Cytochrome P4502E1 primes macrophages to increase TNF-
production in response to lipopolysaccharide
Qi Cao,
Ki M. Mak, and
Charles S. Lieber
Alcohol Research and Treatment Center, Bronx Veterans Affairs Medical Center, and Mount Sinai School of Medicine, Bronx, New York
Submitted 26 August 2004
; accepted in final form 2 March 2005
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ABSTRACT
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Kupffer cells become activated in response to elevated levels of LPS during ethanol feeding, but the role of ethanol in the molecular processes of activation remains unclear. Because cytochrome P4502E1 (CYP2E1) is upregulated in Kupffer cells after ethanol, we hypothesized that this effect primes Kupffer cells, sensitizing them to increase TNF-
production in response to LPS. However, cultured Kupffer cells rapidly lose their CYP2E1. This difficulty was overcome by transfecting CYP2E1 to RAW 264.7 macrophages. Macrophages with stable increased CYP2E1 expression (E2) displayed increased levels of CD14/Toll-like receptor 4, NADPH oxidase and H2O2, accompanied by activation of ERK1/2, p38, and NF-
B. These increases primed E2 cells, sensitizing them to LPS stimuli, with amplification of LPS signaling, resulting in increased TNF-
production. Diphenyleneiodonium, a NADPH oxidase inhibitor, and diallyl sulfide, a CYP2E1 inhibitor, decreased approximately equally H2O2 levels in E2 cells, suggesting that NADPH oxidase and CYP2E1 contribute equally to H2O2 generation. Because CYP2E1 expression also enhanced the levels of the membrane localized NADPH oxidase subunits p47phox and p67phox, thereby contributing to the oxidase activation, it may augment H2O2 generation via this mechanism. H2O2, derived in part from NADPH and CYP2E1, activated ERK1/2 and p38. ERK1/2 stimulated TNF-
production via activation of NF-
B, whereas p38 promoted TNF-
production by stabilizing TNF-
mRNA. Oxidant generation after CYP2E1 overexpression appears to be central to macrophage priming and their sensitization to LPS. Accordingly, CYP2E1 priming could explain the sensitization of Kupffer cells to LPS activation by ethanol, a critical early step in alcoholic liver disease.
reduced nicotinamide adenine dinucleotide phosphate oxidase; diphenyleneiodonium; diallyl sulfide; oxidative stress; nuclear factor-kappaB
KUPFFER CELLS ARE KEY HEPATIC cells that initiate alcoholic liver injury. They become activated in response to elevated levels of LPS after ethanol feeding and produce increased amounts of TNF-
, which cause liver injury (36, 48). According to the current view, the activation process involves a priming phase in which Kupffer cells are sensitized by ethanol, rendering them more responsive to LPS stimuli (1). However, the role of ethanol in this process has yet to be defined.
Previously, we found that ethanol feeding of rats increases cytochrome P4502E1 (CYP2E1) and its catalytic activity 79 times in Kupffer cells (4, 5, 22). In culture, in response to LPS or acetaldehyde, these Kupffer cells released significantly more TNF-
than those of control rats (4, 5). Although the data suggest an involvement of CYP2E1 in the stimulation of TNF-
by LPS, evidence of a direct role for CYP2E1 in this process is still lacking. Cultured Kupffer cells, like primary cultured hepatocytes (51), rapidly lose their CYP2E1. The level was maintained only for 4 h and declined gradually to the level of cells of control rats after 24 h (4, 5). This deficiency has hampered the analysis of the actions of Kupffer cell CYP2E1 on TNF-
production. An alternate approach to evaluate the effects and actions of CYP2E1 in Kupffer cells is to express CYP2E1 in RAW 264.7 cells (a mouse macrophage cell line) that have an undetectable endogenous level of CYP2E1 but share many characteristics with Kupffer cells, in particular LPS signal transduction mediating TNF-
production.
The aims of this study were to determine whether CYP2E1 expression primes macrophages to increase TNF-
production in response to LPS stimuli, and when in the affirmative, to evaluate the signaling mechanisms of this process. To that effect, we assessed the critical mediators of LPS signal transduction, including CD14 and its coreceptor Toll-like receptor 4 (TLR4) and TLR2, NADPH oxidase, and oxidative stress. We also evaluated the capacity of NADPH oxidase and CYP2E1 to generate H2O2 in CYP2E1 transfected macrophages and examined the role of this oxidant in the activation of ERK1/2, p38 MAPKs, and NF-
B, leading to TNF-
production.
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MATERIALS AND METHODS
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Transfection of CYP2El cDNA into RAW 264.7 macrophages.
Human CYP2E1 cDNA (kindly provided by Dr. Arthur Cederbaum, Mount Sinai School of Medicine, New York, NY) was inserted into the EcoR restriction site of a pCI-neo expression vector in the sense orientation to generate the plasmid pCI-neO2E1. For transfection, murine RAW 264.7 cells (American Type Culture Collection, Manassas, VA) were plated onto six-well plates (5 x 105 cells/well) and incubated with 4 µg of pCI-neO2EI or pCI-neo, Effectene, and enhancer (Qiagen, Valencia, CA) for 4 h at 37°C. After washing in PBS, macrophages were kept in RPMI 1640 medium for 48 h. Cells from each well were then subcultured into two flasks (275 ml) for another 24 h. The medium was changed, and geneticin (G418; Fisher, Springfield, NJ) at 1 mg/ml was added for clone selection. The G418-containing medium was changed daily for 5 days until clones were formed. Individual clones were harvested and plated onto 12-well plates in G418-containing medium. The medium was changed daily for 2 wk and established clones were transferred to 100 cm2 flasks and cultured in medium with G418 for two additional weeks. Stable transfectants were selected for analysis of CYP2E1 expression.
CYP2E1 analysis.
