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(Journal of Leukocyte Biology. 2001;69:1060-1066.)
© 2001 by Society for Leukocyte Biology

Phosphatidylcholine-specific phospholipase C regulates activation of RAW264.7 macrophage-like cells by lipopeptide JBT3002

Fahao Zhang, Guiling Zhao and Zhongyun Dong

Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Correspondence: Dr. Zhongyun Dong, Department of Cancer Biology, Box 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail: zdong{at}notes.mdacc.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholipase activities are thought to be involved in the activation of macrophages by lipopolysaccharide (LPS). Because our previous studies showed that the synthetic lipopeptide JBT3002 might activate macrophages via signaling pathways similar to those used by LPS, we investigated whether phospholipase activities are required for activation of macrophages by JBT3002. Treatment of RAW264.7 murine macrophage-like cells with JBT3002 stimulated expression of both inducible nitric oxide synthase (iNOS) and tumor necrosis factor-{alpha} (TNF-{alpha}) in a dose-dependent manner. The JBT3002-induced production of nitric oxide and TNF-{alpha} was significantly inhibited by tricyclodecan-9-yl xanthogenate (D609), a selective inhibitor of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). JBT3002-induced expression of steady-state mRNA for both iNOS and TNF-{alpha} was inhibited by D609. Cells treated with JBT3002 had greater production of diacylglycerol (DAG) in 2 min, which lasted for at least 30 min and could be blocked by D609. Activation of RAW264.7 cells was not affected by butanol, a PC-specific phospholipase D inhibitor, and treatment with JBT3002 did not affect phosphatidic acid formation. RAW264.7 cells treated with DAG analogue 1-oleoyl-2-acetyl-sn-glycerol, in the presence of interferon-{gamma}, produced TNF-{alpha}. These results suggested that activation of RAW264.7 cells by JBT3002 requires PC-PLC activity.

Key Words: signal transduction • immunomodulator • nitric oxide • tumor necrosis factor {alpha}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
JBT3002, an N-acylated derivative of {Psi}-amino-C1-C3-alkanesulfonic acid, is a synthetic analogue of a fragment of a lipopeptide from the outer wall of gram-negative bacteria [1 ]. Preclinical studies showed that oral administration of JBT3002 stimulated expression of interleukin-15 by macrophages in the lamina propria, reduced irinotecan-induced gastrointestinal toxic effects [2 , 3 ], and enhanced efficacy of irinotecan therapy against the growth and metastasis of colon and pancreatic cancers in mice [3 , 4 ]. In vitro studies showed that JBT3002 can up-regulate expression of inducible nitric oxide synthase (iNOS) and metalloproteinases 2 and 9 in murine macrophages [5 6 7 ] and stimulate interleukin-1, interleukin-6, and tumor necrosis factor-{alpha} (TNF-{alpha}) in human monocytes [1 ]. In addition, murine macrophages and human monocytes treated with JBT3002 and interferon-{gamma} became tumoricidal [1 , 5 ]. The molecular mechanisms by which JBT3002 regulates these functions of macrophages, however, remain to be elucidated.

Phospholipases are hydrolytic enzymes that cleave phospholipids. The position of cleavage on the glycerol backbone identifies the phospholipase family and generates unique products, some of which have "second-messenger" function [8 ]. Among the signaling lipid molecules is sn-1, 2-diacylglycerol (DAG), a protein kinase C (PKC) activator [9 10 11 ]. DAG can be generated directly by an action of phosphatidylinositol (PI)-phospholipase C (PI-PLC) [12 ] or phosphatidylcholine (PC)-phospholipase C (PC-PLC) [13 , 14 ]. It can also be formed indirectly by a chain reaction involving PC-phospholipase D (PC-PLD) and phosphatide phosphohydrolase (PAP), in which PC-PLD catalyzes PC, generating phosphatidic acid (PA) that in turn is converted to DAG by PAP [15 ].

