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Originally published online as doi:10.1189/jlb.1105622 on June 5, 2006

Published online before print June 5, 2006
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(Journal of Leukocyte Biology. 2006;80:407-414.)
© 2006 by Society for Leukocyte Biology

Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14

Joseph Cuschieri1, Jens Billgren and Ronald V. Maier

Department of Surgery, University of Washington, Seattle

1Correspondence: Harborview Medical Center, 325 Ninth Avenue, Box 359796, Seattle, WA 98104. E-mail: jcuschie{at}u.washington.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure, as a result of protein kinase C (PKC)-{zeta} activation. However, the mechanism responsible for this remains unknown. The purpose of this study is to determine if LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide produced by phosphatidylcholine-specific phospholipase C (PC-PLC) or CD14. To study this, THP-1 cells were stimulated with LPS. Selected cells were pretreated with the PC-PLC inhibitor D609, exogenous C2 ceramide, CD14 neutralizing antibody, or TLR4 neutralizing antibody. LPS led to production of ceramide, phosphorylation of PKC-{zeta}, and assembly of the TLR4 within lipid rafts. This was followed by activation of the mitogen-activated protein kinase family and the liberation of cytokines. Pretreatment with D609 or CD14 blockade was associated with attenuated LPS-induced ceramide production, TLR4 assembly on lipid rafts, and cytokine production. Pretreatment with TLR4 blockade did not affect LPS-induced ceramide production but was associated with significant attenuation in cytokine production. Treatment with C2 ceramide prior to LPS reversed the inhibitory effects induced by D609 but not of CD14 or TLR4 blockade. C2 ceramide alone induced the activation of PKC-{zeta} and the assembly of TLR4 but was not associated with cytokine liberation. This study demonstrates that TLR4 assembly and activation following LPS exposure require the production of ceramide by PC-PLC, which appears to be CD14-dependent.

Key Words: Key Words: • ceramide • TLR4 • endotoxin • lipid raft • sphingolipids


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gram-negative sepsis remains a leading cause of morbidity and mortality [1 ]. The initiation of sepsis results in a dysregulated activation of the innate immune response leading to the development of adult respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) [2 ]. This altered host response is orchestrated, in part, through the liberation of various inflammatory mediators by the tissue-fixed macrophage in response to Gram-negative bacteria cellular components, such as endotoxin.

Endotoxin, a cell-wall component of Gram-negative bacteria, results in the activation of the tissue-fixed macrophage through the Toll-like receptor 4 (TLR4) complex. Activation of this complex requires the binding of endotoxin to the acute-phase protein, lipopolysaccharide (LPS)-binding protein (LBP) [3 ]. Binding to LBP allows endotoxin to bind to the LPS glycosylphosphatidylinositol (GPI)-anchored recognition receptor, CD14, which is found on lipid rafts [4 ]. Activation of CD14, in turn, is thought to result in the mobilization of TLR4, MD2, and heat shock protein (HSP)70 to lipid rafts, resulting in formation of the TLR4 complex and presentation of endotoxin to TLR4 [5 ]. Previously, we demonstrated that this process is dependent on the activation of the atypical protein kinase C (PKC), PKC-{zeta} [6 ].

This atypical PKC is found abundantly within the tissue-fixed macrophage. The activation of this kinase is known to be regulated by phosphatidylinositol (3,4,5)-triphosphate, ceramide, and phosphatidic acid [7 ]. Ceramide, which is generated by the macrophage in response to endotoxin exposure, has been implicated as directly involved in the macrophage response to endotoxin [8 , 9 ]. However, the full role that ceramide plays in activation of the tissue-fixed macrophage by endotoxin remains unknown.

Ceramide is a lipid second messenger, which is generated from membrane sphingolipids by sphingomyelinase [10 , 11 ]. Previously, activation of sphingomyelinase within the macrophage has been demonstrated to occur through the generation of diacylglycerol (DAG) by phosphatidylcholine-specific phospholipase C (PC-PLC) [12 ]. These independent observations are intriguing, as they indirectly speculate that ceramide generation from sphingolipids found within lipid rafts may be involved in TLR4 complex formation through the activation of PKC-{zeta}.

