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Originally published online as doi:10.1189/jlb.0106053 on September 7, 2006

Published online before print September 7, 2006
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(Journal of Leukocyte Biology. 2006;80:1289-1297.)
© 2006 by Society for Leukocyte Biology

Endotoxin tolerance attenuates LPS-induced TLR4 mobilization to lipid rafts: a condition reversed by PKC activation

Joseph Cuschieri1, Jens Billigren and Ronald V. Maier

Department of Surgery, University of Washington, Seattle, Washington, USA

1 Correspondence: Department of Surgery, University of Washington, Harborview Medical Center, 325 9th Avenue, Box 359796, Seattle, WA 98104, USA. E-mail: jcuschie{at}u.washington.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endotoxin tolerance is characterized by attenuated macrophage activation to subsequent LPS challenge and can be reversed through nonspecific protein kinase C (PKC) activation, and activation by LPS within naïve cells requires the activation of the cell surface receptors CD14 and TLR4 on lipid rafts. The effect of PKC activation and endotoxin tolerance on lipid raft receptor complex assembly is unknown and the focus of this study. Tolerance was induced in THP-1 cells through LPS pre-exposure. Naïve and tolerant cells were stimulated with LPS, with or without PMA pretreatment to activate PKC. TLR4 surface expression and LPS binding were determined by flow cytometry and immunohistochemistry. Cellular and lipid raft protein was analyzed for the presence and activation of the TLR4 complex components. Harvested supernatants were examined for TNF-{alpha} production. Total TLR4 surface expression and LPS binding were not affected by tolerance induction. LPS stimulation of naïve cells resulted in TLR4 and heat shock protein (HSP)70 lipid raft mobilization, MAPK activation, and TNF-{alpha} production. LPS stimulation of tolerant cells was associated with attenuation of all of these cellular events. Although PKC activation by PMA had no effect on naïve cells, it did result in reversal in tolerance-induced suppression of TLR4 and HSP70 lipid raft mobilization, MAPK activation, and TNF-{alpha} production. In addition, the effects associated with PMA were reversed with exposure to a myristoylated PKC-{zeta} pseudosubstrate. Thus, endotoxin tolerance appears to be induced through attenuated TLR4 formation following LPS stimulation. This complex formation appears to be PKC-dependent, and restoration of PKC activity reverses tolerance.

Key Words: macrophage • CD14 • HSP70 • PMA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial endotoxin (LPS) is a complex glycolipid composed of a hydrophilic polysaccharide moiety and hydrophobic domain known as lipid A. Endotoxin is a major component of the outer membrane of Gram-negative bacteria and a potent initiator of inflammation [1 ]. Endotoxin activates the macrophage to produce proinflammatory cytokines, such as TNF-{alpha}. Production of these inflammatory cytokines contributes to the efficient control of growth and dissemination of invading pathogens.

Activation of the macrophage by endotoxin requires LPS to bind to the acute-phase protein LPS-binding protein (LBP). Once bound to LBP, LPS binds to the LPS recognition receptor, CD14 [2 ], which is a GPI-anchored protein that does not have a cytoplasmic domain [3 , 4 ]. This protein is contained within a glycolipid-enriched microdomain in the plasma membrane, termed the lipid raft [5 ]. Following complex binding of LPS-LPB to CD14, assembly of the TLR4 complex, composed of CD14, TLR4, myeloid differentiation protein 2, and heat shock protein (HSP)70, occurs on the lipid raft [6 , 7 ]. Assembly and activation of this complex result in the membrane translocation of the intracellular adaptor protein MyD88, followed sequentially by the intracellular activation of IL-1 associated kinase-1 (IRAK-1) and MAPK [8 , 9 ]. Activation of these pathways, in turn, results in the gene activation and production of TNF-{alpha} and other proinflammatory mediators.

Production of these proinflammatory cytokines is tightly regulated, as excessive production leads to amplified, inflammatory responses and devastating illnesses characteristic of severe septic shock [10 ]. It is interesting that initial survivors of septic shock demonstrate suppressed macrophage response to LPS, a condition reminiscent of in vitro endotoxin tolerance [11 ]. Although this state during septic shock may be protective, late immune suppression as a result of this feedback-controlled down-regulation places the patient at increased risk for the development of nosocomial infections and subsequent morbidity and mortality [12 , 13 ]. Thus, an understanding of the mechanisms that elicit endotoxin tolerance is critical to unraveling the molecular basis of the immune suppression following septic challenge. However, despite numerous studies, these mechanisms remain largely unknown. Whereas inhibition of cell-surface expression of TLR4 has been suggested to underlie LPS tolerance in mouse macrophages, recent work has demonstrated that LPS tolerance does not affect cell surface expression of TLR4 within human cells [14 15 16 17 ]. Despite this lack of TLR4 down-regulation, LPS-tolerant human cell lines, such as THP-1 cells, do exhibit significantly suppressed LPS-induced IRAK activation and diminished IRAK-MyD88 association [18 ]. Thus, these data imply that tolerance induction affects the early phases of intracellular function potentially during TLR4 complex assembly.

