Bacterial lipopolysaccharide (LPS) is a complex glycolipid composed of a hydrophilic polysaccharide moiety and hydrophobic domain known as lipid A (1). Endotoxin is a major component of the outer membrane of gram-negative bacteria and a potent initiator of inflammation. Endotoxin activates the macrophage to produce proinflammatory cytokines, such as tumor necrosis factor (TNF)-α (2). Production of these inflammatory cytokines contributes to the efficient control of growth and dissemination of invading pathogens.
Activation of the macrophage by endotoxin requires the binding of LPS to the acute-phase protein LPS-binding protein (LBP) (3). Binding to LBP forms a complex that facilitates LPS binding to the LPS recognition receptor, CD14 (4). This protein is contained within a glycolipid-enriched microdomain in the plasma membrane termed the lipid raft (5). After complex binding of LPS-LBP to CD14, assembly of the toll-like receptor 4 (TLR4) complex (composed of CD14, heat shock protein 70 (HSP70), TLR4, and MD2) occurs on the lipid raft. Assembly and activation of this complex result in the membrane translocation of the intracellular adaptor protein myeloid differentiation marker 88, followed sequentially by the intracellular activation of the mitogen-activated protein kinases (MAPKs) (6). Activation of these pathways, in turn, results in the gene activation and production of TNF-α and other proinflammatory mediators.
Production of these proinflammatory cytokines is tightly regulated because excessive production leads to amplified inflammatory responses and devastating illnesses characteristic of severe septic shock. Previously, we have demonstrated that autocrine release of oxidants is critical to the regulation of the macrophage (7, 8). Although the mechanism is unknown, we have demonstrated that pretreatment with the antioxidant α-tocopherol succinate results in attenuated endotoxin-mediated proinflammatory mediator production. This seems to result from specific membrane colocalization of α-tocopherol succinate within the lipid bilayer (9).
Recently, it has been demonstrated that lipid raft mobilization of phosphatases, in particular, SH related complex (SRC) homology 2 domain-containing inositol-5-phosphate (SHIP), serves to regulate endotoxin signaling (10). The SHIP is an important phosphatase involved in the dephosphorylation of protein kinase B. The AKT is a regulatory kinase that is activated by phosphatidylinositol 3-kinase (PI3K), which attenuates MAPK signaling (11). Inasmuch as cytoplasmic SHIP results in AKT dephosphorylation, lipid raft mobilization induced by endotoxin results in attenuation of this process, resulting in enhanced MAPK signaling. The role of SHIP, PI3K, and AKT as regulatory kinases involved in endotoxin-mediated signaling is strengthened by observations seen in endotoxin tolerance (12-14). Within these models, enhanced cytoplasmic SHIP, PI3K, and AKT activity can be demonstrated with associated attenuation of endotoxin-mediated MAPK and proinflammatory mediator production.
Based on these observations, we hypothesize that vitamin E in the form of α-tocopherol succinate results in inhibition of SHIP mobilization to lipid rafts, resulting in enhanced AKT activity and downregulation of proinflammatory signaling. Therefore, this study seeks to determine the potential effect of vitamin E on endotoxin-mediated macrophage activation by examining TLR4 receptor complex assembly, intracellular signaling activation, and cytokine liberation. To determine this role, differentiated THP-1 cells were subjected to treatment with maximal pharmacologic doses of vitamin E in the form of α-tocopherol succinate, rather than α-tocopherol because of a previous work demonstrating marked differences in solubility (8).
MATERIALS AND METHODS
Escherichia coli 0111:B4 LPS was obtained from Sigma (St Louis, Mo). Phorbol myristate ([PMA] Upstate Biotechnology, Charlottesville, Va) was dissolved in sterile dimethyl sulfoxide (DMSO). LY294002 and 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate dissolved in DMSO was obtained from Calbiochem (La Jolla, Calif). α-Tocopherol succinate (Sigma) was dissolved in ethanol. Endotoxin contamination of PMA was tested by the limulus amebocyte lysate assay (E-Toxate kit, Sigma) and found to be less than 0.05 ng/mL.
Cell isolation and treatment
Human promonocytic THP-1 cells (American Type Culture Collection, Rockville, Md) were grown in RPMI 1640 (Biowhitaker, Walkersville, Md) supplemented with 10% fetal calf serum (Sigma), 50 U/mL of penicillin, and 50 μg/mL of streptomycin (Cellgro Mediatech Inc, Kansas City, Mo). Cellular differentiation was induced by subjecting cells to 100 ng/mL PMA treatment for 2 days at a concentration of 5 × 106 cells/mL. Cells were then washed and returned to fresh media and serum starved for 24 h before 10% fetal calf serum supplementation and any experimental condition. Selected cells were pretreated with 100 μmol/L α-tocopherol succinate for 1 h. Cells were pretreated with 50 μmol/L LY294002, 50 μmol/L 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, or DMSO, followed by stimulation with 100 ng/mL of LPS for various periods of time, as indicated on the figure legends.