In this study, we set out to determine more fully the role that PC-PLC and ceramide play in TLR4 formation and activation by endotoxin. To investigate the role of PC-PLC, we used the potent, selective inhibitor tricyclodecan-9-yl xanthogenate (D609). Furthermore, we used exogenous ceramide, C2 ceramide, not only to determine the direct effects of ceramide on TLR4 complex formation but also to determine if exogenous ceramide could reverse the effects induced by PC-PLC inhibition. Finally, we set out to determine if these events were CD14- or TLR4-dependent through selective neutralization of CD14 and TLR4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Ultrapure Escherichia coli 0111:B4 LPS was obtained from InvivoGen (San Diego, CA). D609 obtained from Sigma Chemical Co. (St. Louis, MO) was dissolved in sterile H2O at a concentration of 300 mg/ml. C2 ceramide obtained from Sigma Chemical Co. was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM. Endotoxin contamination of D609 and C2 ceramide was tested by the Limulus amebocyte lysate assay (E-TOXATE kit, Sigma Chemical Co.) and found to be <0.05 ng/ml.

Cell isolation and treatment
Human promonocytic THP-1 cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 (BioWhitaker, Walkersville, MD) supplemented with 10% fetal calf serum (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 ng/ml phorbol 12-myristate 13-acetate for 2 days at a concentration of 5 x 106 cells/ml on tissue culture-treated plates. Selected cells were pretreated with 100 µM D609 for 30 min, 10 µg/ml anti-human CD14 antibody (R&D Systems, Minneapolis, MN), 10 µg/ml sheep isotype control (Southern Biotech, Birmingham, AL), 20 µg/ml anti-human TLR4 antibody (eBioscience, San Diego, CA), 20 µg/ml mouse immunoglobulin G2 (IgG2) isotype control (eBioscience), or the vector DMSO. Cells were then stimulated with 100 ng/ml LPS, 16 µM C2 ceramide, or the combination of both for various periods of time as indicated in the figure legends.

Ceramide production
Following LPS stimulation, lipids were extracted from treated, differentiated THP-1 cells and resolved from sphingosine using thin-layer chromatography before acid hydrolysis, conversion to derivatization, and high-pressure ligquid chromatography (HPLC) analysis, as described by Merrill Jr. and Wang [13 ]. Briefly, sphingosine plus 200 pm d-erythro-C20-sphingosine, an internal standard, was extracted as described previously by Bligh and Dyer [14 ]. The chloroform layer was isolated and dried under nitrogen gas. The dried extracts were resuspended in 0.33 ml chloroform and 0.66 ml 0.1 M KOH in methanol. Samples were then incubated for 1 h at 37°C and then rinsed with 1 ml chloroform and 1 ml 1.0 M NaCl. The chloroform phase was washed with NaCl and dried with nitrogen gas. Orthophthaladehyde derivatives were prepared by dissolving the dried samples in 50 ml methanol, followed by the addition of 50 ml o-phthaldialdehyde (OPA) reagent (5 mg OPA in 100 ml ethanol, 9.9 ml 3% boric acid, and 5 ml 2-mercaptoethanol), incubated at room temperature for 5 min, diluted with methanol/water (94/6 v/v), and quantitated by HPLC as described by Merrill Jr. et al. [15 ]. Orthophthaladehyde derivatives were separated on a Beckman UI-transphere C-18 column (Beckmam Coulter, Fullerton, CA) with methanol/water (94/6 v/v) mobile phase at a rate of 1 ml/min. The derivatives were detected using a Thermoseparation Products Spectra System FL3000 fluorescence detector (Thermo Separation Products, San Jose, CA) at 340 nm excitation and 454 nm emission wavelengths.

Lipid raft protein extraction
Following LPS or C2 ceramide stimulation, 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 dithiothreitol (DTT), 200 µM phenylmethylsulfonyl fluoride (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] 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 24 h at 100,000 g at 4°C. Protein at the 5–35% sucrose interface, representing the lipid raft portion, was isolated and resuspended 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 or C2 ceramide stimulation, total cellular protein was extracted 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, 40 mM {alpha}-glycerophosphate). Protein concentration was determined using the Pierce BCA protein assay.