Recently, we have demonstrated that TLR4 complex assembly on lipid rafts following LPS-LPB binding to CD14 requires the activation of the atypical protein kinase C (PKC), PKC-{zeta} [19 ]. It is interesting that PKC activation has been shown to reverse endotoxin tolerance through an undefined mechanism [20 ]. As a result, this study seeks to determine the potential effect of PKC activation by PMA on endotoxin tolerance by examining the surface expression of TLR4, the mobilization of TLR4 and HSP70 to lipid rafts, and the effect on numerous intracellular intermediates involved in the TLR4 pathway in naïve and LPS-tolerant, differentiated THP-1 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Escherichia coli 0111:B4 LPS was obtained from Sigma Chemical Co. (St. Louis, MO). PMA (Upstate Biotechnology, Charlottesville, VA) was dissolved in sterile DMSO. 1-{alpha}, 25-Dihydroxyvitamin D3 (Biomol Research Laboratories, Plymouth Meeting, PA) was dissolved in sterile DMSO. Myristoylated PKC-{zeta} pseudosubstrate obtained from Biosource (Camarillo, CA) was dissolved in sterile H2O at a concentration of 1 mg/ml. Endotoxin contamination of PMA and myristoylated PKC-{zeta} pseudosubstrate was tested by the Limulus amebocyte lysate assay (E-TOXATE kit, Sigma Chemical Co.) and found to be less than 0.05 ng/ml.

Cell isolation and treatment
Human promonocytic THP-1 cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (Sigma Chemical Co.), 50 U/mL penicillin, and 50 µg/mL streptomycin (Cellgro Mediatech Inc., Kansas City, MO). Cellular differentiation was induced by subjecting cells to 100 mM vitamin D3 treatment for 3 days at a concentration of 5 x 106 cells/ml. In addition, peripheral blood monocytes were isolated from whole blood by Ficoll-Paque density gradient from healthy male volunteers. The buff coat was collected, and these cells were allowed to adhere to polysterene tissue-treated culture plates in the presence of RPMI 1640 with 100 mg/ml gentamycin for 2 h at 37°C and 5% CO2. The plates were then washed twice with RPMI 1640 to remove nonadherent cells.

Both cell types were then washed further and returned to fresh media and serum-starved for 24 h prior to supplementation with 10% heat-inactivated adult bovine serum (JRH Bioscience, Lenexa, KA) and any experimental condition. Selected cells were pretreated with 10 ng/ml LPS for 18 h to induce tolerance. Naïve and tolerant cells were then stimulated with 100 ng/ml LPS for various periods of time as indicated on the figure legends, with/without pretreatment of 100 ng/ml PMA for 30 min. In addition, selected cells pretreated with PMA were treated with 60 µM myristoylated PKC-{zeta} for 60 min as indicated in the figure legends.

Flow cytometry
Naïve and tolerant cells treated with or without PMA for 30 min were subjected to TLR4 surface expression analysis by FACScan. Following stimulation, differentiated THP-1 cells were transferred onto ice and washed with ice-cold FACS buffer (10 mM PBS without Ca2+ and Mg2+, 10 mM HEPES, and 0.25% BSA) before fluorescent staining with directly labeled antibodies. Cells were blocked with mouse IgG (50 mg/ml) for 10 min at room temperature and then stained with a 1/10 dilution of FITC-conjugated antimouse IgG mAb against human TLR4 (Serotec, Inc., Raleigh, NC), FITC-conjugated LPS (Sigma Chemical Co.), or istoype control antibody for 45 min at 4°C. Cells were then washed with FACS buffer and fixed using 1x Cellfix (BD Biosciences, Franklin Lake, NJ). Flow cytometry was performed on a dual-laser FACSCaliber (BD Biosciences) using CellQuest software (BD Biosciences).

Immunohistochemistry
Following LPS stimulation, naïve and tolerant cells adherent to coverslips were washed with PBS. Cells were then fixed for 30 min with 3% paraformaldehyde in PBS with calcium and magnesium. Cells were once again washed with PBS and allowed to dry and then quenched with NH4Cl/PBS for 10 min at room temperature. Cells were then washed with PBS without Ca2+ and Mg2+ and blocked with 10% milk and 5% goat serum in PBS. Cells were then stained with rhodamine-labeled cholera toxin (1:300) for 30 min to stain lipid rafts. Cells were then washed and then incubated with rabbit antihuman TLR4 antibody (Abcam, Cambridge, MA) for 30 min. Cells were then washed and blocked with 10% milk and 5% goat serum in PBS for 10 min. Cells were then stained with antirabbit FITC-conjugated antibody (Molecular Probes, Eugene, OR) at 1:500 dilution for 30 min. Cells were then washed, fixed, and examined on an Axiovert 40 microscope (Carl Zeiss, Inc., Thornwood, NY).

