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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-γ
Biochimica et Biophysica Acta 1385 Ž1998. 101–106
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-g Mayumi Nakamura a , Susumu Imaoka a
b,)
, Fumio Amano a , Yoshihiko Funae
b
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan b Laboratory of Chemistry, Osaka City UniÕersity Medical School, Abeno-ku, Osaka, Japan Received 11 December 1997; accepted 17 March 1998
Abstract The presence of P450 in a murine macrophage cell line, RAW264.7, was investigated to clarify the biological role and regulation of P450. Microsomes of RAW264.7 cells were isolated and subjected to immunoblotting with anti-rat CYP2A1, 2B1, and 4A2 antibodies. The microsomes gave staining bands with all these antibodies, suggesting the presence of mouse Cyp2a, 2b, and 4a isoforms in RAW264.7. RAW264.7 cells were treated with typical inducers of P450 Žphenobarbital, clofibrate, b-naphthoflavone and 3-methylcholanthrene.. None of these chemicals induced these P450s. Stimulation of RAW264.7 cells with lipopolysaccharide ŽLPS. and interferon-g ŽINF-g . which increase inducible nitric oxide synthase ŽiNOS. and cytokines in cells decreased Cyp4a protein but not Cyp2a and 2b proteins. To identify P450 isoforms in RAW264.7, we used polymerase chain reaction ŽPCR. primers for mouse Cyp2a4, 2a12, 2b9r10, 4a10, and 4a12. Total RNA was isolated from these cells and converted to cDNA by reverse transcriptase. PCR was done with these primers and the amplified nucleotides were analyzed by a DNA sequencer. Only Cyp2b9r10 and 4a12 primers gave clear bands, although all primers gave clear bands from liver total RNA. Nucleotide sequences of these products amplified by PCR were identical with Cyp2b9 and 4a12. These findings indicate that Cyp2b9 and 4a12 were present in a macrophage cell line, RAW264.7, and the regulation of P450 by inducers and cytokine differed from that in liver. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; Macrophage; Lipopolysaccharide; Interferon-g
Abbreviations: P450 or CYP, cytochrome P450; NO, nitric oxide; NOS, nitric oxide synthase; LPS, lipopolysaccharide; IFNg , interferon-gamma; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PB, phenobarbital; CF, clofibrate; BNF, b-naphthoflavone; 3-MC, 3-methylchoranthrene; DMSO, dimethylsulfoxide; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; GAPDH, glycelaldehyde-3-phosphate dehydrogenase; RT–PCR, reverse transcriptase–polymerase chain reaction ) Corresponding author. Laboratory of Chemistry, Osaka City University Medical School, 1-4-54 Asahimachi, Abeno-ku, Osaka 545, Japan. Fax: q81-6-646-3922.
1. Introduction Nitric oxide Ž NO. is produced by nitric oxide synthase Ž NOS. w1x. NOS catalyzes the formation of NO from L-arginine in two steps w2x. The first step is an N-oxidation of L-arginine with consumption of NADPH and O 2 and formation of N v-hydroxy-Larginine, and the second step an oxidation of N v-hydroxy-L-arginine which also consumes NADPH and
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O 2 and leads to L-citrulline and NO. NOS is a natural fusion protein of NADPH-P450 reductase and cytochrome P450 and has similar characteristics with cytochrome P450 w3x. Recently, Boucher et al. w4x found that CYP3A isoform, a major P450 in liver, contributes to oxidation of N v-hydroxy-L-arginine and suggested that P450 participates in the synthesis of NO in liver. Macrophages produce large amounts of NO. Cytochrome P450 isoforms were detected in lymphocytes and polymorphonuclear leukocytes w5– 7x. The biological role of P450 in macrophage is not known and contribution of P450 to NO synthesis has not been demonstrated. The immune system is a direct target for toxic xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin ŽTCDD. w8x. Many of these chemicals require metabolic activation to reveal toxicity w9x. Previous studies have demonstrated that macrophage metabolizes benzoŽ a.pyrene w10x. Exposure of mice to benzoŽ a.pyrene resulted in an increase in the amounts of some benzoŽ a.pyrene metabolites generated by macrophages w11x. P450 in macrophages may have important roles in cell toxicity of xenobiotics and in the immune system. In this report, we investigated presence of P450 isoforms and induction of these P450s in a murine macrophage cell, RAW264.7 by chemical inducers. Furthermore, we investigated changes in the amount of P450s by treatment of RAW264.7 with lipopolysaccharide ŽLPS. and interferon-g ŽINF-g . and found that the level of Cyp4a12 was decreased by the treatment.