Western blots
Total cellular protein was electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond-enhanced chemiluminescence nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The membrane was blocked for 1 h at room temperature with 1% bovine serum albumin (BSA), 5% BSA, or 5% milk and then incubated with an anti-human dual-phosphorylated Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK; Promega, Madison, WI), anti-human-phosphorylated p38 (Cell Signaling, Beverly, MA), or anti-human dual-phosphorylated extracellular signal-regulated kinase (ERK) 1/2 (Cell Signaling) antibody for 12 h at 4°C, respectively. Blots were then incubated in a horseradish peroxidase (HRP)-conjugated secondary antibody against the primary at room temperature for 1 h. The blot was 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 ERK 1, p38, and JNK1 to confirm equal loading.

Extracted lipid raft protein was run for total TLR4, HSP70, CD14, and Lyn. Gels were run similarly and transferred to nitrocellulose membranes. Following initial blockade in 5% milk, membranes were incubated with anti-human TLR4 (Zymed, San Francisco, CA), anti-human HSP70 (Upstate, Charlottesville, VA), anti-human CD14 (R&D Systems), and anti-human Lyn (Upstate) antibodies overnight at 4°C. Blots were then incubated in a HRP-conjugated IgG secondary antibody against the primary at room temperature for 1 h and developed as described previously.

Tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL)-10 production
Following the treatments described previously, supernatants were harvested under all conditions following 8 h of stimulation. TNF-{alpha} and IL-10 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 and noted to be viable under all conditions including D609 treatment.

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
 
Endotoxin exposure results in ceramide production, which is dependent on PC-PLC activation and endotoxin binding to CD14
Differentiated THP-1 cells were exposed to 100 ng/ml endotoxin. To determine the potential role of PC-PLC on this process, selected cells were pretreated for 30 min with the selective PC-PLC inhibitor D609. Following treatment, cell lysis was performed, and ceramide production was determined by HPLC (Fig. 1 ). Endotoxin stimulation induced production of ceramide over baseline levels. Pretreatment with D609 alone did not result in ceramide production but did result in marked attenuation of endotoxin-mediated production. This attenuated production was similar to unstimulated control cells. The vector DMSO had no effect (data not shown). In addition, the role of CD14 and TLR4 was determined by pretreating cells with anti-human CD14 or anti-human TLR4 to neutralize the binding of endotoxin. CD14 neutralization, similar to D609 pretreatment, resulted in significant attenuation in endotoxin-mediated ceramide production (Fig. 1) . TLR4 neutralization, conversely, did not significantly affect endotoxin-mediated ceramide production, thus implicating PC-PLC and CD14 as critical components involved in endotoxin-mediated ceramide generation.


Figure 1
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  		Figure 1. Effect of PC-PLC inhibition and CD14 blockade on ceramide production. To determine the role of PC-PLC and CD14 on LPS-mediated ceramide production, differentiated THP-1 cells were stimulated with 100 ng/ml LPS. Selected cells were pretreated with 100 µM D609, 10 µg/ml anti-human CD14 antibody, or 20 µg/ml anti-human TLR4 antibody. Lipids were extracted following 5 min of LPS stimulation. Ceramide was determined by HPLC. Values represent the mean ± SEM for three experiments performed separately (*, P<0.05, compared with LPS-treated).

 
Endotoxin-mediated activation of the atypical PKC, PKC-{zeta}, is dependent on PC-PLC-mediated ceramide production and CD14
To determine the role of PC-PLC on the activation of PKC-{zeta} by endotoxin, differentiated THP-1 cells were exposed to endotoxin with selected cells being pretreated with D609 or anti-human CD14 antibody. Following stimulation, cellular protein was harvested and analyzed by Western blot for the phosphorylation of PKC-{zeta} (Fig. 2A ). Endotoxin resulted in the early phosphorylation of PKC-{zeta}. Inhibition of PC-PLC by D609 resulted in marked attenuation in PKC-{zeta} phosphorylation, and the vector DMSO had no significant effect (data not shown). Similarly, neutralization of CD14 resulted in attenuation of endotoxin-mediated PKC-{zeta} phosphorylation but was not affected by mouse IgG2a isotype control antibody (Fig. 2A) .