Several recent studies suggested that phospholipase activities are involved in the activation of macrophages by lipopolysaccharide (LPS) [16 17 18 19 20 ]. For example, PI-PLC and PC-PLC activities were shown to be involved in LPS-induced iNOS expression in RAW264.7 murine macrophage-like cells [17 ]. In human alveolar macrophages, stimulation of PC-PLC by LPS leads to activation of mitogen-activated protein kinases, resulting in expression of TNF-{alpha} [16 ]. Since our previous studies showed that JBT3002 may activate macrophages by mechanisms similar to those used by LPS [1 , 21 ], we investigated whether these phospholipase activities are involved in signaling pathways of JBT3002. To facilitate the interpretation of results, we conducted this study using the RAW264.7 cells that were used by others to study the role of phospholipases in LPS signaling [17 , 18 , 20 ]. Our results indicated that induction of iNOS and TNF-{alpha} expression by JBT3002 requires PC-PLC activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Dulbecco’s modified Eagle’s medium, Hanks’ balanced salt solution, fetal bovine serum, and TRIzol reagent were purchased from Life Technologies (Grand Island, NY). DAG, tricyclodecan-9-yl xanthogenate (D609), 1-oleoyl-2-acetyl-sn-glycerol (OAG), and U73122, a PI-PLC inhibitor (see below), were purchased from BioMol (Plymouth Meeting, PA). JBT3002 was generously provided by Jenner Biotherapies, Inc. (San Ramon, CA). JBT3002 was suspended in Hanks’ balanced salt solution at 1 mg/mL, stored at 4°C and vortexed before each use. PA and a rabbit antibody against ß-actin were purchased from Sigma Chemical Co. (St. Louis, MO). [{alpha}-32P]deoxycytidine 5' triphosphate (3,000 Ci/mmol) was purchased from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). [3H]Myristic acid (2 Ci/mmol) was purchased from ICN Biomedicals (Costa Mesa, CA), and TNF-{alpha} antibodies were from PharMingen (San Diego, CA). Affinity-purified anti-iNOS antibody was raised in rabbits by immunization with a synthetic peptide, corresponding to amino acids 17–31 deduced from the complementary DNA (cDNA) of iNOS (DLKEEKDINNNVKKT). Full-length iNOS cDNA was a generous gift of Carl Nathan (Cornell University Medical College, New York, NY). All reagents used in tissue culture were free of endotoxin, as determined by the Limulus amebocyte lysate assay (sensitivity limit of 0.125 ng/mL) purchased from Associates of Cape Cod Inc. (Woods Hole, MA).

Cell culture
The RAW264.7 murine macrophage-like cell line was purchased from American Type Culture Collection (Manassas, VA). The cells were maintained as monolayer cultures in Dulbecco’s modified Eagle’s medium supplemented with vitamins, sodium pyruvate, nonessential amino acids, L-glutamine, and 5% fetal bovine serum.

Nitrite analysis
Nitrite in culture supernatants was determined by using a microplate assay [22 ]. Briefly, 50 µL of samples were harvested and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% phosphoric acid). The absorbance at 540 nm was measured with a microplate reader (BenchMark; Bio-Rad, Hercules, CA). The nitrite concentration was determined using sodium nitrite as a standard.

TNF-{alpha} analysis
Production of TNF-{alpha} in RAW264.7 cells was determined using the enzyme-linked immunosorbent assay (ELISA). RAW264.7 cells (105/well) were plated in a 96-well plate and stimulated for 4 h at 37°C. The supernatant was harvested and diluted at 1:4 in phosphate-buffered saline. TNF-{alpha} was determined by using the TNF-{alpha}-specific antibody pairs following a recommended protocol from the manufacturer.

Western blot analysis
RAW264.7 cells (2.5 x 106/35-mm-diameter dish) were treated as indicated in the Results section. The cells were washed, scraped into a lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM ethylenediaminetetraacetate, 1 mM phenylmethyl sulfonyl fluoride, 20 µM leupeptin, and 0.15 U/mL of aprotinin) and centrifuged at 12,000 g for 10 min at 4°C. Soluble lysates (20 µg of protein/sample) were denatured, separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. The filters were blocked with 3% bovine serum albumin in Tris-buffered saline (20 mM Tris-HCl, pH 7.5, 150 mM NaCl), probed with iNOS or ß-actin specific antibody (1 µg/mL) in Tris-buffered saline containing 0.1% Tween 20, incubated with horseradish peroxidase-conjugated F(ab')2 of goat anti-rabbit antibody, and visualized using an enhanced chemiluminescence detection system (Amersham-Pharmacia) [23 ]. The bands in the linear range of exposure were recorded with a personal scanner and analyzed by using Scan Analysis software (Biosoft, Ferguson, MO). Each sample measurement was calculated as the ratio of the densities of the iNOS band and the ß-actin band.