2. Materials and methods
2.2. Cell culture A murine macrophage cell line, RAW264.7, was obtained from the American Type Culture Collection ŽRockville, MD.. The cells were maintained by culturing in Ham’s F12 medium ŽDainippon Pharmaceutical, Osaka., supplemented with 50 U penicillinrml, 50 m grml streptomycin Ž Flow Laboratories, Irving Scotland., and 10% heat-inactivated fetal bovine serum ŽGibco, Grand Island, NY. , in a CO 2 incubator Ž5% CO 2 –95% humidified air. at 378C. The cells were seeded onto plastic plate dishes ŽFalcon, a1001, Deckton Dickinson. , and passaged twice a week. 2.3. Induction of inducible nitric oxide synthase ( iNOS ) in macrophages and treatment of macrophages with P450 inducers The cells were seeded at 4 = 10 5 cellsrml onto 60-mm petri dishes ŽFalcon, a1007. in 5 ml of fresh culture medium and then incubated at 378C overnight. The medium was replaced with 5 ml of fresh culture medium containing LPS Ž 100 ngrml. with IFN-g Ž10 Urml. or P450 inducers, PB Ž 0.4 mM. , CF Ž 0.1 mM., BNF Ž 0.01 mM. , 3-MC Ž0.01 mM. and DMSO Ž0.1%.. The cells were further incubated at 378C for 24 h for P450 inducers and 8 h for LPS activation w12x. The cells from 10 plates were collected by centrifugation and washed twice with PBS and homogenized in buffer A containing 0.25 M sucrose, 1 mM EDTA, and 1 mM PMSF in 10 mM HEPES– NaOH ŽpH 7.5.. The homogenates were centrifuged at 750 = g for 5 min at 48C and the supernatant was further centrifuged at 100,000 = g for 60 min at 48C. The pellet, microsomal fraction, was suspended in buffer B containing buffer A and 20% glycerol.
2.1. Chemicals
2.4. Immunoblotting of RAW264.7 microsomes
LPS from Escherichia coli was obtained from Sigma Ž St. Louis, MO. . 125 I-labeled protein A was synthesized as previously described w12x. Phenobarbital ŽPB., clofibrate Ž CF., b-naphthoflavone ŽBNF., 3-methylchoranthrene Ž3-MC., and dimethylsulfoxide ŽDMSO. were obtained from Wako ŽTokyo, Japan.. Recombinant INF-g was kindly supplied by Toray ŽTokyo, Japan..
Fifty micrograms aliquots of microsomes were subjected to SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membrane as described w13x. The proteins on the membrane were reacted with antibodies against CYP2A1, 2B1, and 4A2 and then 125 I-labeled protein A under conditions described previously w12x. Antibodies against rat CYP2A1, 2B1, and 4A2 were prepared
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with Japanese white rabbit Ž Bioteck, Saga, Japan. and were characterized previously w13x. The reacted bands on the membrane were detected with a BAS2000 bioimage analyzer Ž Fuji Film, Tokyo, Japan. . 2.5. RT–PCR and sequence analysis of RAW264.7 RNA Total RNA from RAW264.7 cells was isolated with ISOGEN ŽNippon Gene, Toyama, Japan. according to the manufacturer’s instructions. For reverse transcriptase–polymerase chain reaction, TAKARA RNA PCR Kit ŽTAKARA, Kyoto, Japan. was used including AMV RNA reverse transcriptase. This reaction was done in 20 m l containing 5 m g total RNA according to the manufacturer’s instructions. The mixture was preincubated for 10 min at 308C, then incubated for 30 min at 558C to convert the mRNA to cDNA, and for 5 min at 998C to inactivate the enzyme. PCR reactions were done in a 100-m l final volume consisting of 1 = Taq DNA polymerase buffer Ž50 mM KCl, 10 mM Tris–HCl, pH 9.0, Triton X-100, 1.25 mM MgCl 2 ., 0.2 mM of dNTP, 10 pmol each of forward and reverse primers, and 1 unit of Taq DNA polymerase Ž TAKARA, Kyoto, Japan.. The PCR was done for 30 cycles by sequence temperature, 1 min denaturation at 948C, 1 min annealing at 508C, 2.5 min extension at 728C. The forward and reverse primers for mouse P450s and GAPDH are summarized in Table 1. The primers were prepared from the published nucleotide sequences of Cyp2a4 w14x, 2a12 w15x, 2b9r10 w16x, 4a10 w17x, 4a12 w18x, and GAPDH w19x. PCR products were analyzed by electrophoresis with 2% agarose. If necessary, the band was cut out and the DNA extracted. The nucleotide sequence was analyzed with
Fig. 1. Immunoblotting of a macrophage-like cell line, RAW264.7, with anti-rat P450 antibodies. Macrophage microsomes Ž50 m g of protein. were applied to 7.5% polyacrylamide gel. The proteins were reacted with antibodies against P450 indicated at the right side of the membrane and then the resultant immune complex was reacted with 125 I-labeled protein A. Ab, antibody; PB, phenobarbital-treated; CF, chlofibrate-treated; BNF, b-naphthoflavone-treated; 3-MC, 3-methylcholanthrene-treated; DMSO, dimethylsulfoxide-treated; NT, non-treated.
