QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.004879v1 33/9/1391    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tochigi, Y. Articles by Yokota, H. Search for Related Content PubMed PubMed Citation Articles by Tochigi, Y. Articles by Yokota, H.

ISOFORM-SPECIFIC EXPRESSION AND INDUCTION OF UDP-GLUCURONOSYLTRANSFERASE IN IMMUNOACTIVATED PERITONEAL MACROPHAGES OF THE RAT

Laboratory of Veterinary Biochemistry, Graduate School of Veterinary Medicine (Y.T., S.G., H.Y.), and Laboratory of Biology, Faculty of Environmental System (N.Y.), Rakuno Gakuen University, Ebetsu, Hokkaido, Japan; Expression and Molecular Regulation Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, Tsukisamu-higashi, Toyohira-ku, Sapporo, Japan (Y.T., S.O.); and Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan (S.O.)

(Received April 1, 2005; accepted June 22, 2005)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Phase I drug-metabolizing enzymes such as cytochrome P450 in immunocytes are known to play a role in metabolic activation of toxic and immunosuppressive compounds such as polycyclic aromatic hydrocarbon (PAH). UDP-glucuronosyltransferase (UGT), a drug-metabolizing phase II enzyme, accelerates elimination of these compounds; however, there is little information on the expression and function of UGT in immunocytes. In this study, we investigated the expressions of UGT isoforms in rat peritoneal macrophages and the role of UGT in macrophage functions. Expressions of UGT1A1, 1A6, and 1A7 were observed in macrophages by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. When macrophage cells cultured in plates were exposed to 1-naphthol and 3-hydroxybenzo-[a]pyrene (3-OH-B[a]P), these glucuronides increased in the medium, indicating that macrophages glucuronidated the chemicals. The production of the glucuronides of 1-naphthol and 3-OH-B[a]P was induced by lipopolysaccharide (LPS) treatment of the cultured macrophage cells. Northern blot analysis revealed that UGT1A7 mRNA was induced by LPS treatment. This result is the first evidence that a drug-metabolizing enzyme is induced by immunoactivation. The results indicated that macrophages can detoxify various toxic and immunosuppressive compounds with UGT, and that ability is enhanced by immunoactivation. We propose that macrophages contribute to protection against not only macromolecules as immunocytes but also small molecules such as the immunosuppressive agents PAHs in peripheral blood and interstitial tissues.

Previous studies showed that phase I drug-metabolizing enzymes such as cytochrome P450 (P450) are expressed in lymphocytes and macrophage populations. These enzymes play central roles in the metabolic activation and detoxification of various xenobiotics, including small molecular compounds that cause immunosuppression and allergic reactions. Macrophage populations include alveolar macrophages and Kupffer cells, splenic adherent cells, peritoneal macrophages, and peripheral blood monocytes, which also have P450s (Germolec et al., 1995). P450s catalyze metabolic activation of polycyclic aromatic hydrocarbons (PAHs), including B[a]P. Metabolically activated B[a]P has been reported to show immunosuppressive toxicities toward immunocytes such as monocytes, macrophages, and lymphocytes. B[a]P and dimethylbenzo[a]anthracene inhibit antibody production from murine B-cells (White and Holsapple, 1984). They also suppress mitogenesis of human T lymphocytes (Mudzinski, 1993) and alter B lymphopoiesis through triggering apoptosis of pre-B lymphocytes (Yamaguchi et al., 1997). B[a]P also inhibits differentiation of human monocytes into macrophages (Van Grevenynghe et al., 2003). It is well known that B[a]P is metabolically activated by phase I drug-metabolizing enzymes such as CYP1A1 and CYP1B1, resulting in toxicological effects. Since P450s are expressed in immunocytes, these enzymes, as stated above, play central roles in the metabolic activation of B[a]P.

The elimination of metabolically activated B[a]P and PAHs is accelerated by phase II drug-metabolizing enzymes such as UGT isoforms. Recent studies have shown that UGTs are expressed in immunocytes such as alveolar macrophages (Willey et al., 1996; Yamashiki et al., 2002) and Kupffer cells (Lafranconi et al., 1986; Oesch and Steinberg, 1987; Oesch et al., 1992) and in bone marrow stroma (Ganousis et al., 1992). In addition, glucuronidation activity against B[a]P has been shown in rat lymphocytes, and it has been suggested that lymphocyte UGTs play a role in cytoprotection by eliminating bioactivated B[a]P that may covalently bind to DNA (Hu and Wells, 1994 and 2004). These studies suggest that UGTs function to ward off toxicities of metabolically activated PAHs, including B[a]P, in immunocytes.