RNA isolation and Northern blot analyses
RAW264.7 cells (5 x 106 cells/60-mm-diameter dish) were treated for 2 h (for TNF-{alpha}) or 8 h (for iNOS), and total cellular RNA was extracted using the TRIzol reagent according to the manufacturer’s instructions. For Northern blot analyses, 10 µg/sample of total RNA was fractionated on 1% formaldehyde-agarose gel, transferred onto a GeneScreen nylon membrane (DuPont, Boston, MA), and UV cross-linked at 120,000 µJ/cm2 in a UV chamber (Bio-Rad). The iNOS, TNF-{alpha}, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were detected using cDNA fragments of murine iNOS and TNF-{alpha}, and rat GAPDH labeled by nick translation with {alpha}-[32P]deoxycytosine 5' triphosphate. Filter hybridization (at 65°C) and washes (at 55–60°C with 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS) were performed as described previously [24 ]. The steady-state iNOS and TNF-{alpha} mRNA expressions were quantitated as described above. Each sample measurement was calculated as the ratio of the iNOS or TNF-{alpha} transcript to the GAPDH transcript.

Determination of lipid metabolites
RAW264.7 cells were plated in a 60-mm-diameter dish at a density of 5 x 106 cells/dish and starved overnight in serum-free medium. The cells were labeled with [3H]myristic acid (1.0 µCi/mL) for 3 h, washed, equilibrated for 30 min in medium, and treated as described in Results. The cells were scraped into 400 µL of 0.2% SDS containing 5 mM ethylenediaminetetraacetate, transferred into a glass tube, and extracted on ice for 1 h after addition of 0.5 mL of chloroform, 1 mL of methanol, and carrier lipids (10 µg). After mixing with another 0.5 mL of chloroform and 0.5 mL of NaCl (0.2 M), the extracts were centrifuged at 2,000 g for 5 min, and the lower organic phase was harvested, dried in a vacuum evaporator, and redissolved in 50 µL of chloroform/methanol [2:1 (v/v)]. The lipids (20 µL/sample) were separated on silica plates (Whatman Ltd., Maidstone, Kent, England) by double one-dimensional thin-layer chromatography (TLC) in hexane/diethyl ether/methanol [4:1:1 (v/v/v)], and the up-phase of ethyl acetate/isooctane/acetic acid/water [13:2:3:10 (v/v/v/v)] as described by van Dijk et al. [14 ]. After visualization in iodine vapors, the spots corresponding to DAG and PA were recovered, and radioactivity was measured in a scintillation counter.

Statistical analysis
The experimental results were analyzed for their statistical significance by the two-tailed Student’s t-test. A P value of 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of NO and TNF-{alpha} production by JBT3002
In the first set of experiments, we investigated whether JBT3002 could activate RAW264.7 cells to generate NO and TNF-{alpha}. RAW264.7 cells were incubated for 18–20 h (for NO) or 4 h (for TNF-{alpha}) with increasing concentrations of JBT3002. The NO and TNF-{alpha} in the culture supernatant were measured as described in Materials and Methods. As shown in Figure 1 , RAW264.7 cells did not constitutively produce detectable levels of NO or TNF-{alpha}. Treatment with 0.2–100 ng/mL of JBT3002 induced dose-dependent production of both NO and TNF-{alpha} with a maximal production at 100 ng/mL; therefore, 100 ng/mL of JBT3002 was used for additional studies.



