
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).
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.
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.
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).
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.
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.
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.
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.