Little is known, however, about isoform-specific expression of UGT in immunocytes and its relation to immunofunctions of the cells. In the present study, we investigated expression of UGT isoforms and the ability of glucuronidation of chemicals in rat peritoneal macrophages. We found isoform-specific regulations of UGTs in the immunoactivation phase. The role of UGTs in macrophage functions is discussed in this manuscript.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Restriction endonucleases, other DNA-modifying enzymes, and reagent kits were purchased from Takara Shuzo (Kyoto, Japan), Toyobo (Osaka, Japan), New England Biolabs (Beverly, MA), and Roche Diagnostics (Mannheim, Germany). 1-Naphthol (1-hydroxynaphthalene) and UDP-glucuronic acid (uridine-5'-diphosphoglucuronic acid trisodium salt) were obtained from Wako Pure Chemicals (Osaka, Japan) and Nakarai Chemicals (Kyoto, Japan), respectively. Lipopolysaccharide (LPS) was obtained from List Biological Laboratories Inc. (Campbell, CA). 3-Hydroxybenzo[a]pyrene and benzo[a]pyrene-3-ß-D-glucopyranosuronic acid were obtained from Midwest Research Institute (Kansas City, MO). Other reagents were of the highest grade available. Polyclonal antibodies against phenol UGT were prepared as previously described (Yokota and Yuasa, 1990). The antibodies recognized mainly phenol UGT corresponding to UGT1A6. Peroxidase-conjugated anti-rabbit IgG antibody was obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).

Animals and Preparation of Peritoneal Macrophages. Male Sprague-Dawley rats (9–12 weeks old) were used in this study. The rats were housed under standard conditions and given food and water ad libitum. The rats were killed under anesthesia with 60% urethane (0.5 ml/kg) by exsanguination via carotid arteries. Peritoneal cells were collected and suspended in Hanks' buffer. The cells were cultured in Eagle's minimal essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Invitrogen) and 100 U/ml penicillin–100 µg/ml streptomycin (Sigma-Aldrich, St Louis, MO) and incubated at 37°C in a CO₂ incubator (5% CO₂/95% room air). After 3 h of incubation, nonadhering cells were washed out, and adhering cells were then collected by scraping and used as peritoneal macrophages. Macrophages were identified by means of -naphtyl acetate esterase (nonspecific esterase) staining, and cell viability (over 95%) was determined by a trypan blue exclusion test (Eichner and Smeaton, 1983; Melnicoff et al., 1989).

Preparation of Microsomes and Enzyme Assay. Macrophages and liver were homogenized with 4 volumes of 0.15 M KCl solution containing 1 mM EDTA. The homogenate was centrifuged at 9000g for 15 min, and the supernatant fraction was further centrifuged at 105,000g for 60 min to obtain microsomes. Approximately 30 to 50 µg of microsomal proteins from macrophages were obtained per animal. UGT activities were determined toward various substrates in microsomes, which were activated with 0.05% sodium cholate, in 200 µl of 50 mM Tris-HCl buffer (pH 7.4), 3 mM UDP-glucuronic acid, and 0.5 mM MgCl₂ containing phenolic substrates such as 0.05 mM 1-naphthol, 10 mM 4-methylumbelliferone, and 0.005 mM 3-OH-B[a]P at 37°C. Approximately 50 µg of microsomes of macrophages and liver were used for a single assay. The enzymatic activity was determined by HPLC analysis as described previously (Yokota et al., 1999). Fluorescence of 1-naphthol glucuronide and B[a]P glucuronide were detected with excitation/emission wavelengths of 296/336 nm (1-naphthol glucuronide) and 300/421 nm (B[a]P glucuronide), respectively (Singh and Wiebel, 1979; Yokota et al., 1999). A peak derived from each glucuronide in the HPLC analysis was confirmed by the disappearance of the peak in a sample pretreated with ß-glucuronidase. Protein concentration was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as a standard. Kinetic parameters were estimated by determining enzymatic activities with various concentrations of 1-naphthol (0.00625–0.125 mM). Apparent K_m values were estimated by the Lineweaver-Burk plot of the data.

Immunohistochemical Staining. A cell suspension of peritoneal macrophages was smeared on a coverslip coated with 3-aminopropyltriethoxy silane (Sigma-Aldrich) and fixed for 1 h in 4% paraformaldehyde/phosphate-buffered saline (PBS). After being treated with cold methanol for 10 min, semidried specimens were immersed in PBS with 0.25% bovine serum albumin and 0.1% Triton X-100 (PBS-BT) buffer for 30 min at room temperature. Immunostaining of smeared cells was carried out as follows. After incubation in normal goat serum (Sigma-Aldrich) for 30 min, anti-UGT antibodies/PBS-BT (1:100 dilution) were applied on the coverslip for 2 h at room temperature. After washing three times with PBS-BT, the specimens were treated with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (MP Biomedicals, Irvine, CA)/PBS-BT (1:500 dilution) containing propidium iodide (Sigma-Aldrich) for 1 h at room temperature. The coverslip was mounted on a slide with 70% glycerol containing 5% n-propyl gallate. In control studies for specific immunofluorescence, the specimens were incubated with PBS. Sections of stained cells were examined under an Olympus Fluoview confocal laser-scanning microscope (Olympus, Tokyo, Japan).