CYP2El mRNA expression was analyzed by Northern blot with a human CYP2El cDNA probe (Oxford Biomedical Research, Oxford, MI). The probe was labeled with [32P]dCTP using a random priming DNA labeling kit. Levels of mRNA were quantified by imaging densitometry of bands on X-ray film. CYP2E1 protein content was determined by Western blot analysis, using a rabbit anti-hamster CYP2El IgG as the primary antibody (22). CYP2E1 catalytic activity was measured by determining the rate of p-nitrophenol hydroxylation to 4-nitrocatechol (4-NC) as previously described (4). An equivalent amount of Kupffer cell total RNA or protein lysate from an ethanol-fed rat of a previous study (4) was used as positive control.
Treatment of macrophages.
Transfected macrophages and wild-type macrophages were cultured in serum-free RPMI 1640 medium containing 50 ng/ml LPS (Sigma, St. Louis, MO) and 1 ng/ml LPS binding protein (LBP) (R&D Systems, Minneapolis, MN) (46) plus 20 µM dipheneyleneiodonium (DPI) (Sigma) (47) (LPS/LBP + DPI); 1 mM diallyl sulfide (DAS) (Sigma) (35, 37) (LPS/LBP + DAS); catalase (1,000 U/ml; Sigma) (LPS/LBP + catalase); 1 mM 4-methylpyrazole (4-MP) (Sigma) (LPS/LBP + 4-MP); 30 µM ERK1/2 inhibitor PD-098059 (Sigma) (4, 5) (LPS/LBP + PD-098059); and 10 µM p38 inhibitor SB-203580 (Sigma) (4, 5) (LPS/LBP + SB-203580). DMSO (Sigma), as a vehicle control for PD-098059 and SB-203580, was added to the culture media at a concentration of 2.1 mM when these inhibitors were tested.
TNF-
mRNA and TNF-
protein assays.
TNF-
mRNA was assayed with the mouse Quantikine mRNA kit (R&D Systems), using 10 µg of total RNA isolated from macrophages. Results are expressed as amol/106 cells. TNF-
protein in culture medium was quantified, using the mouse TNF-
ELISA kit (R&D Systems) per the manufacturer's protocol, and data are expressed as ng /ml culture medium.
Western blot analysis of LPS receptors CD14, TLR4, and TLR2.
Aliquots of 20 µg of cell protein lysates were separated on 12% SDS-PAGE. Primary antibodies were mouse anti-CD14 antibody (1:1,000; PharMingen, San Diego, CA) and rabbit anti-TLR 4 and anti-TLR 2 antibodies (1:500; Santa Cruz Biotechnology, Santa Cruz). Immunoreactive proteins were visualized with a mixture of Immuno-Star substrate and enhancer (Bio-Rad, Hercules, CA) and exposed to X-ray film. Intensity of the bands was quantified by imaging densitometry.
Preparation of macrophage membrane and cytosol fractions.
Macrophages (5 x 106) were seeded onto 100 x 20-mm plastic dishes and cultured for 48 h. The cells were treated with DPI or DAS for 30 min, followed by incubation in the presence or absence of LPS/LBP for an additional 30 min. After treatment, cells were washed with ice-cold PBS, scraped off, and pelleted. These were resuspended in 2-ml relaxation buffer [in mM: 100 KCl, 10 HEPES, 3.5 MgCl2, 3 NaCl, 1.2 ethylene glycol tetra-acetic acid, 25 NaF, 5 sodium orthovanadate, 1 p-nitrophenyl phosphate, 1 4-(2-aminoethyl)benzenesulfonyl fluoride, and 2 PMSF, plus 0.5 µM microcystin, 20 µg/ml chymostatin, and 10 µM leupeptin] according to Price et al. (41). Cells were disrupted with a homogenizer and the homogenate was centrifuged at 1,300 rpm for 10 min at 4°C to remove unbroken cells and nuclei. The supernatant was layered on a discontinuous 20/38% sucrose gradient and centrifuged at 204,000 g for 30 min. Cytosol was removed from the top of the gradient, and the membrane fraction was collected at the 20/38% sucrose gradient interface. Fractions were analyzed for NADPH oxidase as described below.
Measurement of NADPH oxidase activity in membrane and cytosol fractions.
Aliquots of the cell membrane or cytosolic protein (0.5 mg) were mixed with (in µM) 100 ferricytochrome c, 10 FAD, 10 GTP, 100 SDS (11), and 10 DPI (38). The mixture was incubated for 10 min at room temperature. NADPH was added to the mixture at a final concentration of 250 µM and incubated for 30 min. The DPI-inhibitable rate of NADPH consumption was used to measure NADPH oxidase activity (47). NADPH consumption was measured by the decrease in absorbance at 340 nm in a Carey Bio100 spectrophotometer. The absorption extinction coefficient used to calculate the amount of NADPH consumed was 6.22 mM/cm. Data are expressed as nanomoles of NADPH per minute per milligram protein.
Western blot analysis of NADPH oxidase p47phox and p67phox subunits.
Aliquots of protein lysates (20 µg) of macrophage membrane and cytosol fractions were separated on 12% SDS-PAGE and then transferred to nitrocellulose membrane. Protein (p47phox and p67phox) was detected with rabbit polyclonal p47phox and p67phox antibodies (kindly provided by Dr. B. M. Babior, Scripps Research Institute, La Jolla, CA). As control for equal protein loading,
-actin was used. Immunoreactive proteins were visualized with a mixture of Immun-Star substrate and enhancer and exposed to X-ray film. Band intensities were quantified by imaging densitometry.
Oxidative stress assessment.
Macrophages (3 x 105) in six-well culture plates were treated for 30 min or 24 h, followed by fluorescent probes (described below) for 30 min in the darkness. Cells were washed once with PBS, trypsinized, and resuspended in PBS. The fluorescence intensity was determined in a spectrofluorometer.
H2O2 generation.
Intracellular H2O2 was assessed by adding 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA), obtained from Molecular Probes (Eugene, OR), to the macrophage culture at a final concentration of 20 µM. The generation of 2',7'-dichlorofluorescein (DCF) by oxidation of DCFH2-DA is proportional to the H2O2 produced (6, 7). DCF fluorescence was measured at 488 nm for excitation and 525 nm for emission.