Lipid raft and nonraft protein extraction
Following LPS stimulation, naïve and tolerant cells were lysed at 4°C in 2 ml 1% Triton X-100 and tris NaCl-EDTA/phosphate (TNE/P) (25 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 µM sodium orthovanadate, 100 µM DTT, 200 µM PMSF, 10 µg/ml leupeptin, 0.15 U/ml aprotinin, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 2.5 µg/ml pepstatin A, and 1 mM benzamidine) for 20 min. Lysate was then mixed with 2.5 ml 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. Lystates were then spun for 18 h at 100,000 g at 4°C. The gradient was then divided into 10 fractions, and Fractions 2–4 represent the lipid raft fraction, and Fractions 6–9 represent the nonraft fraction. Protein within the combined fractions was isolated and resuspened in 200 µl TNE/P. Protein concentration was determined using the Pierce bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL).

Cellular protein extraction
Following LPS stimulation, total cellular protein was extracted from naïve and tolerant cells at 4°C in 500 µl lysis buffer (20 mM Tris, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 1 µM sodium orthovanadate, 100 µM DTT, 200 µM PMSF, 10 µg/ml leupeptin, 0.15 U/ml aprotinin, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 2.5 µg/ml pepstatin A, 1 mM benzamidine, and 40 mM {alpha}-glycerophosphate). Protein concentration was determined using the Pierce BCA protein assay (Pierce).

IRAK immunoprecipitation
Equal amount of cellular protein obtained following the various conditions was used for immunoprecipitation. Polyclonal anti-IRAK antibody (5 µl, Upstate Biotechnology, Inc.) was added to 500 ng isolated cellular protein and incubated at 4°C overnight on a rotator. Fifty percent slurry (50 µl) of prewashed protein G-agarose beads was then added to each sample, followed by incubation for an additional 2 h at 4°C. The samples were spun briefly in a microcentrifuge and washed four times in lysis buffer. Samples were then resuspended in 30 µl lysis buffer for future analysis.

Western blots
Lipid raft and nonraft proteins were electrophoresed in 10% SDS-PAGE and transferred to Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The lipid raft protein membranes were blocked for 1 h with 5% milk and then incubated with an antihuman TLR4 (Zymed, San Francisco, CA), antihuman HSP70 (Upstate Biotechnology), antihuman CD14 (R&D Systems Inc., Minneapolis, MN), antihuman Grb2-associated binder (Gab2; Upstate Biotechnology), antihuman {alpha}-tubulin (Abcam), or antihuman Lyn (Upstate Biotechnology) for 12 h at 4°C. The nonraft protein membranes were blocked for 1 h with 5% milk and then incubated with an antihuman TLR4, antihuman HSP70, antihuman CD14, antihuman {alpha}-tubulin, antihuman Lyn, or antiactin (Zymed) for 12 h at 4°C. Blots were then incubated in a HRP-conjugated secondary antibody against the primary or HRP-conjugated cholera toxin 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).

Total cellular protein was electrophoresed in SDS-PAGE gel and transferred to Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Inc.). The membrane was blocked for 1 h with 1% BSA, 5% BSA, or 5% milk and then incubated with antiphosphorylated JNK/stress-activated protein kinase (SAPK; Promega, Madison, WI), anti-JNK (Santa Cruz Biotechnology, CA), antiphosphorylated p38 (Cell Signaling, Beverly, MA), anti-p38 (Santa Cruz Biotechnology), antiphosphorylated ERK1/2 (Cell Signaling), anti-ERK1 (Santa Cruz Biotechnology), or anti-IRAK-1 (Upstate Biotechnology) antibodies for 12 h at 4°C. Blots were then incubated in a HRP-conjugated secondary antibody against the primary at room temperature for 1 h. The blots were developed and analyzed as described previously. All gels were reblotted for total ERK1, p38, and JNK/SAPK to confirm equal loading.

In a similar manner, the active, phosphorylated form of IRAK was determined using IRAK-immunoprecipitated protein. Gels were run similarly and transferred to nitrocellulose membranes. Following initial blockade in 5% milk, membranes were incubated with an antiphosphothreonine antibody (Zymed) overnight at 4°C. Blots were then incubated in a HRP-conjugated anti-rabbit IgG secondary antibody against the primary at room temperature for 1 h and developed and analyzed as described previously.

Cytokine production
Following the treatments described previously, supernatants were harvested under all conditions following 8 h of stimulation. TNF-{alpha} production was quantitated by an enzyme immunoassay kit (Assay Design, Inc., Ann Arbor, MI), which 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.