Lipid raft protein extraction
After LPS stimulation, cells were lysed at 4°C in 2 mL of 1% Triton X-100 and TNE/P (25 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L EDTA, 1 μmol/L sodium orthovanadate, 100 μmol/L dithiothreitol, 200 μmol/L phenylmethanesulfonyl fluoride, 10 μg/mL leupeptin, 0.15 U/mL aprotinin, 50 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 2.5 μg/mL pepstatin A, and 1 mmol/L benzamidine) for 20 min. Lysate was then mixed with 2.5 mL of 80% sucrose in TNE/P. Samples were then overlaid with 7 mL 35% sucrose in TNE/P and then 3 mL 5% sucrose in TNE/P. Lysates were then spun for 18 h at 100,000g at 4°C. Protein at the 5% to 35% sucrose interface representing the lipid raft portion was isolated and resuspended in 200 μL of TNE/P. Protein concentration was determined using the Pierce BCA protein assay (Pierce, Rockford, Ill).
Cellular protein extraction
After LPS stimulation, total cellular protein was extracted at 4°C in 500 μL of lysis buffer (20 mmol/L Tris, 137 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 1% Triton X-100, 1 μmol/L sodium orthovanadate, 100 μmol/L dithiothreitol, 200 μmol/L phenylmethanesulfonyl fluoride, 10 μg/mL leupeptin, 0.15 U/mL aprotinin, 50 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 2.5 μg/mL pepstatin A, 1 mmol/L benzamidine, and 40 mmol/L α-glycerophosphate). Protein concentration was determined using the Pierce BCA protein assay (Pierce).
Lipid raft protein was electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc, Piscataway, NJ). The membrane was blocked for 1 h with 5% milk and then incubated with either an anti-TLR4 (Zymed, San Francisco, Calif) or anti-SHIP (Upstate Biotechnology) antibody for 12 h at 4°C. Blots were then incubated in a horseradish peroxidase-conjugated antirabbit IgG at room temperature for 1 h. The blots were developed using the SuperSignal chemiluminescent substrate (Pierce) and exposed on Kodak KAR-5 film (Eastman Kodak, Rochester, NY). Densitometry was performed by the NIH.image program (National Institutes of Health, Bethesda, Md) to quantitate optical density. All gels were reblotted for total SRC (Sigma) to confirm equal loading.
Total cellular protein was electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc). The membrane was blocked for 1 h with 1% bovine serum albumin, 5% bovine serum albumin, or 5% milk, and then incubated with an antiphosphorylated Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) (Promega Madison, WI), anti-JNK (Santa Cruz), antiphosphorylated p38 (Cell Signaling, Beverly, Mass), anti-p38 (Santa Cruz), antiphosphorylated extracellular signal-regulated kinase (ERK)-1/2 (Cell Signaling), anti-ERK-1 (Santa Cruz), or antiphosphorylated AKT (Upstate Biotechnology) antibody for 12 h at 4°C. Blots were then incubated in a horseradish peroxidase-conjugated secondary antibody against the primary at room temperature for 1 h. The blots were developed and analyzed as previously described. All gels were reblotted for total ERK-1, p38, JNK/SAPK, and AKT to confirm equal loading.
After the previously described treatments, supernatants were harvested under all conditions after 8 h of stimulation. Production of TNF-α was quantitated by an enzyme immunoassay kit (Assay Design, Inc, Ann Arbor, Mich) that is based on a coated-well, sandwich enzyme immunoassay.
Cell viability and morphologic features
Representative cell populations from each condition were examined under light microscopy. No significant change was noted under any condition. Cell viability was also confirmed by trypan blue exclusion.
Values are expressed as mean ± SEM. Group means are compared by unpaired Student t tests and analysis of variance. P = 0.05 or less was considered significant.
Vitamin E pretreatment of differentiated THP-1 cells does not affect LPS-induced TLR4 receptor mobilization to lipid rafts
First, we sought to determine if vitamin E exposure would affect assembly of the TLR4 receptor complex within differentiated THP-1 cells. Assembly of this complex, composed of TLR4, HSP70, SRC, and other proteins, occurs on lipid rafts after endotoxin exposure (5). Using cultured differentiated THP-1 cells, TLR4 receptor complex assembly was determined by immunoblots of extracted lipid rafts after endotoxin exposure with/without pretreatment with α-tocopherol succinate (Fig. 1). Endotoxin exposure resulted in the mobilization of both TLR4 and HSP70 to lipid rafts. The SRC was constitutively found within lipid rafts, was not affected by endotoxin exposure, and was used as a loading control. Pretreatment with vitamin E in the form of α-tocopherol succinate did not affect lipid raft SRC content or endotoxin-induced mobilization of TLR4 and HSP70.