Superoxide anion generation.
This was determined by the addition of hydroethidine (Molecular Probes) to the macrophage culture at a final concentration 10 µM. Hydroethidine is oxidized by O2 produced in the cells. The loss of fluorescence in the cells is proportional to the superoxide anion generated (6, 7, 37). Hydroethidine fluorescence was measured at 352 nm for excitation and 434 nm for emission.
Lipid peroxidation detection.
This was assessed by the addition of cis-parinaric acid (Molecular Probes) to the macrophage culture at a final concentration of 5 µM. Subsequent to peroxidative stress, cis-parinaric acid is degraded, resulting in decreased fluorescence intensity. The loss of fluorescence is proportional to the lipid peroxidation process (6, 27, 31, 37). Cis-parinaric acid fluorescence was measured at 325 nm for excitation and 413 nm for emission.
Reduced glutathione measurement.
Reduced GSH levels in macrophages were determined using the Cayman (Ann Arbor, MI) Assay Kit according to the manufacturer's instruction.
ERK1/2 and p38 MAPK phosphorylation assays.
These were performed by Western blot analysis using the components provided in the PhosphoPlus p38 and ERK1/2 Antibody Kits (Cell Signaling Technology, Beverly, MA) as previously described (4, 5). Aliquots of macrophage protein lysates (20 µg) were separated on 12% SDS-PAGE. Primary antibodies were rabbit anti-phospho-ERK1/2 or anti-phospho-p38 antibodies that detected activated ERK1/2 or p38. Equal protein loading was controlled by immunoblotting of the corresponding nonphosphorylated ERK1/2 or p38, using anti-ERK1/2 or anti-p38 antibodies. Immunoreactive proteins were detected by chemiluminescence and quantified by imaging densitometry.
EMSA for NF-
B.
Double-stranded NF-
B consensus oligonucleotides were labeled with digoxigenin (DIG) using the DIG Gel Shift Kit (Roche, Indianapolis, IN) as previously described (4, 5). Macrophage nuclear protein extracts (30 µg) were incubated in a reaction buffer containing 4 µl binding buffer, 1 µg poly(dI-dC), 0.1 µg poly-L-lysine and 0.8 ng digoxigenin-labeled oligonucleotides for 15 min at room temperature. The reaction was stopped on ice and 5 µl of loading buffer with bromophenol blue was added to the samples. These were separated on 8% nondenaturing acrylamide gel in 0.5x Tris-borate-EDTA (TBE) buffer and transferred to positively charged nylon membranes in 0.5x TBE buffer. Oligonucleotide mobility was detected with alkaline phosphatase-conjugated DIG antibody and the lumigen CSPD provided in the kit. Signal intensities of NF-
B DNA binding were assessed by imaging densitometry.
Western blot analysis of NF-
B p65 and I
B-
.
Nuclear and cytosolic protein extracts of macrophages were analyzed by Western blot for p65 and I
B-
expression as described previously (4, 5). Rabbit anti-p65 or anti-I
B-
antibodies were used as the primary antibodies. Immunoreactive proteins were visualized with a mixture of Immun-Star substrate and enhancer and exposed to X-ray film. The intensity of the bands was quantified by imaging densitometry.
TNF-
mRNA stability determination.
To assess whether p38 and ERK1/2 MAPK regulate TNF-
gene expression at the posttranscriptional level, E2 cells were treated with LPS/LBP for 1 h to induce TNF-
mRNA, followed by exposure to actinomycin D (10 µg/ml; Sigma) for 20 min to block the transcription (6). The culture medium was changed, and fresh medium containing SB-203580 (10 µM) or PD-098059 (30 µM) was added; 15, 30, 45 and 60 min thereafter, TNF-
mRNA in E2 cells was quantified with the Quantikine TNF-
mRNA kit. The decay time course in the absence or presence of the inhibitors was analyzed.
Protein determination.
Protein contents were determined using the BCA protein assay kit (Pierce Chemicals, Rockford, IL).
Statistics.
Data are reported as means ± SE. Statistical analysis was performed using one-way ANOVA followed by Student-Newman-Keuls post hoc tests for multiple comparisons between treatment groups. P < 0.05 was considered to be significant.
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RESULTS
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Increased CYP2E1 expression in RAW 264.7 macrophages transfected with CYP2E1 cDNA.
Figure 1, A and B illustrates the CYP2E1 contents of a macrophage clone designated E2. There were threefold increases in CYP2E1 mRNA and protein and a twofold increase in catalytic activity relative to those in Kupffer cells of rats fed ethanol for 3 wk (62 vs. 34 pmol 4-NC·min1·mg protein1). No CYP2E1 mRNA and protein and only a negligible catalytic activity were detected in wild-type macrophages or in cells transfected with the empty vector pCI-neo (designated M1); these served as controls. Thus far, stable CYP2E1 expression was maintained in E2 cells for at least 10 passages.

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Fig. 1. cytochrome P4502E1 (CYP2E1) mRNA, protein and catalytic activity in CYP2E1-expressing macrophages shown by Northern blot analysis (A), Western blot analysis (B), and catalytic activity (C). Samples were obtained from a previous study (4). Intensity of the mRNA and protein bands in E2 cells was normalized to that of -actin and GAPDH, respectively, and the values are expressed relative to those of Kupffer cells. Numbers above the blots refer to mean values of 3 individual analyses. CYP2E1 transfection resulted in increased expression of CYP2E1 mRNA, protein, and catalytic activity relative to those in Kupffer cells. No CYP2E1 mRNA and protein and a negligible CYP2E1 catalytic activity were detected in wild-type and M1 macrophages. Wild, wild-type RAW 264.7 cells; M1, macrophages transfected with empty vector; E2(p1), passage 1 CYP2E1 transfected macrophages; E2(p10), passage 10 CYP2E1 transfected cells; KC, equivalent amounts of total RNA and/or protein lysates from Kupffer cells of a rat fed ethanol for 3 wk (positive control). ***P < 0.001 vs. M1 cells.