Statistic analysis
Values are expressed as means ± SEM. Group means are compared by unpaired Student’s t-tests and ANOVA. A probability value of 0.05 or less was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pretreatment of differentiated THP-1 cells with LPS results in attenuated LPS-induced TNF-{alpha} production, which is reversed by PKC activation
In the first series of experiments, we sought to establish conditions under which endotoxin tolerance would be induced reliably in differentiated THP-1 cells. Cultured THP-1 cells were differentiated with vitamin D3 for 72 h. During differentiation, cells obtained the ability to adhere and increased surface expression of CD14. Cells were then serum-starved in fresh media prior to any experimental condition. Serum was added, and selected cells were stimulated with 10 ng/ml LPS for 18 h. Naïve and LPS-pretreated cells were washed and returned to fresh media with serum prior to further LPS exposure.

TNF-{alpha} production was then determined by ELISA. Naïve cells exposed to 100 ng/ml LPS produced TNF-{alpha} (Fig. 1 ). LPS-pretreated cells exposed to 100 ng/ml LPS produced significantly attenuated TNF-{alpha} in comparison with naïve cells and thus, demonstrate the induction of tolerance (Fig. 1) . To determine the effect of PKC activation on tolerance, naïve and tolerant cells were exposed to 100 ng/ml PMA for 30 min prior to subsequent LPS exposure. PMA pretreatment had no effect on endotoxin-mediated TNF-{alpha} production in naïve cells (Fig. 1) . However, PMA pretreatment of tolerant cells restored endotoxin-mediated TNF-{alpha} production, similar to naïve cells and thus, demonstrated that PKC activation can reverse tolerance induction (Fig. 1) .


Figure 1
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  		Figure 1. Impaired, endotoxin-mediated TNF-{alpha} production in tolerant cells is reversed by PKC activation. Differentiated THP-1 cells were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected cells were then treated with 100 ng/ml PMA for 30 min. Naïve and LPS-pretreated cells were stimulated with 100 ng/ml LPS for 8 h. TNF-{alpha} production was then determined by ELISA (R&D Systems). Values represent the mean + SEM for five separately performed experiments (*, P<0.05, compared with naïve, LPS-treated; {dagger}, P<0.05, compared with LPS-pretreated cells treated with LPS without PMA). pg, picogram.

 
Pretreatment of differentiated THP-1 cells with LPS results in attenuated LPS-induced IRAK-1 activation, which is reversed by PKC activation
Demonstrating the effects on endotoxin-mediated TNF-{alpha} production during tolerance with PMA treatment, we next set out to determine the effect on intracellular signaling. Initial endotoxin-mediated intracellular signaling within differentiated THP-1 cells is coordinated through IRAK-1, which becomes phosphorylated and activated following initial binding of LPS to the TLR4 complex. To determine the effects of PKC activation during tolerance, naïve and tolerant cells were pretreated with 100 ng/ml PMA for 30 min prior to LPS stimulation, which led to the phosphorylation of IRAK-1, which was maximal at 7.5 min within naïve cells (Fig. 2 ). Pretreatment of naïve cells with PMA had no significant effect on endotoxin-mediated IRAK-1 phosphorylation (Fig. 2) . Tolerance induced by LPS pretreatment was associated with a significant reduction in endotoxin-mediated IRAK-1 phosphorylation, which was reversed with PMA pretreatment (Fig. 2) . Conversely, total cellular IRAK-1 was affected following LPS stimulation in naïve cells and tolerant cells pretreated with PMA (Fig. 2) . LPS stimulation of these cells resulted in the degradation of IRAK-1. Thirty minutes of PMA treatment, as demonstrated in the first lane of each total cellular IRAK blot, did not affect basal IRAK levels. Tolerance, conversely, was not associated with degradation of IRAK-1 following LPS stimulation. In addition, 18 h of tolerance did not reduce the total cellular IRAK-1 concentration within our experimental model, as demonstrated in the first lane of the tolerance gel (Fig. 2) .


Figure 2
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  		Figure 2. Impaired, endotoxin-mediated IRAK-1 phosphorylation in tolerant cells is reversed by PKC activation. Differentiated THP-1 cells were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected cells were then treated with 100 ng/ml PMA for 30 min. Naïve and LPS-pretreated cells were stimulated with 100 ng/ml LPS for up to 30 min. Cellular protein was harvested, immunoprecipitated for IRAK-1, and analyzed by Western blot for phosphothreonine (p-IRAK-1) and total cellular IRAK. Representative blots were demonstrated from one of four separately performed experiments.