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  		Figure 1. Production of NO and TNF-{alpha} in RAW264.7 cells induced by JBT3002. RAW264.7 cells were plated in a 96-well plate at a density of 105cells/well and then incubated for 18–20 h (for nitrite assay) and 4 h (for TNF-{alpha} assay) with different concentrations of JBT3002. The nitrite and TNF-{alpha} in the culture supernatant were determined as described in Materials and Methods. Data shown are means ± SE of three independent experiments.

 
Effect of D609 on JBT3002-induced iNOS expression
In the next set of experiments, we determined whether the PC-PLC-selective inhibitor D609 [25 , 26 ] could affect expression of iNOS and hence production of NO. RAW264.7 cells were incubated for 18 h in medium, 50 µM D609, and JBT3002 in the presence or absence of 10 µM or 50 µM D609. As shown in Figure 2A , RAW264.7 cells incubated with JBT3002 produced 50.5 ± 5.5 µM NO. D609 did not affect the basal production of NO. JBT3002-induced NO production was inhibited by 51 and 91% when the cells were exposed to 10 and 50 µM D609, respectively. In contrast, JBT3002-induced NO production was not altered by 0.3% butanol, a PC-PLD inhibitor [27 ], or by 10 µM U73122, a PI-PLC inhibitor [28 ]. The treatment of D609 at the concentrations used in this study did not affect the viability of RAW264.7 cells determined by morphology and 3-(4,5-dimethylthiezol-2-yl)-2,5-diphenyltetrazolium bromide staining (data not shown) as described previously [29 ].



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  		Figure 2. Effect of D609 on NO production and iNOS expression in RAW264.7 cells induced by JBT3002. RAW264.7 cells (105/well in 96-well plates) were incubated for 18–20 h in medium alone, medium containing 50 µM D609, or medium containing 100 ng/mL of JBT3002 in the presence or absence of 10 µM or 50 µM D609, 0.3% butanol, or 10 µM U73122 (A). The culture supernatant (50 µL) was removed and analyzed for nitrite as described in Materials and Methods. Data shown are means ± SE of three independent experiments. RAW264.7 cells were treated for 18 h (B) or 8 h (C) with 100 ng/mL of JBT3002 in the presence or absence of 50 µM D609. For Western blot (B), the lysate (20 µg/lane) was separated on 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed with anti-iNOS antibody (1 µg/mL). The immunoreactive bands were detected by incubating the blots with horseradish peroxidase-conjugated F(ab')2 of goat anti-rabbit immunoglobulin G and developed using the enhanced chemiluminescence detection system. For Northern blot (C), total RNA was extracted and separated on 1% agarose gel, transferred to a nylon membrane, and probed with 32P-labeled iNOS and GAPDH cDNA probes. Densitometric quantitation was performed as described in Materials and Methods and is shown in brackets below each lane. These are data from one representative experiment of three. *, P < 0.05 compared with JBT3002 alone.

 
Next, we examined whether D609 altered the expression levels of iNOS protein and mRNA. RAW264.7 cells were incubated with medium or with medium containing D609 or JBT3002 in the presence or absence of D609 for 18 h, and then iNOS protein in the cytosol was identified by Western blotting. As shown in Figure 2B , iNOS protein was not detected in RAW264.7 cells incubated in medium or in medium containing 50 µM D609. High levels of iNOS protein were found in cells stimulated with JBT3002. D609 (50 µM) could attenuate JBT3002-induced iNOS protein expression by 80%. Thus, the effects of D609 on iNOS protein expression correlated directly with the production of NO. Additional studies found that, while D609 reduced the levels of iNOS protein in RAW264.7 cells treated with JBT3002, it did not alter the stability of iNOS protein (data not shown).

Northern blot analysis showed that RAW264.7 cells did not express detectable levels of iNOS mRNA, which was not affected by 50 µM D609. High levels of iNOS mRNA were found in cells treated with JBT3002; the steady-state iNOS mRNA increased by 50-fold. In the presence of 50 µM D609, the induction of iNOS mRNA by JBT3002 was reduced by 88% (Fig. 2C) . This result directly correlated with inhibition of iNOS protein expression. Similar to the results of the stability analysis in Western blotting, we found that D609 did not alter accelerated degradation of iNOS mRNA (data not shown).