Figure 2
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  		Figure 2. Effect of CD14 blockade, PC-PLC inhibition, and ceramide on PKC-{zeta} phosphorylation. (A) To determine the role of PC-PLC or CD14 on LPS-mediated PKC-{zeta} activation, differentiated THP-1 cells were stimulated with 100 ng/ml LPS. Selected cells were pretreated with 100 µM D609, 10 µg/ml anti-human CD14 antibody, or 10 µg/ml mouse IgG2a isotype control antibody. Cellular protein was then extracted following 5 min of LPS stimulation. PKC-{zeta} phosphorylation (p-PKC{zeta}) was determined by immunoblot (upper). Equal loading was determined by immunoblotting for total PKC-{zeta} (lower). (B) To determine the effect of ceramide on PKC-{zeta} activation, differentiated THP-1 cells were stimulated with 16 µM C2 ceramide. Selected cells were pretreated with 100 µM D609. PKC-{zeta} phosphorylation (upper) and total PKC-{zeta} (lower) were then determined by subjecting extracted cellular protein to immunoblot analysis.

 
To determine if the attenuation in endotoxin-mediated PKC-{zeta} activation by D609 was a result of attenuated ceramide production, differentiated THP-1 was exposed to endogenous ceramide, with or without D609 pretreatment (Fig. 2B) . Similar to endotoxin, ceramide exposure alone resulted in the phosphorylation of PKC-{zeta}, which when induced by ceramide, was not affected by D609 pretreatment or the vector DMSO (data not shown), thus suggesting that PKC-{zeta} phosphorylation is dependent on ceramide production and that endotoxin-mediated PKC-{zeta} phosphorylation is dependent on PC-PLC and CD14-dependent ceramide production.

Endotoxin-induced lipid raft receptor clustering is dependent on PC-PLC-mediated ceramide production and CD14
Demonstrating the effects on PKC-{zeta} activation, we next set out to determine if lipid raft clustering of TLR4 components was affected by PC-PLC inhibition or CD14 blockade. To determine the role of PC-PLC and CD14 on lipid raft clustering, differentiated THP-1 cells were exposed to endotoxin, with or without D609, anti-human CD14 neutralizing antibody, or sheep IgG isotype control antibody. Following stimulation, lipid raft protein was extracted and analyzed by Western blot for TLR4, HSP70, CD14, and Lyn (Fig. 3A ). Endotoxin induced the lipid raft recruitment of TLR4 and HSP70. CD14 and Lyn were constitutively found on lipid rafts. Inhibition of PC-PLC or blockade of CD14 resulted in a marked attenuation of endotoxin-mediated lipid raft receptor clustering of TLR4 and HSP70. Treatment with sheep IgG isotoype control antibody had no effect on endotoxin-induced TLR4 and HSP70 lipid raft recruitment.


Figure 3
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  		Figure 3. Effect of CD14 blockade, PC-PLC inhibition, and ceramide on lipid raft receptor clustering. (A) To determine the role of PC-PLC on LPS-mediated TLR4 clustering on lipid rafts, differentiated THP-1 cells were stimulated with 100 ng/ml LPS. Selected cells were pretreated with 100 µM D609, 10 µg/ml anti-human CD14 antibody, or 10 µg/ml mouse IgG2a isotype control antibody. Lipid raft protein was harvested following 10 min of LPS stimulation. HSP70, TLR4, and Lyn were determined by immunoblot. (B) To determine the effect of ceramide on TLR4 clustering on lipid rafts, differentiated THP-1 cells were stimulated with 16 µM C2 ceramide. Selected cells were pretreated with 100 mM D609. HSP70, TLR4, and Lyn were then determined by subjecting extracted lipid raft protein to immunoblot analysis.

 
To determine if the attenuation in endotoxin-mediated lipid raft receptor clustering by D609 was a result of attenuated ceramide production, differentiated THP-1 was exposed to exogenous ceramide, with or without D609 pretreatment (Fig. 3B) . Similar to endotoxin, ceramide exposure resulted in the lipid raft mobilization of TLR4 and HSP70. This lipid raft receptor clustering induced by ceramide was not affected by D609 pretreatment. These findings are similar to those demonstrated for PKC-{zeta} phosphorylation.