 
Pretreatment of differentiated THP-1 cells with LPS results in attenuated LPS-induced MAPK activation, which is reversed by PKC activation
Downstream to IRAK-1 phosphorylation and activation is the activation of the MAPK family composed of ERK1/2, p38, and JNK/SAPK. Demonstrating the effects on IRAK-1 during tolerance and following PKC activation, we next set out to determine the effect on the phosphorylation and activation of each of the MAPK members during similar states. Endotoxin treatment of naïve cells led to the phosphorylation and activation of each of the MAPKs, and maximal activation occurred following 30 min of stimulation (Fig. 3 ). PKC activation by PMA had no significant effect on endotoxin-mediated activation of any of the MAPKs, similar to IRAK-1 (Fig. 3) . Tolerance induced by LPS pretreatment was associated with significant reduction in endotoxin-mediated MAPK activation, which was reversed completely for ERK1/2 and JNK/SAPK by PMA pretreatment but only partially reversed by p38 (Fig. 3) .


Figure 3
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  		Figure 3. Impaired, endotoxin-mediated MAPK activation in tolerant cells is reversed by PKC activation. Differentiated THP-1 cells were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected cells were then treated with 100 ng/ml PMA for 30 min. Naïve and LPS-pretreated cells were stimulated with 100 ng/ml LPS for up to 60 min. Cellular protein was harvested and analyzed by Western blot for the phosphorylation and total cellular content of (A) ERK1/2, (B) p38, and (C) JNK/SAPK. A representative blot was demonstrated from one of five separately performed experiments.

 
Pretreatment of differentiated THP-1 cells with LPS or PMA does not affect surface expression of TLR4 or binding of LPS
Demonstrating the significant effects on proximal intracellular signaling and TNF-{alpha} production during endotoxin tolerance and restoration through PKC activation, we next set out to determine if any potential effect occurred on TLR4 surface expression under the various conditions studied. Using FACScan and immunoblotting of cellular proteins, no change in surface expression or cellular protein was noted following LPS pretreatment or with PMA (Figs. 4 and 5 , respectively). In a similar manner, we set out to determine if tolerance was associated with attenuated binding of endotoxin. Using FACScan, no change in binding of LPS was noted under these conditions (Fig. 6 ).


Figure 4
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  		Figure 4. Surface expression of TLR4 is not affected during tolerant states. Differentiated THP-1 cells were treated for 18 h with 10 ng/ml LPS to induce tolerance. Selected cells were treated with 100 ng/ml PMA for 30 min. TLR4 surface expression was then examined in control and tolerant cells with appropriate isotype control antibody by FACScan.

 

Figure 5
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  		Figure 5. Total cellular TLR4 is not affected during tolerance induction or PKC activation. Differentiated THP-1 cells were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected cells were then treated with 100 ng/ml PMA for 30 min. Cellular protein was harvested and analyzed by Western blot for the TLR4. A representative blot was demonstrated from one of five separately performed experiments.

 

Figure 6
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  		Figure 6. Binding of LPS was not affected during tolerant states. Differentiated THP-1 cells were treated for 18 h with 10 ng/ml LPS to induce tolerance. Selected cells were treated with 100 ng/ml PMA for 30 min. FITC-LPS binding was then examined in control and tolerant cells with FACScan.

 
Pretreatment of differentiated THP-1 cells with LPS results in attenuated LPS-induced lipid raft mobilization of TLR4 and HSP70, which is reversed by PKC activation
Despite not demonstrating any significant effect on TLR4 surface expression, we next set out to determine if tolerance was associated with any effect on TLR4 assembly on lipid rafts. To study this, the mobilization of TLR4 and HSP70 to the lipid raft was examined by isolating lipid raft fractions and analyzing them for TLR4 and HSP70. Minimal TLR4 and HSP70 were present within the lipid raft prior to endotoxin exposure. Exposure of endotoxin for only 5 min resulted in a significant increase in TLR4 and HSP70 protein within the lipid raft (Fig. 7A ). This mobilization was not affected by pretreatment with PMA. Tolerance, however, induced by endotoxin pretreatment resulted in significant attenuation of endotoxin-mediated mobilization of TLR4 and HSP70 to lipid rafts (Fig. 7A) . This attenuated mobilization during tolerance was reversed by PMA pretreatment, similar to the previous effects demonstrated on endotoxin-mediated intracellular signaling and proinflammatory mediator production. In addition, nonraft fractions were analyzed for TLR4 components. TLR4 and HSP70 were found abundantly within the nonraft protein fraction (Fig. 7B) . Endotoxin exposure resulted in minimal changes in the nonraft composition of TLR4 or HSP70 under naïve or tolerant conditions. This lack of change demonstrates that these proteins remain mostly nonraft-associated and inactive. CD14, unlike TLR4 and HSP70, was found mostly in the lipid raft fraction, and minimal amounts were found within the nonraft fraction. Specificity of raft and nonraft fractions was verified further by consistent detection of Lyn, in addition to CD14 within the raft fraction and identification of Gab2, actin, and {alpha}-tubulin only within the nonraft fraction.