Immunoblot Analysis. Microsomal protein samples (10 µg) were subjected to SDS-polyacrylamide slab gel electrophoresis. The polypeptide bands thus separated were transferred to a nitrocellulose membrane, and immunoreactive bands were detected using polyclonal antibodies against UGT1A6 (Yokota and Yuasa, 1990).

RT-PCR. Total RNA was isolated from peritoneal macrophages by using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany). DNase digestion was performed in all RNA preparations before RT-PCR. cDNA was synthesized from total RNA with Superscript II (Invitrogen) reverse transcriptase. The coding regions of the respective cDNA species were amplified by PCR with oligonucleotide primers that were designed by reference to the sequences of rat UGT1A family members (Grams et al., 2000). A UGT1A1 sense primer, 5'-TGGTGTGCCGGAGCTCATGTTCG-3'; UGT1A2 sense primer, 5'-GGAAGAATATCAGCGGGAAATACTGGGC-3'; UGT1A3 sense primer, 5'-ATTTTCTCTGAAGTTAGTTCTACAG-3'; UGT1A5 sense primer, 5'-GTGGTCTTTGAAACAGGCAACTATGTG-3'; UGT1A6 sense primer, 5'-CCTCAGTGAACGCGGACACGAC-3'; UGT1A7 sense primer, 5'-CAGTTGGCAGCTGGGAAAACCA-3'; and UGT1A8 sense primer, 5'-GGCACATGG-GAAAGTCGTTGA-3', were designed from their isoform-specific regions. An antisense primer, 5'-CTGGAATCTCTGAGACCATGGATC-3', was designed from the UGT1A family common region. A UGT2B family sense primer, 5'-GGAAGAATTTGTTCAGAGC-3', and an antisense primer, 5'-AACAGCTGCTCCTTTGGC-3', were designed from the common region of the family. An MRP1 sense primer, 5'-ACCTGCGCTTCAAGATCACC-3'; MRP1 anitisense primer, 5'-GCCAGAGATCAGTTCACACC-3'; MRP2 sense primer, 5'-CCAGACATGGTGAGATCCAG-3'; MRP2 antisense primer, 5'-CACTGGGAGAACTCCTTTCG-3'; MRP3 sense primer, 5'-CCATGACCTGCGTTCACAAC-3'; and MRP3 antisense primer, 5'-AGGCGAGTCCTGCATCTTTG-3', were synthesized for amplification of MRP cDNA. All cDNA bands amplified by PCR were sequenced by a model 310 sequencer (Applied Biosystems, Foster City, CA).

Detection of UGT Activity in Cells. Isolated peritoneal macrophages were plated on dishes in a fresh medium. After cell culturing for 12 h, 1-naphthol was added to the medium to a final concentration of 0.04 mM. Medium samples were periodically collected, and 1-naphthol glucuronide concentration in the medium was measured by HPLC (Yokota et al., 1999). LPS (0.1 and 1 µg/ml of culture medium) was added to the culture medium to investigate immunostimulation and preincubated for 24 h. After preincubation with or without LPS, 1-naphthol (final concentration, 0.04 mM) or 3-OH-B[a]P (final concentration, 2.5 µM) was added to the culture medium. 3-OH-B[a]P was dissolved by DMSO. The final concentration of DMSO in the culture medium did not exceed 0.05% in these assays. After 24 h, collected culture media were assayed for 1-naphthol glucuronide and 3-OH-B[a]P glucuronide by HPLC, and NO was assayed using a Grass Reagent System (Promega, Madison, WI).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Immunohistochemical staining of UGTs in rat peritoneal macrophages. Immunohistochemical staining of rat peritoneal macrophage cells was performed as described under Materials and Methods using polyclonal antibodies against a UGT isoform corresponding to UGT1A6. Confocal laser-scanning microscopic images of peritoneal macrophages (A and D), alveolar macrophages (B and E), and monocyte cells in whole blood cells (C and F) are shown. The red signal of propidium iodide shows cell nuclei. The green signal indicates the existence of UGT. No signals were detected in the cells stained only with the second antibody (D–F). Bar indicates 10 µm.