Vitamin E pretreatment of differentiated THP-1 cells results in attenuated LPS-induced MAPK activation
After assembly of the TLR4 complex, endotoxin binds to TLR4, leading to a conformational change and intracellular signal transduction through the MAPK composed of ERK-1/2, p38, and JNK/SAPK. Demonstrating the lack of effect by vitamin E on TLR4 receptor complex assembly, we next set out to determine the effect of vitamin E on the phosphorylation and activation of each of the MAPK during similar states. Endotoxin treatment led to the phosphorylation and activation of each of the MAPK, with maximal activation occurring after 30 min of stimulation (Fig. 2). Pretreatment with vitamin E led to a significant attenuation of endotoxin-mediated activation of each of the MAPK.
Vitamin E pretreatment results in enhanced activation of LPS-induced AKT phosphorylation, a condition that is reversed by PI3K inhibition
Demonstrating the attenuation in endotoxin-mediated MAPK activation without any effect on TLR4 receptor complex assembly, we next set out to determine if vitamin E had any affect on the MAPK counter-regulatory kinase, AKT. Endotoxin exposure resulted in the activation of AKT, demonstrated by an increase in the phosphorylated state (Fig. 3). Vitamin E pretreatment was associated with a significant increase in endotoxin-mediated phosphorylation of AKT. To determine if this increase in AKT activity was induced through PI3K, selective inhibition of PI3K was performed by pretreating selected cells with LY294002. Pretreatment with LY294002 or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate resulted in attenuation of endotoxin-mediated AKT phosphorylation with or without vitamin E pretreatment.
Vitamin E pretreatment of differentiated THP-1 results in attenuated LPS-induced phosphatase mobilization to lipid rafts
Demonstrating the attenuation in endotoxin-mediated MAPK activation and increase in AKT phosphorylation, we next set out to determine if vitamin E had any effect on AKT regulatory phosphatase, SHIP. Inasmuch as the regulation of AKT signaling by SHIP is a result of lipid raft mobilization of this protein, isolated lipid raft protein was investigated under the various conditions for SHIP. Endotoxin exposure resulted in the mobilization of SHIP to the lipid raft (Fig. 4). Pretreatment with vitamin E was associated with a significant attenuation in SHIP mobilization to lipid rafts by endotoxin.
Vitamin E pretreatment results in attenuated LPS-induced TNF-α production, a condition that is reversed by PI3K inhibition
Demonstrating the overall effects by vitamin E on intracellular signaling, we finally sought to determine if vitamin E exposure would result in any changes in proinflammatory mediator production in the form of TNF-α. Endotoxin resulted in the production of TNF-α from differentiated THP-1 cells (Fig. 5). Pretreatment with vitamin E, similar to the effect on MAPK signaling, was associated with a significant attenuation in TNF-α production. To determine if this was modulated through selective increases in AKT activity through PI3K activity, inhibition for PI3K/AKT was done with LY294002, and AKT was done with 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate. Cells pretreated with LY294002 were associated with an attenuation of LPS-induced TNF-α production, but did result in a reversal in the attenuation in LPS-induced TNF-α production by vitamin E. Cells pretreated with 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate were not associated with an effect on LPS-induced TNF-α production, but did result in a reversal in the attenuation in LPS-induced TNF-α production by vitamin E.
Sepsis remains one of the leading causes of morbidity and mortality among critically ill patients (15). Sepsis is associated with an overly aggressive inflammatory response, demonstrated by a marked liberation of cytokines. This exaggerated cytokine release results in the physiological symptoms attributed to sepsis. Previously, we have demonstrated that oxidants play a critical role in the liberation of cytokines, such as TNF-α, from tissue-fixed macrophages.
Tumor necrosis factor-α is an important mediator during sepsis and endotoxemia. Increased TNF-α levels during sepsis are predictive of the relative risk of acute respiratory distress syndrome development (16). Previous work in our laboratory has demonstrated that TNF-α production within macrophage is significantly attenuated by the use of antioxidants, such as vitamin E. The current study verifies the previous report by showing an inhibition of TNF-α production after LPS stimulation by vitamin E in the form of α-tocopherol succinate. Demonstrating this consistent attenuation in endotoxin-mediated TNF-α production, we set out to determine the potential mechanism in which antioxidants regulate endotoxin-mediated macrophage activation.