Figure 7
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  		Figure 7. Impaired, endotoxin-mediated TLR4 assembly on lipid rafts in tolerant cells is reversed by PKC activation. Differentiated THP-1 cells were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected cells were then treated with 100 ng/ml PMA for 30 min. Naïve and LPS-pretreated cells were stimulated with 100 ng/ml LPS for 5 min. (A) Lipid raft protein harvested and analyzed by Western blot for TLR4, HSP70, CD14, Gab2, {alpha}-tubulin, and Lyn. (B) Nonraft protein was harvested and analyzed by Western blot for TLR4, HSP70, CD14, {alpha}-tubulin, Lyn, and actin. Representative blots were demonstrated from one of three separately performed experiments.

 
These findings suggest that tolerance is associated with attenuation in TLR4 assembly on lipid rafts. These effects were reversed completely by nonspecific PKC activation by PMA. Having previously demonstrated the importance of PKC-{zeta} on this receptor complex formation, we set out to determine if the effects attributed to PMA are PKC-{zeta}-dependent [19 ]. To study this, we subjected cells to pre-exposure with a myristoylated PKC-{zeta} pseudosubstrate. Pretreatment with this pseudosubstrate reversed all the effects induced by PMA, implicating PKC-{zeta} rather than other PKC family members as the key kinase involved in tolerance regulation (Fig. 8A and 8B ).


Figure 8
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  		Figure 8. PMA-induced tolerance reversal of endotoxin-mediated TLR4 assembly on lipid rafts is PKC-{zeta}-dependent. Differentiated THP-1 cells were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected tolerant cells were then treated with 100 ng/ml PMA for 30 min or 60 µM myristoylated PKC-{zeta} pseudosubstrate for 60 min followed by PMA. Naïve and LPS-pretreated cells were stimulated with 100 ng/ml LPS for 5 min. (A) Lipid raft protein harvested and analyzed by Western blot for TLR4, HSP70, CD14, Gab2, {alpha}-tubulin, and Lyn. (B) Nonraft protein was harvested and analyzed by Western blot for TLR4, HSP70, CD14, {alpha}-tubulin, Lyn, and actin. Representative blots were demonstrated from one of three separately performed experiments.

 
The findings demonstrated for differentiated THP-1 were not only specific to this cell line, but in fact, similar results were demonstrated in human peripheral blood monocytes (Fig. 9 ). As demonstrated, isolated lipid raft protein from nonstimulated peripheral blood monocytes contains little TLR4 or HSP70. Following endotoxin exposure, TLR4 and HSP70 are mobilized to the lipid raft. Similar to differentiated THP-1 cells, tolerance induced by LPS pretreatment is associated with a significant attenuation in TLR4 and HSP70 mobilization to the lipid raft. This condition is reversed with PMA pretreatment. Equal loading of raft fractions was insured by equal detection of GM1-ganglioside under each experimental condition.


Figure 9
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  		Figure 9. Impaired, endotoxin-mediated TLR4 assembly is not unique to differentiated THP-1 cells. Isolated human peripheral blood monocytes were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected cells were then treated with 100 ng/ml PMA for 30 min. Naïve and LPS-pretreated cells were stimulated with 100 ng/ml LPS for 5 min. Lipid raft protein was harvested and analyzed by Western blot for TLR4, HSP70, and GM1-ganglioside. Representative blots were demonstrated from one of two separately performed experiments.

 
These findings were verified using immunohistochemistry. As demonstrated in Figure 10A , endotoxin resulted in the lipid raft TLR4 redistribution. Within unstimulated cells, a diffuse membrane pattern for the lipid raft is demonstrated. This diffuse pattern becomes polymerized, forming macrodomains. TLR4, conversely, appears localized evenly within the plasma membrane and upon subsequent endotoxin exposure, appears to become associated with lipid rafts, as demonstrated on the composite image, with the fluorescence merge appearing yellow. PMA pretreatment had no effect on these redistribution events. Similar to the immunoblots, tolerance induced by endotoxin pretreatment resulted in significant attenuation of TLR4 and lipid raft colocalization (Fig. 10B) . This effect was reversed by pretreatment with PMA and therefore, implicates attenuated PKC activation and TLR4 complex assembly as the critical events associated with the induction of endotoxin tolerance.


Figure 10
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  		Figure 10. Lipid raft mobilization of TLR4. Differentiated THP-1 cell colocalization of TLR4 and lipid rafts was determined by immunohistochemistry. (A) Naïve and (B) tolerant cell cellular expression of lipid rafts (red) and TLR4 (green) was determined. Colocalization was determined by overlaying images, resulting in yellow fluorescence.