Statistical Analysis. Statistical analysis of data in Fig. 5 was performed by using analysis of variance between groups with the statistical software KaleidaGraph (version 3.6; Abelbeck/Synergy, Reading, PA). When statistically significant differences were found, the data were further analyzed by Bonferroni's multiple comparison test. Statistical analysis of data in Fig. 6 was performed by using Student's t test. In each analysis, values of p < 0.05 were considered to be statistically significant.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Effect of LPS on UGT activities in rat peritoneal macrophages. Adherent peritoneal macrophages were prepared and cultured on plates, and LPS (0.1 µg/ml and 1 µg/ml) was added to the culture medium. After 24 h of preincubation, the culture medium was changed to a fresh medium containing LPS and 1-naphthol (final concentration, 0.04 mM) or 3-OH-B[a]P (final concentration, 2.5 µM). After 24 h of incubation, 1-naphthol glucuronide (A), 3-OH-B[a]P glucuronide (B), and NO (C) in the medium were assayed as described under Materials and Methods. Each value is the mean ± S.E. of three independent experiments. *, p < 0.05 and **, p < 0.01 with respect to the control (saline). , p < 0.01 with respect to no substrate.

View larger version (54K): [in this window] [in a new window]   FIG. 6. Effect of LPS on mRNA expression of UGT isoforms in rat peritoneal macrophages. mRNAs of major UGT isoforms were analyzed by Northern blotting as described under Materials and Methods. Total RNAs prepared from rat peritoneal macrophages cultured with 1 µg/ml LPS for 24 h were electrophoresed, blotted, and hybridized with probes UGT1A1 (A), UGT1A6 (B), UGT1A7 (C), and GAPDH (D). Top, typical results of Northern blotting analysis; bottom, data obtained from the densitometric analysis of Northern blotting experiments by NIH Image. The amount of mRNA was normalized with 18S rRNA amounts. Each value is the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to the control (saline).

View larger version (31K): [in this window] [in a new window]   FIG. 2. Western blot analysis for peritoneal macrophages. The contents of UGT protein in microsome fractions of the liver and peritoneal macrophages were compared. The three lanes on the left were loaded with liver microsomes; the three lanes on the right were loaded with peritoneal macrophage microsomes. Each lane was loaded with 10 µg of protein. Open delta symbol indicates a positive band corresponding to UGT protein.

	Results

Top Abstract Materials and Methods Results Discussion References

In addition, UGT activities in the microsomal fractions of rat peritoneal macrophages were detected (Table 1). UGT activities were detected in the microsomal fractions of rat peritoneal macrophages toward 1-naphthol, 4-metylumbelliferon, and 3-OH-B[a]P, which are glucuronidated by phenol UGTs such as UGT1A5, 1A6, and 1A7 (Table 1). The levels of UGT activities toward these substrates in peritoneal macrophages were lower than those in the liver. These results correspond to the results shown in Fig. 2. Furthermore, the K_m value of microsomal UGT activities against 1-naphthol in peritoneal macrophages was smaller than that in hepatic microsomes (peritoneal macrophages, 0.0224 ± 0.0023; liver, 0.0508 ± 0.001).

Peritoneal macrophages were prepared from rats and cultured in the medium as described under Materials and Methods. 1-Naphthol was added to the culture medium, and the metabolites in the medium were analyzed by HPLC. An HPLC peak of 1-naphthol glucuronide was observed in the culture medium 48 h after the addition of the substrate to the culture plate of peritoneal macrophages, as shown in Fig. 3A. The glucuronide concentrations in the medium increased with increase in incubation time and were proportional to the amounts of cells (Fig. 3B). These results indicate that the peritoneal macrophages on the plate can take up 1-naphthol from the medium and glucuronidate the xenobiotics, and the resultant glucuronide was excreted into the culture medium.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Glucuronidation of 1-naphthol and excretion of its glucuronide from rat peritoneal macrophages. Peritoneal macrophage cells were prepared from the rat as described under Materials and Methods. Macrophages were cultured on plates and exposed to 1-naphthol (final concentration, 0.04 mM). The culture media after 6, 12, 24, and 48 h were analyzed by HPLC as described under Materials and Methods. The HPLC chromatogram shown in A indicates the production of 1-naphthol glucuronide in the culture medium. The glucuronide was confirmed by deconjugation using ß-glucuronidase. The product concentrations in the medium are shown in B (open squares, 6 x 106 cells/plate; closed squares, 3 x 106 cells/plate). Data are shown as means ± S.E. of three independent experiments.

cDNAs of various UGT isoforms were amplified by RT-PCR using specific primers as described under Materials and Methods. The amplification of cDNAs is shown in Fig. 4, A (peritoneal macrophages) and B (liver). Four UGT1 isoforms, UGT1A1, UGT1A5, UGT1A6, and UGT1A7, and UGT2B family members were expressed in the rat liver, whereas peritoneal macrophages expressed only three isoforms, UGT1A1, UGT1A6, and UGT1A7 (Fig. 4, A and B). To investigate the significance of UGT1A6 and UGT1A7 conjugation in macrophages, the excretion of 1-naphthol glucuronide from the cells was studied as follows. Multidrug resistance-associated proteins MRP1 (Nunoya et al., 2003), MRP2 (Ito et al., 1998), and MRP3 (Hirohashi et al., 1999) have been reported to transport xenobioticglucuronide conjugate through cell membranes. These transporter mRNAs were amplified using specific primers as described under Materials and Methods, and the results are shown in Fig. 4C. These three transporter mRNAs were observed by RT-PCR in the rat liver, and MRP1 and MRP3 were also detected in rat peritoneal macrophages (Fig. 4C), suggesting that these MRPs participate in the excretion of 1-naphthol glucuronide from the macrophages.