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Effects of increased CYP2E1 expression on TNF-
production.
These were studied in the absence or presence of LPS stimuli. In the absence of LPS/LBP, E2 cells contained about six times more TNF-
mRNA and released seven times more protein into the culture media than M1 or wild-type cells (Fig. 2), suggesting that E2 cells are primed to produce more TNF-
. LPS/LBP treatment upregulated TNF-
mRNA and increased its protein in E2, M1, and wild-type macrophages, but the treatment resulted in higher levels of the mRNA (60%) and protein (
100%) in E2 cells compared with similarly treated M1 or wild-type cells. These results demonstrate that overexpression of CYP2E1 sensitizes macrophages to increase TNF-
production in response to LPS stimuli.
To ensure that the phenomenon of CYP2E1 sensitization was not related to a clonal artifact, we determined TNF-
production in two additional macrophage clones (E3 and E1) with CYP2E1 catalytic activity of 34.5 and 41.1 pmol 4-NC·min1·mg protein1, respectively. Table 1 shows that these clones, like the E2 cells, generated increased amounts of TNF-
compared with control M1 cells, whether in the absence of LPS/LBP (E3, 335%, P < 0.001; E1, 421%, P < 0.001; E2, 792%, P < 0.001) or when stimulated with LPS/LBP (E3, 35%, P < 0.05; E1, 64%, P < 0.01; E2, 95%, P < 0.001). There was a positive correlation between CYP2E1 catalytic activities and TNF-
levels, whether in the absence or presence of LPS/LBP. These data clearly indicate that increased TNF-
production in macrophages is not a clonal artifact but a result of CYP2E1 expression.
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Table 1. Effects of increasing CYP2E1 catalytic activity on TNF- production in CYP2E1 transfected RAW 264.7 macrophage clones
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Effects of increased CYP2E1 expression on CD14 and TLR 4.
To study the possible mechanisms involved, we first evaluated the cell surface receptor CD14 and its coreceptor TLR4, which transduce LPS signals (9, 18, 45), leading to the generation of oxidants (28, 55). By Western blot analysis, in the absence of LPS/LBP, a higher level of CD14 was seen in E2 than in M1 cells (Fig. 3). Treatment with LPS/LBP upregulated CD14 in both E2 and M1 cells, but the level was higher in E2 (65%) than in similarly treated M1 cells. A smaller, but significant, increase was observed for TLR4, whether in the absence of LPS/LBP or in their presence. TLR2 was expressed equally by these macrophages and the levels were not altered by LPS/LBP. Thus enhancement of CD14 and TLR4 after CYP2E1 transfection could be a mechanism by which macrophages are sensitized to LPS stimuli.

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Fig. 3. Increased CD14 and TLR4 levels in CYP2E1-expressing macrophages with or without LPS/LBP treatment. Levels of the LPS receptors in macrophages cultured in the absence or presence of LPS/LBP for 24 h were analyzed by Western blot. Densitometric units are expressed as fold change relative to M1 macrophages (control) assigned a value of 1. CD14 and TLR4 levels in E2 cells were higher than those in M1 cells, whether in the absence or presence of LSP/LBP. TLR2 expression was not affected. Values for wild-type cells were identical to those of M1 cells (data not shown).
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Effects of increased CYP2E1 expression on NADPH oxidase activity and its inhibition by DPI and DAS.
We also assessed the activity of NADPH oxidase, which is a plasma membrane-associated multicomponent enzyme that catalyzes the transfer of electron from NADPH to molecular oxygen, producing O2, which undergoes dismutation to H2O2 (2). In the absence of LPS/LBP, NADPH oxidase activity in the membrane fraction of E2 cells was 2.5 times higher than that in M1 cells (Fig. 4A). DPI, an inhibitor of NADPH oxidase complex (12, 17, 38), inhibited most of the oxidase activity in E2 and M1 cells, consistent with the action of the inhibitor. DAS, a CYP2E1 inhibitor (3, 54), halved the oxidase activity in E2 cells, suggesting involvement of CYP2E1, at least in part, in the stimulation of NADPH oxidase activity in E2 cells, but it had no effect on the oxidase activity in M1 cells, reflecting the absence of CYP2E1. LPS/LBP treatment raised the oxidase activity in both E2 and M1 cells compared with corresponding untreated cells (Fig. 4, A and B), but the treatment doubled the activity in E2 cells compared with similarly treated M1 cells (Fig. 4B). DPI abolished the LPS-stimulated oxidase activity in both E2 and M1 cells, whereas DAS reduced partially the activity in E2 cells and had no such effect on M1 cells. No, or only a negligible, amount of NADPH oxidase activity was detected in the cytosol of corresponding macrophages (data not shown), in accordance with the membrane localization of the oxidase (2, 41, 43). These data suggest that the increase of NADPH oxidase activity after CYP2E1 expression is another mechanism by which macrophages are sensitized to LPS stimuli, leading to oxidant generation.

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Fig. 4. Effects of dipheneyleneiodonium (DPI) and diallyl sulfide (DAS) on plasma membrane-associated NADPH oxidase activity in CYP2E1-expressing macrophages. E2 and M1 macrophages were treated with or without the inhibitors for 30 min, followed by incubation in the absence (A) or presence (B) of LPS/LBP for an additional 30 min. NADPH oxidase activity in the membrane fraction of macrophages was measured as DPI-inhibitable NADPH consumption by the cells. E2 cells had increased NADPH oxidase activity compared with corresponding M1 cells, whether treated or not with LSP/LBP. DPI prevented the increases in both E2 and M1 cells, whereas DAS partially diminished the increase in E2 cells and had no such effect on M1 cells. **P < 0.01 and ***P < 0.001 vs. M1 cells (without inhibitors) in A or B.
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Increased NADPH oxidase activity is accompanied by upregulation of p47phox and p67phox and effects of DPI and DAS.