 
Tolerance is associated with attenuated PKC-{zeta} activation
Demonstrating attenuated mobilization of TLR4 into lipid rafts during tolerance and reversal with PKC activation, we next set out to explore the potential effect on the PKC isoform, thought to be in part responsible for this event, PKC-{zeta}. To determine the effect of tolerance on PKC-{zeta}, naïve and tolerant cells exposed to 100 ng/ml LPS were examined by immunoblot for the phosphorylation of PKC-{zeta} (Fig. 11 ). Endotoxin exposure led to the phosphorylation of PKC-{zeta}. This phosphorylation event was not enhanced by PMA pretreatment but was activated by PMA alone. Tolerance, however, was associated with a marked attenuation of endotoxin-mediated PKC-{zeta} phosphorylation, which was reversed and maintained by PMA pretreatment.


Figure 11
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  		Figure 11. Impaired, endotoxin-mediated PKC-{zeta} phosphorylation occurs in tolerant cells. Differentiated THP-1 cells were pretreated for 18 h with medium or 10 ng/ml LPS, washed with PBS, and returned to fresh medium. Selected cells were then treated with 100 ng/ml PMA for 30 min. Naïve and LPS-pretreated cells were stimulated with 100 ng/ml LPS for 1–2 min. Cellular protein was harvested and analyzed by Western blot for the phosphorylation of PKC-{zeta}. Representative blots were demonstrated from one of four separately performed experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Major stress response, including sepsis, remains one of the leading causes of morbidity and mortality among critically ill patients. A patient may succumb to an excessive immunoinflammatory response to the inciting event or recover often with a state of immune hyporesponsiveness, which has been termed immunoparalysis or immunodepression [21 ]. Innate immune cells obtained from patients in a state of immunoparalysis are less responsive to ex vivo inflammatory stimulation, resulting in attenuated production of proinflammatory mediators [22 ]. This response is thought to be an excessive, compensatory mechanism, which may be beneficial to the patient in preventing a rogue inflammatory response that is responsible for systemic inflammatory response syndrome, acute respiratory distress syndrome, and multiorgan dysfunction syndrome. Unfortunately, prolonged or excessive immunosuppression of the innate immune system has been correlated with increased risk of infection, prolonged Intensive Care Unit stay, and death [23 ]. This compensatory mechanism provides the opportunity for a nosocomial infection to become invasive, leading to secondary sepsis, further dysregulation of the immune response, and eventual organ dysfunction [24 ].

Although a significant amount of work has been dedicated toward these clinical states, the molecular mechanisms involved, for the most part, remain unknown. Recently, it has been demonstrated that critically ill patients with increased risk for morbidity and mortality during sepsis have depressed ex vivo endotoxin-mediated mononuclear cell function [22 ]. To further elucidate the potential mechanisms involved during this state in vitro and in vivo, endotoxin tolerance models have been created. The consistent finding within these models, as well as the one used within this study, is attenuated inflammatory mediator production in response to endotoxin stimulation following a prior nonlethal exposure, which is characteristic of the clinical state.

Endotoxin activates the macrophage through complex binding with LBP and the specific GPI-anchored cell membrane receptor, CD14. Although surface expression of CD14 has not been demonstrated to be affected by tolerance induction, binding of endotoxin to the cell surface following the induction of tolerance has not been investigated previously [18 ]. Based on our current observation, no change in cell surface binding of endotoxin was demonstrated with tolerance induction. Thus, we focused on subsequent mechanisms known to be activated within the macrophage following initial binding of endotoxin to CD14.

Following LPS/LPB binding to CD14, TLR4 and HSP70 are mobilized to lipid rafts and subsequently, complex with CD14 and other membrane components to form GPI-mediated receptor complexes [6 ]. Assembly of this complex results in a conformational change within TLR4, leading to the membrane recruitment of MyD88 and IRAK-1, which leads to eventual activation of the MAPK family, consisting of ERK1/2, p38, and JNK/SAPK. Activation of these various pathways eventually leads to up-regulation of gene transcription and translation, leading to increased inflammatory mediator production and release. Endotoxin tolerance is associated with attenuation of all of these signaling events, as demonstrated in this study.

As a result of the limited effect on LPS binding and the attenuation in the initial steps in intracellular signaling following tolerance induction, we set out to determine if endotoxin tolerance within our model was associated with changes in the TLR4. Despite observations by Nomura and colleagues [14 ], demonstrating down-regulation of surface expression TLR4 in C57BL/6J mice peritoneal macrophages following tolerance induction, we were not able to demonstrate any change in surface expression or cellular content of TLR4 following tolerance induction. Other investigators have also demonstrated similar findings [16 , 25 26 27 28 ]. Although surface expression of TLR4 was unchanged, endotoxin-mediated TLR4 mobilization to lipid rafts was attenuated significantly during tolerance. This lack of mobilization is intriguing, as recent data by us and others have suggested that complex formation on these cholesterol and sphingolipid-rich microdomains is essential to eventual activation of the macrophage in response to endotoxin [6 , 19 , 29 ].