an autosequencer Ž 377A, Perkin-Elmer. after fluorescent labeling.
3. Results 3.1. Effects of P450 inducers on RAW264.7 cells Three kinds of antibodies against rat CYP2A1, 2B1, and 4A2 were used. Cyp2a and 2b isoforms
Table 1 The primers used for PCR Cyp
Sense
Anti-sense
2a4 2a12 2b9r10 4a10 4a12 GAPDH
5X-TCT GTT GCT GAT GAA GTA CC-3X 5X-CCT GCT ACT AAT GAA GCA TC-3X 5X-CAG AGT TCC ATC ACC AGA AC-3X 5X-CTA ATG GTG CGT ACA GAT TG-3X 5X-TTG GCC ACA AAT CCT GAA CAT-3X 5X-CAG CCT CGT CCC GTA GAC AA-3X
5X-TTG TCA TCT AGG AAG TGC TT-3X 5X-TTG TGA TCC AGG AAG TGC TG-3X 5X-CTG TGG ATC ATG TAG AGC TG-3X 5X-AAT GGC CTG GAG GTA GGT CC-3X 5X-AAG ACC CTG GTG CAA ATC GA-3X 5X-GTA GTT GAG GTC AAT GAA GGG-3X
The primers were prepared from published nucleotide sequences of Cyp2a4 w14x, 2a12 w15x, 2b9r10 w16x, 4a10 w17x, 4a12 w18x, and GAPDH w19x. Primers for Cyp2b9r10 recognized both Cyp2b9 and 2b10.
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3.2. Changes in the leÕels of P450 by actiÕation of RAW264.7 by LPS and IFN-g LPS and INF-g strongly induce iNOS in macrophages. RAW264.7 cells were treated with LPS and INF-g . Changes in the protein levels of Cyp2a, 2b and 4a were investigated Ž Fig. 2. . Cyp2a and 2b levels did not change but Cyp4a levels decreased significantly. Fig. 2. Changes in the levels of P450 by treatment with LPS and INF-g . Microsomes were isolated from non-treated RAW264.7 cells or those treated with LPS and INF-g . Levels of P450 were assayed by immunoblotting. The results are expressed as relative values with the control levels set at 100%. The values are expressed as mean"SD of four different preparation of microsomes. )Significantly different from the non-treated; p- 0.05.
were first isolated from mouse liver or kidney w14–16x and Cyp4a is a major form in extrahepatic tissue such as kidney w17,18x. The band at similar mobility with a purified rat P450 was detected with each antibody, suggesting the presence of Cyp2a, 2b, and 4a in RAW264.7 microsomes ŽFig. 1.. PB decreased Cyp2b but other inducers including CF, BNF, 3-MC, and DMSO did not affect the levels of P450 in RAW264.7 cells.
Fig. 3. RT–PCR of P450 mRNA in RAW264.7 cells and mouse liver. Total RNA was isolated from RAW264.7 without treatment ŽNT., those treated with LPS and INF-g ŽLPSqINF., and mouse liver. RNA Ž5 m g for cells and 0.5 m g for liver. was converted to cDNA with reverse transcriptase and amplified by PCR with the primers shown in Table 1.