Endotoxin-induced intracellular mitogen-activated protein kinase (MAPK) activation is dependent on PC-PLC-induced ceramide production
Demonstrating the effects on lipid raft receptor clustering, we set out to determine the effect of PC-PLC activation and ceramide production on intracellular activation of the MAPK family, composed of ERK 1/2, p38, and JNK/SAPK. To determine the effects, differentiated THP-1 cells were exposed to endotoxin, with or without D609 pretreatment (Fig. 4 ). Following treatment, cellular protein was extracted and analyzed by Western blot for the phosphorylation of ERK 1/2, p38, and JNK/SAPK. Endotoxin led to the phosphorylation of each member of the MAPK family. Pretreatment with D609 followed by endotoxin exposure led to near-abolishment of MAPK signaling. Similarly, blockade of CD14 resulted in similar attenuation in endotoxin-mediated MAPK activation, and sheep IgG isotype control antibody was associated with no effect on endotoxin-mediated MAPK activation (data not shown).


Figure 4
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  		Figure 4. Effect of PC-PLC inhibition and ceramide on endotoxin-mediated MAPK activation. To determine the role of PC-PLC and ceramide production on LPS-mediated MAPK activation, consisting of ERK 1/2, p38, and JNK/SAPK, differentiated THP-1 cells were stimulated with 100 ng/ml LPS, 16 µM C2 ceramide, or both. Selected cells were pretreated with 100 µM D609. Cellular protein was then extracted following 30 min of stimulation. Phosphorylation of each of the MAPK was then determined by immunoblot. Equal loading was verified by total ERK 1, p38, and JNK 1 (data not shown).

 
To determine the role of ceramide on MAPK signaling, differentiated THP-1 cells were exposed to exogenous ceramide without endotoxin. Ceramide exposure led to the phosphorylation of ERK 1/2 and JNK/SAPK. However, it was not associated with the activation of p38. Exogenous ceramide treatment of D609-pretreated cells was associated with activation of all MAPK family members, including p38, by endotoxin.

Endotoxin-induced proinflammatory mediator production is dependent on PC-PLC activation, CD14, and TLR4
Demonstrating the effects on intracellular signaling, we set out to determine the role of PC-PLC, ceramide, CD14, and TLR4 on inflammatory mediator production. To determine this effect, differentiated THP-1 cells were exposed to endotoxin, with or without D609, anti-human CD14 neutralizing antibody, anti-human TLR4 neutralizing antibody, sheep IgG isotype control antibody, or mouse IgG2a isotype control antibody pretreatment. Harvested supernatants were then analyzed by enzyme-linked immunosorbent assay (ELISA) for the production of TNF-{alpha} and IL-10 (Figs. 5 and 6 , respectively). Endotoxin led to the production of TNF-{alpha} and IL-10. The production of TNF-{alpha} and IL-10 was not affected by pretreatment with either isotype control antibody. Pretreatment with D609, CD14 blockade, or TLR4 blockade, similar to the effect on lipid raft receptor complex formation and intracellular signaling, was associated with a significant attenuation of endotoxin-mediated production of both inflammatory mediators.


Figure 5
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  		Figure 5. Effect of CD14 blockade, PC-PLC inhibition, and ceramide on endotoxin-mediated TNF-{alpha} production. To determine the role of PC-PLC and ceramide production on LPS-mediated TNF-{alpha}, differentiated THP-1 cells were stimulated with 100 ng/ml LPS, 16 µM C2 ceramide, or both. Selected cells were pretreated with 100 µM D609 (A) or blocking antibodies to CD14 or TLR4 (B). Supernatants were harvested and analyzed by ELISA (R&D Systems). Values represent the mean ± SEM for five separately performed experiments (*, P<0.05, compared with LPS-treated, similar conditions; {dagger}, P<0.05, compared with similar condition without ceramide).

 

Figure 6
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  		Figure 6. Effect of CD14 blockade, PC-PLC inhibition, and ceramide on endotoxin-mediated IL-10 production. To determine the role of PC-PLC and ceramide production on LPS-mediated IL-10, differentiated THP-1 cells were stimulated with 100 ng/ml LPS, 16 µM C2 ceramide, or both. Selected cells were pretreated with 100 µM D609 (A) or blocking antibodies to CD14 or TLR4 (B). Supernatants were harvested and analyzed by ELISA (R&D Systems). Values represent the mean ± SEM for five separately performed experiments (*, P<0.05, compared with LPS-treated, similar conditions; {dagger}, P<0.05, compared with similar condition without ceramide).