Vitamin E in the form of α-tocopherol succinate includes an aromatic chromanol head and a 16-carbon hydrocarbon tail. The antioxidant function is localized to a phenolic hydroxyl group on the chromanol head, whereas the hydrocarbon tail is important for rapid uptake and localization within the cell membrane (17). This localization within the membrane is critical to the biological effects attributed to α-tocopherol succinate. In addition to the antioxidant effects, α-tocopherol succinate also results in inhibition of various kinases, including protein kinase C (PKC) (18, 19). These data suggest that the effect of α-tocopherol succinate is caused by membrane localization of this lipid, but the full effect that this lipid has on membrane trafficking and intracellular activation remains unknown.
Endotoxin or LPS activates the macrophage through complex binding with LPB and the specific glycosol phosphatidylinositol-anchored cell membrane receptor, CD14. Binding of endotoxin to LPB and CD14, in turn, results in mobilization of TLR4, MD2, and HSP70 to lipid rafts, resulting to glycosol phosphatidylinositol-receptor complex assembly. Assembly of this complex on these membrane microdomains is PKC-ζ dependent (20). Although α-tocopherol succinate is localized to the membrane and affects PKC activation, α-tocopherol succinate pretreatment, as demonstrated in our study, did not affect endotoxin-mediated mobilization of TLR4 or HSP70. Thus, it seems that the effect of α-tocopherol succinate is not caused by alterations in membrane receptor trafficking.
After TLR4 receptor coclustering, a conformational change within the cytoplasmic domain of TLR4 occurs resulting in intracellular signaling. Although several signaling pathways become activated, the MAPK pathways seem critical to proinflammatory phenotypic differentiation and subsequent TNF-α production (21-23). Although TLR4 coclustering on lipid rafts was not affected by α-tocopherol succinate, this study demonstrated significant attenuation of endotoxin-mediated MAPK signaling by α-tocopherol succinate. This attenuation in MAPK signaling was characterized by attenuated activation of all major MAPK members.
Regulation of the MAPK is complex but is thought to occur, in part, through the activation of PI3K/AKT (24). Although somewhat controversial, recent reports have suggested that PI3K/AKT plays a negative regulatory role in MAPK signaling (10). This suppression of MAPK signaling occurs through the activation of the regulatory protein AKT. The AKT is a serine/threonine kinase that is activated by PI3K and influences the activation status of the MAPK through regulation of the upstream MAPK activators, MEK kinase 3 and Raf-1 kinase (25). Our current observations support a role for PI3K/AKT in the regulation of macrophage activation by antioxidants. As demonstrated, AKT activation is enhanced by antioxidant exposure. Although attenuation in PI3K by LY294002 was associated with reversal of the effects induced by vitamin E, treatment with LY294002 was associated with an attenuation in the overall release in TNF-α because of the other signaling roles attributed to PI3K in LPS-mediated signaling. Selective attenuation in AKT by 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, on the other hand, did not significantly affect LPS-mediated signaling but was associated with reversal in the effects induced by vitamin E. Taken together, these data suggest that vitamin E affects LPS-induced macrophage activation through upregulated PI3K/AKT activation.
Although the regulation of PI3K and AKT is incompletely understood, recent work by Fang et al. (10) have suggested that endotoxin-mediated SHIP mobilization to lipid rafts is important in the regulation of both PI3K and AKT. The SHIP is an inositol phosphatase that is found within the cytosol during unstimulated conditions. On stimulation, SHIP mobilizes to the lipid raft that is verified in our study. This mobilization in SHIP to the lipid raft serves to dephosphorylate AKT, resulting in unabated MAPK signaling. Dissociation of SHIP, on the other hand, from the lipid raft results in PI3K phosphorylation of AKT, resulting in attenuation in MAPK signaling. Similar to these previous observations, we demonstrate that endotoxin-mediated SHIP mobilization to the lipid raft is inhibited by α-tocopherol succinate. Consistent with this attenuated mobilization is increased activation of AKT and attenuated activation of the MAPK. The role of PI3K in this process is suggested by reversal of the effects of α-tocopherol succinate by the specific PI3K inhibitor, LY294002.
Taken together, these data demonstrate the regulatory role that cellularly produced oxidants play in the control of intracellular signaling and cytokine liberation (Fig. 6). Antioxidant exposure results in attenuation of proinflammatory mediator production through inhibition of SHIP mobilization to the lipid raft. This attenuated mobilization leads to increased AKT activation, resulting in attenuation of MAPK signaling and liberation of inflammatory cytokines. These molecular data are intriguing when combined with previous clinical studies, demonstrating a reduction in the development of multiple organ dysfunction in critically ill patients treated with antioxidants. Thus, these data provide more insight into the potential mechanism in which antioxidants may serve to modulate the immune response. As a result, it seems that appropriate treatment of critically ill patients with antioxidants may serve to regulate the immune response and potentially prevent the development of dysregulated immune responses.
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