3.3. Detection of P450 mRNA in RAW264.7 To confirm the presence of P450 in RAW264.7, RT–PCR with total RNA from the cells was done using the primers indicated in Table 1. The primers of GAPDH, Cyp2b9r10, and Cyp4a12 gave clear bands ŽFig. 3.. Other sets of primers did not give
Fig. 4. Nucleotide sequences of fragments amplified from RAW264.7 mRNA by RT–PCR. The lower lines are results in this study. The numbers indicated in the left side are positions from published data w16,18x. The nucleotides underlined were used for PCR primers, which were also used for sequence analysis.
M. Nakamura et al.r Biochimica et Biophysica Acta 1385 (1998) 101–106
bands of the sizes predicted from the cDNA sequences although they gave clear bands when total RNA from mouse liver was used Ždata not shown.. The bands of RT–PCR products from RAW 264.7 RNA were cut out from the gels and the DNA extracted. The nucleotide sequence was analyzed. The sequences of the bands from Cyp2b9r10 and Cyp4a12 were identical with mouse Cyp2b9 and Cyp4a12 cDNA sequences although substitution of some nucleotides in Cyp4a12 was seen Ž Fig. 4. . The primers from Cyp2a4 and 2a12 gave no band although an anti-rat CYP2A1 antibody gave a clear band, suggesting the presence of another subfamily of Cyp2a isoform in RAW264.7 cells. The results from RT–PCR qualitatively suggested that treatment with LPS and INF-g decreased Cyp4a12 mRNA levels as well as protein levels.
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Although liver has Kupffer cells which are of macrophage-lineage, the major constituent of the liver cells is the parenchymal cells that are known to be an abundant source of P450 and responsible for the drug metabolism. This might explain the difference in the distribution of P450 as well as the response of P450 isoforms when certain drugs or biological response modifiers like LPS and INF-g are administered to liver or macrophages. Based on the variety of responses of macrophages to the pathological and pharmacological reagents, studies on P450 species in macrophages, especially in the process of macrophage activation, will lead to new findings on the role of P450 in regulation of macrophage functions.
References 4. Discussion In this study, we found that Cyp2b9 and 4a12 were present in a murine macrophage cell line, RAW264.7. The presence of CYP1A1 in macrophages was already reported and macrophages have aryl hydrocarbon hydroxylation activity which is responsible for CYP1A1 activity w5,11x. TCDD which suppresses the immune system is a strong inducer of CYP1A1 w8,20x. The P450 in macrophages may have an important role in immunosuppression of chemicals such as TCDD. The biological role of Cyp2b9 and 4a12 in RAW264.7 is not clear. CYP2B forms metabolize many drugs efficiently in liver w21x. CYP4A forms metabolize fatty acids and prostaglandins in kidney w22x. Macrophages oxidize low density lipoprotein w23x. CYP4A in macrophages may oxidize lipoprotein. PB is a strong inducer of Cyp2b forms in liver w5x but it did not induce Cyp2b9 protein in RAW264.7. CF is an inducer of Cyp4a forms w24x but it did not induce Cyp4a12 protein. LPS or INF-g usually suppresses the expression of P450, although suppression levels of P450 are different w25–27x. Regulation of P450s in macrophage is different from that in liver. The response to LPS and INF-g was also different between liver and macrophage-like cell line, RAW264.7.
w1x D.S. Bredt, P.M. Hwang, C.E. Glatt, C. Lowenstein, R.R. Reed, S.H. Snyder, Nature 351 Ž1991. 714–718. w2x D.J. Stuehr, M. Ikeda-Saito, J. Biol. Chem. 267 Ž1992. 20547–20550. w3x K.A. White, M.A. Marletta, Biochemistry 31 Ž1992. 6627– 6631. w4x J.L. Boucher, A. Genet, S. Vadon, M. Delaforge, Y. Henry, D. Mansuy, Biochem. Biophys. Res. Commun. 187 Ž1992. 880–886. w5x D.R. Germolec, N.H. Adams, M.I. Luster, Biochem. Pharmacol. 50 Ž1995. 1495–1504. w6x Y. Soh, H.M. Rhee, D.H. Sohn, B.J. Song, Biochem. Biophys. Res. Commun. 227 Ž1996. 541–546. w7x A. Mahnke, P.H. Roos, W.G. Hanstein, G.G. Chabot, Biochem. Pharmacol. 51 Ž1996. 1579–1582. w8x M.P. Holsapple, N.K. Snyder, S.C. Wood, D.L. Morris, Toxicology 69 Ž1991. 219–255. w9x S.N. Wickramasinghe, Clin. Lab. Haematol. 9 Ž1987. 271– 280. w10x T.C. Peterson, Biochem. Pharmacol. 36 Ž1987. 3911–3916. w11x G.S. Ladics, T.T. Kawabata, A.E. Munson, K.L. White Jr., Toxicol. Appl. Pharmacol. 116 Ž1992. 258–266. w12x T. Noda, F. Amano, J. Biochem. ŽTokyo. 121 Ž1997. 38–46. w13x S. Imaoka, Y. Terano, Y. Funae, Arch. Biochem. Biophys. 278 Ž1990. 168–178. w14x E.J. Squires, M. Negishi, J. Biol. Chem. 263 Ž1988. 4166– 4171. w15x M. Noshiro, M. Lakso, K. Kawajiri, M. Negishi, Biochemistry 27 Ž1988. 6434–6443. w16x M. Iwasaki, R.L. Lindberg, R.O. Juvonen, M. Negishi, Biochem. J. 291 Ž1993. 569–573. w17x C.J. Henderson, T. Bammler, C.R. Wolf, Biochim. Biophys. Acta 1200 Ž1994. 182–190. w18x D.R. Bell, N.J. Plant, C.G. Rider, L. Na, S. Brown, I.