Effect of D609 on JBT3002-induced TNF-{alpha} expression
To determine whether the suppression by D609 was selective toward the expression of iNOS, we next investigated the effect of D609 on expression of TNF-{alpha}. RAW264.7 cells were incubated for 4 h in medium, medium containing 50 µM D609, or medium containing JBT3002 in the presence or absence of 10 µM or 50 µM D609. TNF-{alpha} protein in the culture supernatant was measured by ELISA (Fig. 3A ). RAW264.7 cells constitutively released 41.2 ± 5.6 pg/106 cells of TNF-{alpha}. Cells incubated with JBT3002 produced 1,550 ± 185 pg/106 cells of TNF-{alpha}. D609 did not affect the basal production of TNF-{alpha}, but it did suppress JBT3002-induced TNF-{alpha} production in a dose-dependent manner: 10 µM and 50 µM D609 reduced the production by 50 and 90%, respectively. Butanol and U73122 also did not affect JBT3002-induced TNF-{alpha} production. Consistent with the effect on TNF-{alpha} production, D609 suppressed JBT3002-induced expression of TNF-{alpha} mRNA by 86% (Fig. 3B) . This result suggested that attenuation of JBT3002-induced TNF-{alpha} expression by D609 also occurred at the mRNA level.



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  		Figure 3. Effect of D609 on TNF-{alpha} production and TNF-{alpha} mRNA expression in RAW264.7 cells induced by JBT3002. RAW264.7 cells (105/well) in 96-well plates were incubated for 4 h in medium alone, medium containing 50 µM D609, or medium containing 100 ng/mL of JBT3002 in the presence or absence of 10 µM or 50 µM D609, 0.3% butanol, or 10 µM U73122 (A). TNF-{alpha} in the culture supernatants was measured by ELISA. Data shown are means ± SE of duplicate cultures. This is one representative experiment of three. RAW264.7 cells (5 x 106/dish) in a 60-mm-diameter plate were incubated for 2 h with 100 ng/mL of JBT3002 in the presence or absence of 50 µM D609 (B). Total cellular RNA was extracted and subjected to Northern blot analysis as described in Materials and Methods, using mouse TNF-{alpha} or GAPDH cDNA probes. Densitometric quantitation was performed as described in Materials and Methods and is shown in brackets below each lane. This is one representative experiment of three. *, P < 0.05, compared with JBT3002 alone.

 
DAG and PA production in RAW264.7 cells induced by JBT3002
Since DAG and PA are the two principal products released by phospholipase, we next investigated whether they were formed in RAW264.7 cells stimulated with JBT3002. RAW264.7 cells were labeled for 3 h with 1.0 µCi/mL of [3H]myristic acid under conditions in which 85% of the radioactivity is incorporated into PC [30 ]. Figure 4A shows the time course of DAG and PA formation. JBT3002 induced a 2- to 2.5-fold increase of DAG production at 2 min of stimulation, and the elevated production continued for at least 30 min. The JBT3002-enhanced production of DAG was dose dependent; it was observed in cells treated with 10 ng/mL of JBT3002 and reached maximum in cells treated with 100 ng/mL of JBT3002 (Fig. 4B) . JBT3002 had no effect on PA formation at any time point (Fig. 4A) or concentration (Fig. 4B) tested.



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  		Figure 4. The time and dose course of DAG and PA formation in RAW264.7 cells induced by JBT3002. RAW264.7 cells (5 x 106/dish) in a 60-mm-diameter plate were labeled for 3 h with 1.0 µCi of [3H]myristic acid (A). The cells were treated in medium with (closed symbols) or without (open symbols) 100 ng/mL of JBT3002 for the indicated time periods. Lipids were extracted as described in Materials and Methods, and the levels of radioactive DAG and PA were determined by TLC. RAW264.7 cells (5 x 106/dish) in a 60-mm-diameter plate were labeled for 3 h with 1.0 µCi of [3H]myristic acid (B). The cells were treated for 10 min in medium containing different concentrations of JBT3002. Lipids were extracted as described in Materials and Methods, and the levels of radioactive DAG and PA were determined by TLC. Data shown are means ± SE of duplicate experiments. This is one representative experiment of three.