View larger version (48K): [in this window] [in a new window]   FIG. 4. Detection of UGT1A, UGT2B, and MRP transcripts in rat liver and peritoneal macrophages by RT-PCR. Total RNA was prepared from peritoneal macrophages and liver of the rat. RT-PCR was performed as described under Materials and Methods. The transcripts of parts of UGT isoforms such as UGT1A1, UGT1A2, UGT1A3, UGT1A5, UGT1A6, UGT1A7, UGT1A8, and UGT2B family members as well as GAPDH are shown in respective lanes in the peritoneal macrophages (A) and liver (B). The three major MRP transcripts were also amplified from total RNA of rat peritoneal macrophages and liver (C).

We also investigated the regulation of UGT in macrophages by immunostimulation. UGT activities toward 1-naphthol, 3-OH-B[a]P, and NO production in the macrophage cells were assayed after LPS (final concentrations, 0.1 and 1 µg/ml) had been added to the culture medium. The results are shown in Fig. 5. 1-Naphthol glucuronide, 3-OH-B[a]P glucuronide, and NO production levels were increased with the increase in the amount of LPS. NO has been used as an indicator of immunoactivation, because NO production from immunocytes is increased by immunoactivation. mRNA expressions of the UGT isoforms UGT1A1, UGT1A6, and UGT1A7 were analyzed by Northern blotting using specific probes as described under Materials and Methods. In LPS-stimulated macrophage cells, UGT1A7 mRNA was specifically induced by 1.7-fold, whereas UGT1A1 was only slightly induced. In contrast, UGT1A6 was reduced in the cells (Fig. 6). These results suggested that the immunoactivation of macrophages alter the expression of these UGT isoforms in an isoform-specific manner. Interestingly, the expression levels of the UGT isoforms in macrophages differed greatly from those in normal liver. Expression levels of UGT1A6 and 1A7 in normal and immunoactivated macrophages were remarkably higher then those in the liver. UGT2B family members were not expressed in immunoactivated macrophages (data not shown).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we found the expression of UGT isoforms UGT1A1, UGT1A6, and UGT1A7 in macrophage cells by the determination of enzymatic activities (Table 1), immunohistochemical study (Fig. 1), Western blot analysis (Fig. 2), and RT-PCR (Fig. 4). When 1-naphthol, a substrate of UGT1A6 (Burchell and Coughtrie, 1989), and 3-OH-B[a]P, a substrate of UGT1A7 (Grove et al., 1997), were added to culture medium of rat peritoneal macrophages, both glucuronides were observed in the medium (Figs. 3 and 5). MRP1 and MRP3, which are members of a large family of ATP-binding cassette proteins, transport many anionic conjugates, such as estradiol glucuronide (Hirohashi et al., 1999; Nunoya et al., 2003), across the cell membrane. MRP1 and MRP3 have been shown to be expressed in various tissues (Langmann et al., 2003). In blood cells, MRP1 has been reported to be expressed in erythrocytes (Flens et al., 1996). We also found the expression of these MRP mRNAs in rat peritoneal macrophages (Fig. 4C). These results suggested that many xenobiotics were oxidized by P450 and glucuronidated by UGT isoforms in macrophages and that the resultant glucuronides were excreted into the medium by the MRPs. These results imply that macrophages play a role not only as scavenger cells for macromolecules such as proteins but also in metabolism of small molecules in blood.