NADPH oxidase is activated by its p47phox and p67phox subunits that are translocated from the cytosol to the plasma membrane (2, 41). Therefore, we assessed their expression. The Western blot data in Fig. 5 revealed that p47phox and p67phox were present predominantly in the cytosolic fraction of macrophages, as expected, and no apparent changes were revealed in E2 or M1 cells. By contrast, in the membrane fraction, changes of p47phox and p67phox in E2 and M1 cells were seen to match those of the oxidase activity (see Fig. 4), whether in the absence of LPS/LBP (Fig. 5A) or in their presence (Fig. 5B), and whether treated with DPI or with DAS. These data demonstrate that increased CYP2E1 expression enhances expression of the membrane localized p47phox and p67phox.

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Fig. 5. Effects of DPI and DAS on NADPH oxidase subunits p47phox and p67phox in macrophage membrane fraction. Top: membrane localized p47phox and p67phox in M1 and E2 cells (treated according to conditions described in Fig. 4) were analyzed by Western blot using polyclonal p47phox and p67phox antibodies. Representative Western blots and histograms of data from 3 separate analyses are shown. Bottom: densitometric units are expressed as fold change relative to M1 cells in the absence or presence of LPS/LBP. Equal protein loading was controlled by immunoblotting of -actin. E2 cells had increased p47phox and p67phox levels in the membrane fraction compared with corresponding M1 cells whether in the absence or presence of LPS/LBP. DPI prevented the increases in both E2 and M1 cells, whereas DAS partially diminished the increase in E2 cells and had no such effect on M1 cells. No changes were detected in the cytosolic fraction. **P < 0.01 and ***P < 0.001 vs. M1 cells (without inhibitors) in (A) or in (B).
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CYP2E1 catalytic activity in CYP2E1-expressing macrophages is not affected by LPS/LBP.
Unlike NADPH oxidase, CYP2E1 catalytic activity in E2 cells was not stimulated by LPS/LBP (data not illustrated). The catalytic activity, however, was abolished by DAS, in accordance with the results observed in alcohol-fed animals (34) as well as in hepatoma (8) and hepatic stellate cells (37) transfected with CYP2E1. By contrast, DPI had no such effect. These data illustrate the specificity of actions of the inhibitors.
Effects of increased CYP2E1 expression on oxidant generation.
Because oxidants mediate LPS signal transduction, leading to TNF-
production in macrophages (42), we determined their generation. In the absence of LPS/LBP, intracellular H2O2 levels of E2 cells in culture were 2 and 3 times higher than those of M1 cells after 30 min and 24 h, respectively, but O2, lipid peroxidation and GSH levels were not affected by CYP2E1 expression (Fig. 6). LPS/LBP treatment for 30 min elevated the level of H2O2 (80%) as well as those of O2 (75%) and of lipid peroxidation (35%) in E2 cells compared with similarly treated M1 cells. Similar trends of increases were seen at 24 h after LPS/LBP. At this time, the GSH level in E2 cells became significantly lowered (20%) compared with M1 cells. These results demonstrate that CYP2E1 overexpression enhances oxidative stress in macrophages and sensitizes them to generate increased amounts of O2 and H2O2 in response to LPS stimuli.

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Fig. 6. Effects of CYP2E1 overexpression on oxidative stress in macrophages with or without LPS/LBP treatment. E2 and M1 macrophages cultured in the absence or presence of LPS/LBP for 0.5 h and 24 h were analyzed for intracellular H2O2 and O2 formation and lipid peroxidation using fluorescent probes. Increased 2',7'-dichlorofluorescein (DCF) fluorescence is proportional to increased H2O2 generation. Decreased fluorescence of hydroethidine and cis-parinaric acid reflected increased O2 formation and lipid peroxidation, respectively. Values are expressed relative to M1 cells at 0.5 h, assigned a value of 1. In the absence of LPS/LBP, only H2O2 generation was increased in E2 cells at both 30 min and 24 h. After LPS/LBP, H2O2 and O2 formation and lipid peroxidation were increased in E2 cells at 0.5 and 24 h, while GSH level was reduced in E2 cells at 24 h.
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Generation of H2O2 by NADPH oxidase and CYP2E1 in macrophages expressing CYP2E1: effects of DPI, DAS, catalase, and 4-MP.
To determine whether NADPH oxidase and CYP2E1 are a source of H2O2, we measured its formation in macrophages treated with or without DPI or DAS. As shown in Fig. 7, A and B, whether in the absence or presence of LPS/LBP, DPI reduced H2O2 formation in both E2 and M1 cells to nearly one-half, suggesting an NADPH-mediated process. DAS halved the level in E2 cells, suggesting a CYP2E1-mediated process, but it had no such effect on M1 cells, consistent with the absence of CYP2E1. These data suggest that NADPH oxidase and CYP2E1 contribute equally to the generation of H2O2 in CYP2E1-expressing macrophages. Whereas CYP2E1 is not a source of H2O2 in macrophages which do not express CYP2E1, NADPH contributes to at least half of the oxidant in these cells. Fig. 7, A and B also shows that catalase, an antioxidant enzyme, abolished H2O2 formation in both E2 and M1 cells, confirming the production of H2O2 in macrophages.

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Fig. 7. Effects of DPI, DAS, catalase, and 4-MP on H2O2 generation. E2 and M1 macrophages were treated with the inhibitors for 30 min, followed by incubation in the absence (A) or presence (B) of LPS/LBP. After 30 min, H2O2 generation was determined by DCF fluorescence. Data are expressed relative to M1 cells without the inhibitor, assigned a value of 1. DPI decreased H2O2 levels in both E2 cells and M1 cells in the absence of LPS/LBP or in their presence. DAS decreased H2O2 level in E2 cells treated with or without LPS/LBP, but it had no such effect on M1 cells. Catalase completely suppressed H2O2 generation. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. M1 cells (without inhibitors) in (A) or (B). C: 4-MP decreased the level of H2O2 in E2 cells, but not in M1 cells, whether in the absence or presence of LPS/LBP.