 
Effect of D609 on JBT3002-induced DAG formation
Next we assessed the effects of D609 on production of DAG. RAW264.7 cells were labeled with 1.0 µCi of [3H]myristic acid for 3 h, followed by an incubation for 30 min in medium or medium containing 10 µM or 50 µM D609. Then the cells were stimulated with JBT3002 (100 ng/mL) for 10 min. The total lipids were extracted, and production of DAG was determined by TLC. Figure 5A shows cellular DAG content on a TLC plate; clearly, formation of DAG in RAW264.7 cells was enhanced by JBT3002, which could be blocked by the presence of 50 µM D609. Quantitative analysis of [3H]-labeled DAG revealed that the basal level of DAG was 1.98 ± 0.35 x 104 cpm/107 cells, and the level was not affected by D609. After treatment with JBT3002, DAG production was increased 2.5-fold. The addition of 10 µM and 50 µM D609 reduced the JBT3002-induced DAG formation by 48% and 93%, respectively (Fig. 5B) .



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  		Figure 5. Effect of D609 on DAG formation in RAW264.7 cells induced by JBT3002. RAW264.7 cells (5 x 107/dish) in a 100-mm-diameter plate were treated for 10 min with medium or JBT3002 (100 ng/mL) in the absence or presence of 50 µM D609. All extracted lipids (50 µL) were separated on a silica plate and visualized in iodine vapors (A). RAW264.7 cells (5 x 106/dish) in a 60-mm-diameter plate were labeled for 3 h with 1.0 µCi of [3H]myristic acid (B). After preincubation with 10 µM or 50 µM D609 for 30 min, the cells were treated for an additional 10 min with 100 ng/mL of JBT3002. Lipids were extracted as described in Materials and Methods, and the levels of radioactive DAG were determined by TLC. Data shown are means ± SE of duplicate measurement. Similar results were obtained in three separate experiments._art>

 
In the final experiment, we directly examined the potential role of DAG in stimulation of RAW264.7 cells. RAW264.7 cells were incubated with OAG, a cell membrane-permeable analog of DAG, in the presence or absence of 10 U/mL of interferon (IFN)-{gamma} (Fig. 6 ). Whereas OAG alone was a weak TNF-{alpha} inducer, it did stimulate TNF-{alpha} production when used in combination with low doses of IFN-{gamma}.



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  		Figure 6. OAG-induced production of TNF-{alpha} in RAW264.7 cells. RAW264.7 cells (1x105/well) in 96-well plate were incubated for 8 h with medium or 400 µM OAG (OAG was added two times at 0 and 4 h, respectively) in the presence or absence of 10 U/ml IFN-{gamma}. TNF-{alpha} in the culture supernatants was measured by ELISA. Data shown are means ± SE of six independent experiments. *P < 0.05 compared with medium alone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we examined the role of PC-PLC in the activation of RAW264.7 murine macrophage-like cells induced by JBT3002. We found that the JBT3002-induced production of NO and TNF-{alpha} in RAW264.7 cells was almost completely inhibited by 50 µM D609, the PC-PLC-selective inhibitor [25 , 26 ].Western and Northern blot analyses revealed that D609 could almost completely block JBT3002-induced iNOS protein and mRNA expression and TNF-{alpha} mRNA expression. The TLC analysis showed that RAW264.7 cells constitutively produced basal levels of DAG and PA. JBT3002 enhanced DAG formation in a time- and dose-dependent manner, and this formation was completely inhibited by D609. JBT3002 did not affect the PA formation in RAW264.7 cells. Finally, OAG, an analogue of DAG, stimulated TNF-{alpha} production when it was used in combination with IFN-{gamma}. These data suggest that PC breakdown by PC-PLC is involved in JBT3002-induced activation of RAW264.7 cells.