The specificity of these finding was verified through the use of several controls. It has been well established that HSP70 and TLR4 are recruited to lipid rafts following endotoxin stimulation [6 ]. In addition, several recent studies have suggested that HPS70 at low levels is constitutively found within rafts [30 , 31 ]. This was the case within our study. However, TLR4 is not found constitutively but recruited following endotoxin stimulation. In addition to these receptor proteins, Lyn, CD14, and GM1-gangliosides are found strictly within the raft fraction [30 , 32 33 34 35 36 ]. However, other proteins, such as Gab2 and {alpha}-tubulin, during SDS-PAGE electrophoresis, are found only within the nonraft fraction [6 , 30 , 37 ]. Therefore, based on all these controls, it appears that the mobilization of TLR4 following endotoxin stimulation represented in these immunblots is not a result of selective contamination.

Although this study demonstrates colocalization of TLR4 and lipid rafts following endotoxin, the mechanism responsible for this still remains unknown. This study does demonstrate that lipid rafts are distributed throughout the cell, and upon endotoxin stimulation, these rafts become polarized, forming macrodomains. Gulbins and Kolesnick [38 ] previously described the formation of these lipid raft macrodomains. As a result, changes in lipid raft fluidity occur, resulting in the recruitment of receptor proteins, such as TLR4, which allows an interaction of TLR4 with CD14. This allows TLR4 to interact with LPS, resulting in TLR4 activation. During endotoxin tolerance, this polarization and redistribution of lipid rafts to form these surface macrodomains are attenuated. Although this may be a result of membrane ruffling, as previously demonstrated by Glebov and Nichols [39 , 40 ] in T cells, the exact mechanism remains unknown within monocytes and macrophages. In fact, recent evidence suggests that the recruitment of proteins in rafts occurs during ceramide generation, leading to gel-phase fluidity and macrodomain formation within the raft [38 ]. However, unlike the previous reports by Glebov and Nichols [39 , 40 ] in T cells, this study demonstrates that receptor proteins, such as TLR4 and HSP70, are recruited to the lipid raft following stimulation.

Despite this inability to define the mechanism of lipid raft reorganization, we have demonstrated previously that the activation of the atypical PKC, PKC-{zeta}, is essential toward the lipid raft mobilization of TLR4 [19 ]. In the current model, induction of tolerance was associated with attenuation in subsequent PKC-{zeta} phosphorylation by endotoxin, which was not a result of change in total cellular content of PKC-{zeta}. Attenuated activation of PKC-{zeta} is intriguing, as previous data by West and colleagues [20 ] have suggested that nonspecific, pharmacological activation of the PKC family can reverse tolerance induction.

Pretreatment of tolerant cells with PMA reversed not only inflammatory mediator production, as previously demonstrated, but also reversed the effects of tolerance on early upstream signaling as a result of IRAK-1 and MAPK activation in response to endotoxin. Having demonstrated a return of endotoxin-mediated, intracellular signaling, we investigated the effect of PMA on TLR4 and HSP70 mobilization to lipid rafts. Pretreatment with PMA, similar to the effect on intracellular signaling, resulted in reversal of tolerance-induced inhibition of TLR4 and HSP70 mobilization to lipid rafts. This reversal of tolerance by PMA and its known effect on PKC-{zeta} support the essential role of PKC-{zeta} on this mobilization event.

Although this study provides further insight into the mechanism of tolerance, the mechanism in which PKC-{zeta} activity is attenuated during tolerance induction remains unknown. A potential mechanism, however, centers on the regulation of PKC activity by the phosphatase SHIP. Recently, SHIP has been demonstrated to be essential to the activation of the macrophage in response to endotoxin through regulation of AKT activity [41 ]. Tolerance induction appears to occur following endotoxin exposure through a TGF-β-driven pathway in which Sma- and Mad-related protein activation leads to increased production of SHIP [42 , 43 ]. This increased cellular content of SHIP serves to attenuate subsequent endotoxin responsiveness, resulting in tolerance. Potentially, this increased cellular content of SHIP may serve to attenuate PKC-{zeta} activity and membrane mobilization. This potential mechanism is consistent with data showing that SHIP serves to regulate PKC membrane mobilization and activation during mast cell degranulation [44 ].

In summary, this paper provides further insight into the molecular mechanisms in which endotoxin tolerance occurs. Although this in vitro model clearly demonstrates that endotoxin tolerance is associated with attenuated PKC-{zeta} activity and TLR4 assembly, a similar finding has not been identified in vivo. This study suggests a potential mechanism in which recently demonstrated SHIP activity could regulate PKC-{zeta} activity and contribute to the mechanisms underlying endotoxin tolerance. It is therefore plausible to use these novel insights to determine the potential mechanisms in vivo and identify therapeutic targets to modify the dysregulated, inflammatory response during sepsis.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant KO8 GM68816-01.

Received January 24, 2006; revised July 4, 2006; accepted August 5, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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