 
In a similar manner, the effect of ceramide was determined by treating differentiated THP-1 cells with ceramide. This resulted in no significant inflammatory mediator production. However, treatment of D609-treated cells with exogenous ceramide re-established endotoxin-mediated inflammatory mediator production but not for cells treated with anti-human CD14 or anti-human TLR4 neutralizing antibodies.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Endotoxin, a major constituent of the outer membrane of Gram-negative bacteria, activates the tissue-fixed macrophage. Although this activation is important for eradication of invasive Gram-negative organisms through the local production of inflammatory mediators, overproduction of these factors during septic states is believed important in the pathogenesis of sepsis-induced ARDS and MODS [16 ].

Activation of the tissue-fixed macrophage by endotoxin requires the binding of LPS to LBP. Binding to LBP allows LPS to bind to the LPS recognition receptor, CD14, which is a GPI-anchored protein that does not have a cytoplasmic domain. This protein is thought to be contained within lipid raft microdomains on the plasma membrane [5 , 17 , 18 ]. Lipid rafts are sphingolipid-enriched membrane domains and are believed to be critical sites for receptor complex assembly and signal transduction. Constitutively found on lipid rafts are various members of the SRC family of kinases, such as Lyn [19 , 20 ]. Although the full role of these kinases remains unknown, the SRC family members play critical roles in endotoxin-mediated signaling within the macrophage [21 ].

Although complex binding of LPS-LPB to CD14 is essential toward activation by endotoxin of the tissue-fixed macrophage, intracellular activation occurs through receptor complex assembly of CD14, TLR4, and MD2 [22 ]. It appears that based on our data and that by Triantafilou and colleagues [5 ] that TLR4 found on the plasma membrane and HSP70 found intracellularly migrate into the lipid raft following exposure to endotoxin. Although the complete mechanism responsible for the migration is unknown, previous data from us suggest that it is dependent on activation of the atypical PKC, PKC-{zeta} [6 ]. Although the potential mechanism in which CD14 could result in PKC-{zeta} activation remains unknown, activation of this atypical PKC appears to be dependent on ceramide production [23 ].

Therefore, this study was performed to determine the role that ceramide generation plays in the activation of the macrophage by endotoxin. We hypothesized that endotoxin binding to CD14 would lead to the activation of PC-PLC and generation of DAG, which would then result in the activation of sphingomyelinase, leading to the generation of ceramide. Formation of ceramide would result in activation of PKC-{zeta}, leading to TLR4 complex assembly and subsequent intracellular activation and inflammatory mediator production.

To study this, we first set out to determine if ceramide was generated within our in vitro model. Using differentiated THP-1 cells, we were able to demonstrate that ceramide was produced early following endotoxin exposure. The generation of ceramide by endotoxin appeared dependent on PC-PLC activation, as inhibition of PC-PLC by D609 resulted in a marked attenuation in ceramide production. These findings are consistent with previous observations by Monick and colleagues [12 ], demonstrating PC-PLC-dependent DAG and ceramide production in human alveolar macrophages in response to endotoxin. In addition, production of ceramide by endotoxin is dependent on binding of endotoxin to CD14 but not TLR4, as demonstrated through the blocking of endotoxin binding to CD14, and TLR4, through the selective use of neutralizing antibodies.

Demonstrating the role of PC-PLC and CD14 in endotoxin-mediated ceramide production, we next set out to determine if PC-PLC activation and endotoxin binding to CD14 played a role in the activation of PKC-{zeta} by endotoxin. We were able to demonstrate that endotoxin exposure resulted in the phosphorylation of PKC-{zeta}, and the phosphorylation of this kinase was dependent on binding of endotoxin to CD14 and PC-PLC activation, resulting in the generation of ceramide. Although not previously demonstrated for CD14, Monick and colleagues [12 ] demonstrated previously the dependence of PC-PLC on endotoxin-mediated activation of PKC-{zeta} as it related to the activation of the MAPK ERK 1/2 [12 ].