It is known that activities of drug-metabolizing enzymes are suppressed by immunoactivation. The expression of an isoform of hepatic cytochrome P450 was reduced by LPS or cytokines such as interleukin-1ß and tumor necrosis factor- in in vivo and in vitro assays (Barker et al., 1992; Abdel-Razzak et al., 1993; Muntane-Relat et al., 1995). Ke et al. (2001) reported that tumor necrosis factor-- and LPS-induced down-regulation of CYP1A1 expression was mediated by nuclear factor-B and aryl hydrocarbon receptor interaction. A UGT isoform was also reduced by interleukin-1ß treatment in the liver (Ferrari et al., 1993). It has been reported that P450s in macrophages were also reduced by immunoactivations using LPS (Nakamura et al., 1998); however, there is little information on drug-metabolizing enzymes in immunocytes. We first showed the specific expression of UGT1A1, 1A6, and 1A7 in macrophages. In these UGT isoforms, mRNA of UGT1A7 in rat peritoneal macrophages was specifically increased by LPS treatment (Fig. 6C). In addition, glucuronidation of 3-OH-B[a]P, which is mainly catalyzed by UGT1A7 (Grove et al., 1997), was also increased by LPS treatment (Fig. 5). These results showed that UGT1A7 was induced by immunoactivation of macrophages. This is the first report of a drug-metabolizing enzyme being induced by immunoactivation in immunocytes. We also revealed that UGT1A1, 1A6, and 1A7 were differently regulated in the phase of immunoactivation (Fig. 6). These isoform-specific regulations may indicate that an individual isoform of UGTs in macrophage cells plays an independent role. In the experiment for which results are shown in Fig. 5, we measured NO production from macrophages to confirm immunoactivation. Although NO production was increased by only LPS treatment, it was further increased by the addition of a UGT substrate, 1-naphthol, or 3-OH-B[a]P (Fig. 5C). A previous study also showed that 17ß-estradiol increased NO production through the induction of inducible NO synthase (You et al., 2003). These results clearly indicate that UGT activities in macrophages are induced by immunoactivation of macrophages, which is synergistically enhanced by substrates.

3-OH-B[a]P, which we used for measurement of UGT activities, is a well known chemical causing immunosuppression in immunocytes (White and Holsapple, 1984; Mudzinski, 1993; Yamaguchi et al., 1997; Van Grevenynghe et al., 2003). Many studies have suggested that UGTs play a role in cell protection against PAHs. Hu and Wells (1992) showed by using lymphocytes from UGT1A-deficient rats (Gunn rats) that UGTs contribute to the reduction of the covalent binding of B[a]P to DNA. Our experiment showed that the ability of glucuronidation against 3-OH-B[a]P was increased by the activation of immunity (Fig. 5). This response would be a protective mechanism against immunosuppressive chemicals in macrophages. Therefore, our results suggest that the regulation of metabolic enzymes and the metabolism of chemicals in macrophages are important in the study of immunosuppressive chemicals.

Exogenous chemicals such as drugs and endocrine disrupters taken into a living subject and endogenous materials such as hormones are mainly metabolized in the liver and digestive tract. However, drug-metabolizing enzymes are expressed in many organs, including immunocytes, and it is thought that they contribute to the metabolism of xenobiotics at the local sites; however, functions of drug-metabolizing enzymes in immunocytes such as macrophages are not well known. In this study, we demonstrated the isoform-specific expressions of UGTs and their glucuronidation activities in macrophage cells. Although the number of macrophage cells was small, UGT1A6 and UGT1A7 mRNA were expressed in normal peritoneal macrophages at levels 5- and 10-fold higher than those in a normal liver, respectively (Fig. 6, B and C).

This finding may indicate differences in stabilities of these mRNAs in each organ. However, the differences between the expressions of these mRNAs in the liver and peritoneal macrophages indicate that macrophage UGTs may play a role different from that of liver UGTs. For example, some hormones contribute to the regulation of cell functions in immunocytes. Macrophage UGTs may be related to the regulation of cell functions through hormone metabolisms, because UGT1A family members are known to glucuronidate several hormones.

In summary, we showed detailed expression profiles of UGT and glucuronidation activities in rat peritoneal macrophage cells. We also showed immunoinducibility of the UGT1A7 gene for the first time. We conclude that macrophages contribute to protection against not only macromolecules as immunocytes but also small molecules such as immunosuppressive agents in peripheral blood and interstitial tissues. We propose here that the regulation of drug-metabolizing enzymes is an important factor in the study of immunosuppressive agents such as PAH in immunocytes.

	Footnotes

 
This work was supported in part by grants-in-aid for High Technological Research Center (Rakuno Gakuen University) from the Ministry of Education, Science, Sports and Culture of Japan.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.004879.

ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; 3-OH-B[a]P, 3-hydroxybenzo[a]pyrene; P450, cytochrome P450; PAH, polycyclic aromatic hydrocarbon; B[a]P, benzo[a]pyrene; LPS, lipopolysaccharide; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; RT, reverse transcriptase; PCR, polymerase chain reaction; MRP, multidrug resistance-associated protein; DMSO, dimethyl sulfoxide; DIG, digoxigenin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BT, 0.25% bovine serum albumin and 0.1% Triton X-100.