PC can be catalyzed by either a PC-PLC, generating the PKC activator DAG, or by a PC-PLD, deriving PA that can be converted to DAG by a PAP action [15 ]. It has been suggested that PC-PLC and PI-PLC but not PC-PLD mediate LPS-induced activation of PKC, resulting in the expression of iNOS and NO release in RAW264.7 cells [17 ]. On the other hand, it was shown that production of NO in J774.1 macrophages induced by the combination of IFN-{gamma} and LPS requires PC-PLC but not PI-PLC and PC-PLD [13 ]. The PC-PLC pathway was also linked to LPS-induced activation of mitogen-activated protein kinases in human alveolar macrophages [16 ]. We concluded that JBT3002-induced activation of RAW264.7 cells requires PC-PLC but not PI-PLC and PC-PLD activities. This conclusion is based on the following observations. First, JBT3002-induced expression of iNOS and TNF-{alpha} was suppressed in a dose-dependent manner by the PC-PLC-selective inhibitor D609 at concentrations not affecting PC-PLD activity [14 , 31 ]. Second, JBT3002 induced DAG formation in a dose-dependent manner, and the formation was completely abolished by D609. Third, JBT3002-induced production of NO and TNF-{alpha} was not affected by the PI-PLC inhibitor U73122 or the PC-PLD inhibitor butanol. Finally, JBT3002 did not alter PA formation.

The fourth potential pathway that generates DAG from PC is mediated by sphingomyelin synthase (SMS) [32 ]. SMS activity can also be blocked by D609 at the concentrations used in the current study [33 ]. However, the kinetics of DAG formation by SMS and by PC-PLC differ substantially. In this study, we found that PC-PLC activity can be stimulated in 2 min and last for at least 30 min. On the other hand, SMS-mediated DAG formation is a slow process, in which DAG formation can be detected after 15–30 min and lasts for several hours [33 ]. Furthermore, whereas the generation of DAG by SMS uses ceramides as a substrate [33 ], treatment of cells with a C2-ceramide suppressed JBT3002-induced activation of RAW264.7 cells (data not shown). Therefore, the pattern of DAG formation in RAW264.7 cells induced by JBT3002 does not match with that catalyzed by SMS. Nonetheless, further studies are needed to determine whether the SMS contributed to activation of RAW264.7 cells by JBT3002 in our system.

Recent studies show that toll-like receptors (TLRs) are responsible for recognition of a variety of bacterium-related products by monocytes/macrophages, leading to activation of transcription nuclear factor (NF)-{kappa}B and expression of proinflammatory molecules, such as TNF-{alpha} [34 35 36 ]. Our preliminary studies showed that JBT3002-induced activation of NF-{kappa}B in several lines of tumor cells of nonmonocyte/macrophage lineage could be enhanced by transfection with TLR2 cDNA, which could be partially attenuated by the presence of D609 (data not shown). However, in the absence or presence of JBT3002, we were unable to show TNF-{alpha} expression in TLR2-transfected cells, suggesting that activation of other signaling pathways is necessary for induction of TNF-{alpha} by JBT3002 [37 ]. This notion is further supported by our observation on induction of TNF-{alpha} production by OAG. Production of TNF-{alpha} in RAW264.7 cells could be induced by OAG, which provides direct evidence for a role of DAG in JBT3002-mediated TNF-{alpha} induction. However, we noticed that exogenous OAG was a very weak stimulus in comparison with JBT3002. Although this difference could result from degradation of OAG in culture medium, it is more likely that induction of TNF-{alpha} expression by JBT3002 is mediated by activation of multiple signaling pathways.

In summary, we have shown that the PC-PLC-selective inhibitor D609 could suppress JBT3002-induced production of NO and TNF-{alpha} in RAW264.7 macrophage-like cells. This inhibition directly correlated with reduced expression levels of iNOS and TNF-{alpha} mRNA. JBT3002 could increase DAG formation, which could also be suppressed by D609. These data indicate that PC-PLC activity is involved in activation of RAW264.7 cells by JBT3002.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grant RPC-98-332-01 from the American Cancer Society and The University of Texas start-up funds.

We thank Dr. Isaiah J. Fidler for helpful discussions and Michael S. Worley for critical editorial comments. We also thank Jenner Biotherapies, Inc. (San Ramon, CA), for kindly providing the JBT3002.

Received September 22, 2000; revised January 30, 2001; accepted January 31, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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