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Because DAS may possess antioxidant properties (39), an additional experiment was performed using 4-MP, an inhibitor of CYP2E1 catalytic activity without antioxidant action (52). Fig. 7C shows that 4-MP reduced H2O2 formation to the same extent as DAS in E2 cells, whether in the absence or presence of LPS stimuli, but not in M1 cells which do not express CYP2E1, supporting the conclusion that DAS inhibits H2O2 formation through its action on CYP2E1 as a competitive inhibitor of CYP2E1 (3, 54) and not through its antioxidant effect.
Activation of ERK1/2 and p38 MAPK and its inhibition by DPI, DAS, and catalase.
Having shown that H2O2 formation is increased in CYP2E1-expressing macrophages, we determined its role in the LPS signal transduction pathways. Because ERK1/2 and p38 are activated by LPS (4, 15, 19, 20, 21) through CD14/TLR4 (16, 44), their activation was assessed. Western blot analyses in Fig. 8 show that, whether in the absence of LPS/LBP (A) or in their presence (B), phosphorylation of ERK1/2 and p38 was greater in E2 than M1 cells, in accordance with the higher levels of H2O2 in E2 cells. DPI, which inhibited NADPH oxidase and H2O2 formation, decreased ERK1/2 and p38 activation in both E2 and M1 cells. DAS, which inhibited CYP2E1 and H2O2 formation, also decreased ERK1/2 and p38 activation in E2 cells, but it had no such effect on M1 cells, reflecting the absence of CYP2E1. Phosphorylation of ERK1/2 and p38 in E2 and M1 cells was dependent on H2O2 because it was abolished by catalase. Activation of ERK1/2 was blocked by PD-098059 (13) and that of p38 by SB-203580 (10), in accordance with the actions of these inhibitors. Likewise, p38 activation was blocked by SB-203580, but not PD-098059. We conclude from these data that, in CYP2E1 expressing macrophages, H2O2 derived in part from NADPH and CYP2E1 (see Fig. 7) activates ERK1/2 and p38. These signaling pathways are amplified in response to LPS stimuli.

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Fig. 8. Effects of DPI, DAS, and catalase on p38 and ERK1/2 phosphorylation. E2 and M1 cells were treated with or without DPI, DAS, catalase, p38 inhibitor SB-203580 (SB), and ERK1/2 inhibitor PD-098059 (PD) for 30 min, followed by incubation in the absence (A) or presence (B) of LPS/LBP for 30 min. Macrophage protein lysates were analyzed for p38 and ERK1/2 phosphorylation by Western blot. Densitometric units are normalized to that of M1 cells (with no inhibitor or LPS/LBP), assigned a value of 1. Numbers above the blots refer to mean values of 3 separate analyses. E2 cells had increased p38 and ERK1/2 phosphorylation, whether in the absence or presence of LPS/LBP. DPI attenuated the phosphorylation in both E2 and M1 cells. DAS decreased the phosphorylation in E2 but not in M1 cells. Catalase abolished p38 and ERK1/2 phosphorylation in both E2 and M1 cells. Inhibition of p38 phosphorylation by SB and of ERK1/2 phosphorylation by PD illustrates the specificity of the kinase activation. DMSO, vehicle control for SB and PD, had no effect on the kinase phosphorylation (data not shown).
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NF-
B activation and its inhibition by DPI, DAS, and catalase and the ERK1/2 inhibitor PD-098059.
To examine whether ERK1/2 and p38 participate in the activation of NF-
B in LPS signaling pathways, mediating TNF-
production, we assessed NF-
B activation. Fig. 9 shows that, whether in the absence or presence of LPS/LBP, NF-
B DNA binding was greater in E2 than M1 cells. DPI and DAS decreased the binding equally in E2 cells, suggesting involvement of NADPH oxidase and CYP2E1 in this process. Whereas DPI also reduced NF-
B activation in M1 cells, DAS had no such effect, consistent with the absence of CYP2E1 expression in M1 cells. Catalase blocked NF-
B activation in both E2 and M1 cells, suggesting involvement of H2O2. Importantly, NF-
B activation was totally suppressed by the ERK1/2 inhibitor PD-098059, indicating an ERK1/2-mediated process. NF-
B activation was not affected by the p38 inhibitor SB-203580. The activation was accompanied by translocation of NF-
B into the nucleus (where it regulates TNF-
gene expression), as revealed by an increase in nuclear p65 expression with a slight decrease in cytoplasmic I
B-
expression; these changes were more evident after LPS/LBP treatment than in untreated cells. We conclude from these data that NF-
B is activated by an H2O2-mediated ERK1/2 signaling mechanism. This process is increased in CYP2E1 expressing cells and is amplified in response to LPS stimuli.

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Fig. 9. Effects of DPI, DAS, catalase, p38 inhibitor SB, and ERK1/2 inhibitor PD on NF- B activation. M1 and E2 macrophages were treated with or without the inhibitors for 30 min, followed by incubation in the absence (A) or presence (B) of LPS/LBP for 30 min. Macrophage nuclear protein extracts were analyzed for NF- B DNA binding by EMSA. Expression of nuclear p65 and cytoplasmic I B- was analyzed by Western blot. Densitometric units are expressed relative to that of M1 cells (no inhibitor or LPS/LBP) assigned a value of 1. Numbers above the blots refer to mean values of 3 separate analyses. E2 macrophages had increased NF- B DNA binding, whether in the absence or presence of LPS/LBP. Increases were lowered by DPI, DAS, and catalase. DPI also decreased the binding levels in M1 cells, whereas DAS had no such effect. While PD completely blocked NF- B DNA binding in E2 and M1 cells, SB had no such effect. Changes in nuclear expression of p65 were more evident in LPS/LBP-treated macrophages cells than in untreated ones. DMSO used as vehicle control for SB and PD had no effect on NF- B DNA binding (data not shown).