Address correspondence to: Dr. Hiroshi Yokota, Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan. E-mail: h-yokota{at}rakuno.ac.jp

	References

Top Abstract Materials and Methods Results Discussion References

 


Abdel-Razzak Z, Loyer P, Fautrel A, Gautier JC, Corcos L, Turlin B, Beaune P, and Guillouzo A (1993) Cytokines down-regulate expression of major cytochrome P-450 enzymes in adult human hepatocytes in primary culture. Mol Pharmacol 44: 707–715.[Abstract]

Barker CW, Fagan JB, and Pasco DS (1992) Interleukin-1 beta suppresses the induction of P4501A1 and P4501A2 mRNAs in isolated hepatocytes. J Biol Chem 267: 8050–8085.[Abstract/Free Full Text]

Burchell B and Coughtrie MWH (1989) UDP-glucuronosyltransferases. Pharmacol Ther 43: 261–289.[CrossRef][Medline]

Coffman BL, Green MD, King CD, and Tephly TR (1995) Cloning and stable expression of a cDNA encoding a rat liver UDP-glucuronosyltransferase (UDP-glucuronosyltransferase 1.1) that catalyzes the glucuronidation of opioids and bilirubin. Mol Pharmacol 47: 1101–1105.[Abstract]

Eichner RD and Smeaton TC (1983) Agar accumulates in rat peritoneal macrophages elicited with thioglycollate broth. Scand J Immunol 18: 259–263.[CrossRef][Medline]

Ferrari L, Jouzeau JY, Gillet P, Herber R, Fener P, Batt AM, and Netter P (1993) Interleukin-1 beta differentially represses drug-metabolizing enzymes in arthritic female rats. J Pharmacol Exp Ther 264: 1012–1020.[Abstract/Free Full Text]

Flens MJ, Zaman GJ, van der Valk P, Izquierdo MA, Schroeijers AB, Scheffer GL, van der Groep P, de Haas M, Meijer CJ, and Scheper RJ (1996) Tissue distribution of the multidrug resistance protein. Am J Pathol 148: 1237–1247.[Abstract]

Fournel-Gigleux S, Sutherland L, Sabolovic N, Burchell B, and Siest G (1991) Stable expression of two human UDP-glucuronosyltransferase cDNAs in V79 cell cultures. Mol Pharmacol 39: 177–183.[Abstract]

Ganousis LG, Goon D, Zyglewska T, Wu KK, and Ross D (1992) Cell-specific metabolism in mouse bone marrow stroma: studies of activation and detoxification of benzene metabolites. Mol Pharmacol 42: 1118–1125.[Abstract]

Germolec DR, Adams NH, and Luster MI (1995) Comparative assessment of metabolic enzyme levels in macrophage populations of the F344 rat. Biochem Pharmacol 50: 1495–1504.[CrossRef][Medline]

Grams B, Harms A, Braun S, Strassburg CP, Manns MP, and Obermayer-Straub P (2000) Distribution and inducibility by 3-methylcholanthrene of family 1 UDP-glucuronosyltransferases in the rat gastrointestinal tract. Arch Biochem Biophys 377: 255–265.[CrossRef][Medline]

Grove AD, Kessler FK, Metz RP, and Ritter JK (1997) Identification of a rat oltipraz-inducible UDP-glucuronosyltransferase (UGT1A7) with activity towards benzo[a]pyrene-7,8-dihydrodiol. J Biol Chem 272: 1621–1627.[Abstract/Free Full Text]

Harding D, Fournel-Gigleux S, Jackson MR, and Burchell B (1988) Cloning and substrate specificity of a human phenol UDP-glucuronosyltransferase expressed in COS-7 cells. Pro Natl Acad Sci USA 85: 8381–8385.[Abstract/Free Full Text]

Hirohashi T, Suzuki H, and Sugiyama Y (1999) Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3). J Biol Chem 274: 15181–15185.[Abstract/Free Full Text]

Hu Z and Wells PG (1992) In vitro and in vivo biotransformation and covalent binding of benzo[a]pyrene in Gunn and RHA rats with a genetic deficiency in bilirubin uridine diphosphate-glucuronosyltransferase. J Pharmacol Exp Ther 263: 334–342.[Abstract/Free Full Text]

Hu Z and Wells PG (1994) Modulation of benzo[a]pyrene bioactivation by glucuronidation in lymphocytes and hepatic microsomes from rats with a hereditary deficiency in bilirubin UDP-glucuronosyltransferase. Toxicol Appl Pharmacol 127: 306–313.[CrossRef][Medline]

Hu Z and Wells PG (2004) Human interindividual variation in lymphocyte UDP-glucuronosyltransferases as a determinant of in vitro benzo[a]pyrene covalent binding and cytotoxicity. Toxicol Sci 78: 32–40.[Abstract/Free Full Text]

Ito K, Suzuki H, Hirohashi T, Kume K, Shimizu T, and Sugiyama Y (1998) Functional analysis of a canalicular multispecific organic anion transporter cloned from rat liver. J Biol Chem 273: 1684–1688.[Abstract/Free Full Text]

Ke S, Rabson AB, Germino JF, Gallo MA, and Tian Y (2001) Mechanism of suppression of cytochrome P-450 1A1 expression by tumor necrosis factor-alpha and lipopolysaccharide. J Biol Chem 276: 39638–39644.[Abstract/Free Full Text]