Re-establishing the relationship among PC-PLC, ceramide, and PKC-{zeta}, we next set out to determine if PC-PLC or ceramide is involved in lipid raft mobilization of HSP70 and TLR4. We were able to demonstrate that endotoxin-mediated HSP70 and TLR4 lipid raft mobilization were dependent on PC-PLC activation. In addition, we were able to demonstrate that endotoxin binding to CD14 was critical to these events and that ceramide alone could result not only in the activation of PKC-{zeta} but also the formation of this lipid raft cluster. These novel findings provide further insight into the mechanisms responsible for the formation of this receptor complex. It is consistent with our data demonstrating the role of CD14, PC-PLC, and ceramide on PKC-{zeta} activation.

Establishing the role of CD14, PC-PLC, and ceramide on TLR4 complex formation, we next set out to define more definitively the role of these factors on MAPK activation. Inhibition of binding to CD14 or PC-PLC was associated with marked attenuation in the activation of all members of the MAPK family by endotoxin. This inhibition was reversed if ceramide and endotoxin exposure were simultaneous. It is interesting that ceramide alone only led to robust activation of ERK 1/2 and minimal activation of JNK/SAPK. This selective effect by ceramide provides indirect evidence that it is involved in the activation of these kinases through a PKC-dependent pathway, such as raf-1 and/or MAPK kinase [24 ]. This selective activation by ceramide of ERK 1/2 is consistent with previous data from alveolar macrophages [12 ].

In addition to these effects, endotoxin binding to CD14 and TLR4 and activation of PC-PLC were demonstrated to be important to subsequent inflammatory mediator production. TNF-{alpha} and IL-10 production, in response to endotoxin, was significantly attenuated if cells were pretreated with anti-human CD14 neutralizing antibody, anti-human TLR4 neutralizing antibody, or D609. This attenuation in cytokine production was reversed if exogenous ceramide was administered in D609-pretreated cells but not in CD14- and TLR4-blocked cells. This is obviously consistent with our data demonstrating that endotoxin binding to CD14 and PC-PLC activation is central to the production of ceramide, the formation of the TLR4 complex, and subsequent intracellular activation of the MAPK family.

Our data thus provide a novel mechanism by which lipid raft clustering occurs on lipid rafts. The formation of the TLR4 complex, which is dependent on PKC-{zeta}, requires the initial binding of endotoxin to CD14, followed by the production of ceramide through the activation of PC-PLC. Although the mechanism responsible for the activation of PC-PLC by CD14 remains unknown, it appears that the receptor complex formation induced by this particular pathway allows CD14 to present endotoxin to TLR4, leading to intracellular activation and inflammatory mediator production. Taken together, these findings suggest potential new and novel, therapeutic targets, which may serve to regulate the macrophage during Gram-negative sepsis.