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Effects of DPI, DAS, catalase, p38 inhibitor SB-203580, and ERK1/2 inhibitor PD-098059 on TNF-
production in CYP2E1-expressing macrophages.
DPI, DAS, catalase, p38 inhibitor SB-203580, and ERK1/2 inhibitor PD-098059 on TNF-
production in CYP2E1-expressing macrophages were evaluated to verify the involvement of NADPH oxidase, CYP2E1, H2O2, p38, and ERK1/2 in LPS-induced TNF-
production. The data in Fig. 10 show that, whether in the absence or presence of LPS/LBP, DPI diminished TNF-
concentrations in the culture media of E2 and M1 cells to one-half. DAS equally decreased TNF-
production in E2 cells, but it had no such effect on M1 cells. Catalase, SB-203580 and PD-098059 reduced TNF-
production in both E2 and M1 cells, with the strongest effect seen with catalase. These data support the conclusion that the stimulation of TNF-
production by LPS in CYP2E1-expressing macrophages is mediated in part by H2O2, derived from NADPH oxidase and CYP2E1, via p38 and ERK1/2 signaling pathways.
p38 MAPK stabilizes TNF-
mRNA.
The data of Figs. 9 and 10 revealed that p38 increases TNF-
production that is independent of NF-
B activation. Accordingly, we evaluated whether p38 acts at the posttranscriptional level by stabilizing TNF-
mRNA. Fig. 11 shows, in LPS/LBP-stimulated E2 cells, that the p38 inhibitor SB-203580 shortened the half-life of TNF-
mRNA from 46 to 16 min, suggesting that p38 stimulates TNF-
mRNA expression through stabilization of its message. The ERK1/2 inhibitor PD-098059 had no effect on TNF-
mRNA decay.
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DISCUSSION
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Our findings are summarized in Fig. 12. CYP2E1 overexpression in macrophages resulted in increased levels of CD14 and its coreceptor TLR4, NADPH oxidase (including its activity and its membrane-localized p47phox and p67phox subunits), and H2O2, accompanied by activation of ERK1/2, p38, and NF-
B, resulting in TNF-
production. Increased CYP2E1 expression primes macrophages, sensitizing them to increase TNF-
production in response to LPS stimuli. These act through CD14/TLR4 or through NADPH oxidase to generate increased amounts of H2O2, with amplification of ERK1/2 and p38 signaling pathways. ERK1/2 activates NF-
B to increase TNF-
production, while p38 stabilizes TNF-
mRNA, promoting TNF-
production.
Central to CYP2E1 priming of macrophages and their sensitization to LPS stimuli is the induction of oxidative stress. Increased oxidative stress in CYP2E1-expressing macrophages amplifies LPS signaling pathways, leading to TNF-
production. Oxidant generation and its mediation of signaling cascades have been proposed to play a role in the sensitization of Kupffer cells to increase TNF-
production in response to LPS after ethanol (1, 49). Kono and colleagues observed that oxidative stress and liver pathology induced by enteral ethanol administration were lacking in rats given DPI (24) and in p47phox knockout mice with NADPH deficiency (25). These results emphasize the predominant role for Kupffer cell NADPH oxidase in the generation of oxidants and the resulting liver damage during alcohol administration. Consistent with this notion, we found an enhanced NADPH oxidase activity in CYP2E1-expressing macrophages with increased generation of oxidants. Our data indicate that, in addition to NADPH oxidase, CYP2E1 is a source of oxidants in CYP2E1-expressing macrophages treated or not with LPS/LBP. Indeed, CYP2E1 contributes as much as NADPH oxidase to H2O2 formation. Furthermore, because CYP2E1 expression also resulted in enhanced expression of the membrane-localized p47phox and p67phox, which paralleled the changes of the NADPH oxidase activity and thereby contributed to the oxidase activation, it may augment H2O2 generation via this mechanism. However, CYP2E1 is not a source of H2O2 in macrophages that do not express CYP2E1, whereas NADPH oxidase is the predominant source of H2O2 in these cells, whether in the absence or presence of LPS stimuli. The remaining sources of H2O2 were not identified, but they could be xanhine oxidase and nitric oxide synthase, which have been shown to be sensitive to DPI inhibition (12, 32, 38). However, contributions of oxidants from these factors are not considered to be major in alcohol-fed animals (24).
Data in Fig. 7 also suggest that a significant portion of H2O2 formation in CYP2E1-expressing macrophages does not appear to be derived from either the NADPH or the CYP2E1 pathway. It is likely that this relates to the upregulation of CD14/TLR4 (28, 55), but how CYP2E1 expression affects the signal transduction from these receptors to the formation of oxidants is not known (see Fig. 12). In Kupffer cells, an upregulation of CD14 and TLR4 has been reported after ethanol administration (45, 46), but the involvement of CYP2E1 in this process was not evaluated in these studies. It has been suggested that these LPS receptors could generate oxidants via NADPH oxidase in response to LPS (49). This is actually possible in view of the rise of NADPH oxidase in CYP2E1-expressing macrophages. TLR2 expression in macrophages is not affected by CYP2E1, which is consistent with the suggestion that this receptor does not contribute to LPS signal transduction (40, 45, 56).