Lafranconi WM, Glatt H, and Oesch F (1986) Xenobiotic metabolizing enzymes of rat liver nonparenchymal cells. Toxicol Appl Pharmacol 84: 500–511.[CrossRef][Medline]

Langmann T, Mauerer R, Zahn A, Moehle C, Probst M, Stremmel W, and Schmitz G (2003) Real-time reverse transcription-PCR expression profiling of the complete human ATP-binding cassette transporter superfamily in various tissues. Clin Chem 49: 230–238.[Abstract/Free Full Text]

Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275.[Free Full Text]

Melnicoff MJ, Horan PK, and Morahan PS (1989) Kinetics of changes in peritoneal cell populations following acute inflammation. Cell Immunol 118: 178–191.[CrossRef][Medline]

Mudzinski SP (1993) Effects of benzo[a]pyrene on concavalin A-stimulated human peripheral blood mononuclear cells in vitro: inhibition of proliferation but no effect on parameters related to the G1 phase of the cell cycle. Toxicol Appl Pharmacol 119: 166–174.[CrossRef][Medline]

Muntane-Relat J, Ourlin JC, Domergue J, and Maurel P (1995) Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2 and CYP3A4 in human hepatocytes in primary culture. Hepatology 22: 1143–1153.[CrossRef][Medline]

Nakamura M, Imaoka S, Amano F, and Funae Y (1998) P450 isoforms in a murine macrophage cell line, RAW264.7 and changes in the levels of P450 isoforms by treatment of cells with lipopolysaccharide and interferon-gamma. Biochim Biophys Acta 1385: 101–106.[CrossRef][Medline]

Nunoya K, Grant CE, Zhang D, Cole SP, and Deeley RG (2003) Molecular cloning and pharmacological characterization of rat multidrug resistance protein 1 (mrp1). Drug Metab Dispos 31: 1016–1026.[Abstract/Free Full Text]

Oesch F, Arand M, Coughtrie MW, Burchell B, and Steinberg P (1992) The distribution of UDP-glucuronosyltransferases in rat liver parenchymal and nonparenchymal cells. Biochem Pharmacol 43: 731–737.[CrossRef][Medline]

Oesch F and Steinberg P (1987) A comparative study of drug-metabolizing enzymes present in isolated rat liver parenchymal, Kupffer and endothelial cells. Biochem Soc Trans 15: 372–373.[Medline]

Singh J and Wiebel FJ (1979) A highly sensitive and rapid fluorometric assay for UDP-glucuronyltransferase using 3-hydroxybenzo[a]pyrene as substrate. Anal Biochem 98: 394–401.[CrossRef][Medline]

Van Grevenynghe J, Rion S, Le Ferrec E, Le Vee M, Amiot L, Fauchet R, and Fardel O (2003) Polycyclic aromatic hydrocarbons inhibit differentiation of human monocytes into macrophages. J Immunol 170: 2374–2381.[Abstract/Free Full Text]

White KL Jr and Holsapple MP (1984) Direct suppression of in vitro antibody production by mouse spleen cells by the carcinogen benzo[a]pyrene but not by the noncarcinogenic congener benzo[e]pyrene. Cancer Res 44: 3388–3393.[Abstract/Free Full Text]

Willey JC, Coy E, Brolly C, Utell MJ, Frampton MW, Hammersley J, Thilly WG, Olson D, and Cairns K (1996) Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells. Am J Respir Cell Mol Biol 14: 262–271.[Abstract]

Yamaguchi K, Near RI, Matulka RA, Shneider A, Toselli P, Trombino AF, and Sherr DH (1997) Activation of the aryl hydrocarbon receptor/transcription factor and bone marrow stromal cell-dependent preB cell apoptosis. J Immunol 158: 2165–2173.[Abstract]

Yamashiki N, Yokota H, Sakamoto M, and Yuasa A (2002) Presence of phenol UDP-glucuronosyltransferase in bovine alveolar macrophages and bronchial epithelial cells. Toxicology 176: 221–227.[CrossRef][Medline]

Yokota H, Iwano H, Endo M, Kobayashi T, Inoue H, Ikushiro S, and Yuasa A (1999) Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver. Biochem J 340: 405–409.

Yokota H and Yuasa A (1990) Increase of a form of UDP-glucuronyltransferase glucuronizing various phenolic xenobiotics and the corresponding translatable mRNA in 3-methylcholanthrene-treated rat liver. J Biochem (Tokyo) 107: 92–96.[Abstract/Free Full Text]

You HJ, Kim JY, and Jeong HG (2003) 17 Beta-estradiol increases inducible nitric oxide synthase expression in macrophages. Biochem Biophys Res Commun 303: 1129–1134.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.004879v1 33/9/1391    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tochigi, Y. Articles by Yokota, H. Search for Related Content PubMed PubMed Citation Articles by Tochigi, Y. Articles by Yokota, H.