Received November 1, 2005; revised January 31, 2006; accepted April 3, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hurley, J. C. (2003) Endotoxemia and Gram-negative bacteremia as predictors of outcome in sepsis: a meta-analysis using ROC curves J. Endotoxin Res. 9,271-279[CrossRef]
  2. Dellinger, R. P., Carlet, J. M., Masur, H., Gerlach, H., Calandra, T., Cohen, J., Gea-Banacloche, J., Keh, D., Marshall, J. C., Parker, M. M., Ramsay, G., Zimmerman, J. L., Vincent, J. L., Levy, M. M. (2004) Surviving sepsis campaign guidelines for management of severe sepsis and septic shock Intensive Care Med. 30,536-555[CrossRef][Medline]
  3. Alexander, C., Rietschel, E. T. (2001) Bacterial lipopolysaccharides and innate immunity J. Endotoxin Res. 7,167-202[CrossRef]
  4. Finberg, R. W., Re, F., Popova, L., Golenbock, D. T., Kurt-Jones, E. A. (2004) Cell activation by Toll-like receptors: role of LBP and CD14 J. Endotoxin Res. 10,413-418[CrossRef]
  5. Triantafilou, M., Miyake, K., Golenbock, D. T., Triantafilou, K. (2002) Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation J. Cell Sci. 115,2603-2611[Abstract/Free Full Text]
  6. Cuschieri, J., Umanskiy, K., Solomkin, J. (2004) PKC-{zeta} is essential for endotoxin-induced macrophage activation J. Surg. Res. 121,76-83[CrossRef][Medline]
  7. Wang, Y. M., Seibenhener, M. L., Vandenplas, M. L., Wooten, M. W. (1999) Atypical PKC {zeta} is activated by ceramide, resulting in coactivation of NF-{kappa}B/JNK kinase and cell survival J. Neurosci. Res. 55,293-302[CrossRef][Medline]
  8. Monick, M. M., Mallampalli, R. K., Carter, A. B., Flaherty, D. M., McCoy, D., Robeff, P. K., Peterson, M. W., Hunninghake, G. W. (2001) Ceramide regulates lipopolysaccharide-induced phosphatidylinositol 3-kinase and Akt activity in human alveolar macrophages J. Immunol. 167,5977-5985[Abstract/Free Full Text]
  9. Pfeiffer, A., Bottcher, A., Orso, E., Kapinsky, M., Nagy, P., Bodnar, A., Spreitzer, I., Liebisch, G., Drobnik, W., Gempel, K., et al (2001) Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts Eur. J. Immunol. 31,3153-3164[CrossRef][Medline]
  10. Abdel Shakor, A. B., Kwiatkowska, K., Sobota, A. (2004) Cell surface ceramide generation precedes and controls Fc{gamma}RII clustering and phosphorylation in rafts J. Biol. Chem. 279,36778-36787[Abstract/Free Full Text]
  11. Kolesnick, R. (1994) Signal transduction through the sphingomyelin pathway Mol. Chem. Neuropathol. 21,287-297[Medline]
  12. Monick, M. M., Carter, A. B., Gudmundsson, G., Mallampalli, R., Powers, L. S., Hunninghake, G. W. (1999) A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages J. Immunol. 162,3005-3012[Abstract/Free Full Text]
  13. Merrill, A. H., Jr, Wang, E. (1986) Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s) J. Biol. Chem. 261,3764-3769[Abstract/Free Full Text]
  14. Bligh, E. G., Dyer, W. J. (1959) A rapid method of total lipid extraction and purification Can. J. Biochem. Physiol. 37,911-917
  15. Merrill, A. H., Jr, Wang, E., Mullins, R. E., Jamison, W. C., Nimkar, S., Liotta, D. C. (1988) Quantitation of free sphingosine in liver by high-performance liquid chromatography Anal. Biochem. 171,373-381[CrossRef][Medline]
  16. Pinsky, M. R. (2004) Dysregulation of the immune response in severe sepsis Am. J. Med. Sci. 328,220-229[CrossRef][Medline]
  17. Dai, Q., Zhang, J., Pruett, S. B. (2005) Ethanol alters cellular activation and CD14 partitioning in lipid rafts Biochem. Biophys. Res. Commun. 332,37-42[CrossRef][Medline]
  18. Gekara, N. O., Weiss, S. (2004) Lipid rafts clustering and signaling by listeriolysin O Biochem. Soc. Trans. 32,712-714[CrossRef][Medline]
  19. Radeva, G., Sharom, F. J. (2004) Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells Biochem. J. 380,219-230[CrossRef][Medline]
  20. Young, R. M., Holowka, D., Baird, B. (2003) A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation J. Biol. Chem. 278,20746-20752[Abstract/Free Full Text]
  21. Meng, F., Lowell, C. A. (1997) Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn J. Exp. Med. 185,1661-1670[Abstract/Free Full Text]
  22. Fitzgerald, K. A., Rowe, D. C., Golenbock, D. T. (2004) Endotoxin recognition and signal transduction by the TLR4/MD2-complex Microbes Infect. 6,1361-1367[CrossRef][Medline]
  23. Ghosh, S., Bhattacharyya, S., Das, S., Raha, S., Maulik, N., Das, D. K., Roy, S., Majumdar, S. (2001) Generation of ceramide in murine macrophages infected with Leishmania donovani alters macrophage signaling events and aids intracellular parasitic survival Mol. Cell. Biochem. 223,47-60[CrossRef][Medline]
  24. Pintus, G., Tadolini, B., Posadino, A. M., Sanna, B., Debidda, M., Carru, C., Deiana, L., Ventura, C. (2003) PKC/Raf/MEK/ERK signaling pathway modulates native-LDL-induced E2F-1 gene expression and endothelial cell proliferation Cardiovasc. Res. 59,934-944[Abstract/Free Full Text]



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