Kupffer cell CYP2E1 is catalytically active (26, 35, 50) and is upregulated after chronic ethanol consumption (4, 5, 22). The seven- to ninefold induction is of the same relative magnitude as in hepatocytes, but in absolute amounts, the CYP2E1 content is 10 times lower than in hepatocytes of the same animals (22). Our previous studies (4, 5) with cultured Kupffer cells of ethanol-fed rats suggest a possible involvement of CYP2E1 and oxidant formation in the stimulation of TNF-
production by LPS or acetaldehyde. It is now possible to attribute these processes to the induction of CYP2E1 by ethanol, which primes Kupffer cells and sensitizes them to LPS stimuli in accordance with the mechanism demonstrated in CYP2E1-expressing macrophages. Thus upregulation of CYP2E1 in conjunction with increased NADPH oxidase in Kupffer cells with increased production of oxidants may contribute to the development of alcoholic liver injury by supplementing the primary role of CYP2E1 in hepatocytes, which is also upregulated by ethanol (reviewed in Ref. 30). In support of this view were studies that demonstrated that, after intragastric ethanol administration in rats, chemical inhibition of hepatic CYP2E1 induction with DAS and phenethyl isothiocynate resulted in reduced liver pathology, including early changes of steatosis and lipid peroxidation (34). Furthermore, CYP2E1 transgenic mice were found to develop more hepatic steatosis than nontransgenic animals after enteral ethanol feeding (33). At variance with this is the observation that in CYP2E1-deficient mice, liver steatosis and oxidant generation associated with enteral ethanol administration were not diminished, which led to the conclusion that CYP2E1 and its capacity to generate oxidants are not involved in early alcoholic liver injury in that model (23). In CYP2E1 knockout mice, other P450 isoforms, including CYP4A (29), that are upregulated in the absence of CYP2E1 in the knockouts, may play a compensatory role in the induction of alcoholic liver injury. Despite these differing views, it is germane to take into account the contribution of Kupffer cell CYP2E1 relative to that of hepatocyte CYP2E1 in the analysis of alcoholic liver injury.
We found that ERK1/2 and p38 were activated in CYP2E1-expressing macrophages and that these signaling pathways were amplified when the macrophages were stimulated with LPS/LBP. These processes were mediated by H2O2 derived, in part, from CYP2E1 and NADPH oxidase (see Fig. 7 and 8). ERK1/2 stimulated TNF-
production via activation of NF-
B, while p38 promoted TNF-
production by a mechanism involving stabilizing of TNF-
mRNA. These findings are consistent with our observations and that of others in Kupffer cells of ethanol-fed rats in response to LPS (4, 5, 21) and support the conclusion that activation of NF-
B with increased TNF-
production plays a central role in alcoholic liver (36).
In addition to the regulation of TNF-
production, ERK1/2 and p38 could also contribute, at least in part, to the increase in NADPH oxidase activity, because it has been shown by El Benna et al. (14) that, in human neutrophils, the NADPH oxidase subunit p47phox was phosphorylated by ERK1/2 and p38, but not by JNK, another member of MAPKs. This remains to be investigated in CYP2E1-expressing macrophages. We did not observe activation of JNK in either macrophages after CYP2E1 transfection or Kupffer cells of ethanol-fed rats, whether or not stimulated with LPS (unpublished data), whereas this kinase was found to be activated in the RALA rat hepatocyte cell line overexpressing CYP2E1 (31). In these cells, JNK was shown to participate in the mediation of CYP2E1 sensitization of cell death induced by TNF-
, in association with oxidant stress. CYP2E1 overexpression in hepatoma cells also resulted in activation of p38 and ERK1/2 (53). Activation of p38, but not ERK1/2 in these hepatoma cells appears to promote arachidonic acid-induced oxidative stress and cell toxicity by affecting mitochondrial membrane potential and by modulating NF-
B activation. Collectively, these results demonstrate that, whether in macrophages, hepatocytes, or hepatoma cells, CYP2E1 overexpression elicits activation of MAPK signal transduction pathways that regulate diverse cellular functions, including cytokine generation, inflammation, cell toxicity, and apoptosis.
Our investigation with E2 cells demonstrated the involvement of CYP2E1 in the priming of macrophages, sensitizing them to increase TNF-
production in response to LPS. Because the CYP2E1 catalytic activity in E2 cells was two times that in Kupffer cells from alcohol-fed rats, we wondered whether the sensitization also occurred in macrophages when CYP2E1 was expressed at a level similar to that in Kupffer cells after ethanol feeding. The data in Table 1 show that cells of the E3 clone, which had a catalytic activity equivalent to that of Kupffer cells, produced TNF-
at a level that was statistically significantly higher than control cells, whether in the absence or presence of LPS. These results demonstrated the physiological relevance of CYP2E1 transfection in macrophages to CYP2E1 induction in Kupffer cells in vivo by ethanol. It is particularly worth noting that TNF-
generation correlated positively and significantly with increasing CYP2E1 catalytic activities, which clearly indicates that stimulated TNF-
production is a result of CYP2E1 expression and is not related to a clonal artifact. The data of LPS signaling mechanisms are derived from experiments with E2 cells. It is likely that similar results would be found in E3 cells with perhaps a different level of significance statistically, because these cells, like E2 cells, are sensitized to generate increased amounts of TNF-
when stimulated with LPS, although less than E2 cells.
In conclusion, because CYP2E1 is upregulated in Kupffer cells after ethanol feeding, CYP2E1 priming could explain the mechanisms by which Kupffer cells are sensitized to LPS by ethanol, a critical step in early alcoholic liver injury. The signaling mechanisms associated with LPS in Kupffer cells after ethanol consumption can be reproduced in E2 macrophages. Admittedly, these studies do not address other aspects of Kupffer cell responses to alcohol, including their phagocytic activity and immunomodulatory functions, but nonetheless, CYP2E1 expressing RAW 264.7 macrophages, which are readily amenable to genetic manipulation, offer a starting point for further refinement of our understanding of the biochemical and toxicological properties associated with Kupffer cell CYP2E1 in alcoholic liver disease.
 |
GRANTS
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This study was supported, in part, by National Institute on Alcohol Abuse and Alcoholism Grant AA-11115, the Department of Veterans Affairs, and the Kingsbridge Research and the Christopher D. Smithers Foundations.
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ACKNOWLEDGMENTS
|
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We thank Dr. Jingxiang Bai, Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, for help in establishing CYP2E1 macrophage clones.
Portions of this work were presented at the Annual Meeting of Research Society on Alcoholism, June 2630, 2004, Vancouver, BC, Canada and published in abstract form in Alcohol Clin Exp Res 28: 123A, 2004.
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FOOTNOTES
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Address for correspondence: C. S. Lieber, Alcohol Research Center, Veterans Affairs Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468 (E-mail: liebercs{at}aol.com)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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