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w19x w20x w21x
w22x
M. Nakamura et al.r Biochimica et Biophysica Acta 1385 (1998) 101–106 Ateitalla, S.K. Acharya, M.H. Davies, E. Elias, N.A. Jenkins, D.J. Gilbert, N.G. Copeland, C.R. Elcombe, Biochem. J. 294 Ž1993. 173–180. D.E. Sabath, H.E. Broome, M.B. Prystowsky, Gene 91 Ž1990. 185–191. R.K. Dooley, M.P. Holsapple, Immunopharmacology 16 Ž1988. 167–180. R.W. Nims, R.A. Lubet, in: C. Ioannides ŽEd.., Cytochromes P450: Metabolic and Toxicological aspect, CRC Press, London, 1996, pp. 135–160. B.G. Lake, D.F.V. Lewis, in: C. Ioannides ŽEd.., Cytochromes P450: Metabolic and Toxicological aspect, CRC Press, London, 1996, pp. 271–297.
w23x M.I. Dushkin, N.K. Zenkov, E.B. Menshikova, E.N. Pivovarova, G. Lyubimov, N.N. Volsky, Atherosclerosis 114 Ž1995. 9–18. w24x Y. Kikuta, E. Kusunose, K. Endo, S. Yamamoto, K. Sogawa, Y. Fujii-Kuriyama, M. Kusunose, J. Biol. Chem. 268 Ž1993. 9376–9380. w25x E.T. Morgan, Mol. Pharmacol. 36 Ž1989. 699–707. w26x L.C. Knickle, D.F. Spencer, K.W. Renton, Biochem. Pharmacol. 44 Ž1992. 604–608. w27x H. Sakai, T. Okamoto, Y. Kikkawa, J. Pharmacol. Exp. Ther. 263 Ž1992. 381–386.
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-g Mayumi Nakamura a , Susumu Imaoka a
b,)
, Fumio Amano a , Yoshihiko Funae
b
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan b Laboratory of Chemistry, Osaka City UniÕersity Medical School, Abeno-ku, Osaka, Japan Received 11 December 1997; accepted 17 March 1998
Abstract The presence of P450 in a murine macrophage cell line, RAW264.7, was investigated to clarify the biological role and regulation of P450. Microsomes of RAW264.7 cells were isolated and subjected to immunoblotting with anti-rat CYP2A1, 2B1, and 4A2 antibodies. The microsomes gave staining bands with all these antibodies, suggesting the presence of mouse Cyp2a, 2b, and 4a isoforms in RAW264.7. RAW264.7 cells were treated with typical inducers of P450 Žphenobarbital, clofibrate, b-naphthoflavone and 3-methylcholanthrene.. None of these chemicals induced these P450s. Stimulation of RAW264.7 cells with lipopolysaccharide ŽLPS. and interferon-g ŽINF-g . which increase inducible nitric oxide synthase ŽiNOS. and cytokines in cells decreased Cyp4a protein but not Cyp2a and 2b proteins. To identify P450 isoforms in RAW264.7, we used polymerase chain reaction ŽPCR. primers for mouse Cyp2a4, 2a12, 2b9r10, 4a10, and 4a12. Total RNA was isolated from these cells and converted to cDNA by reverse transcriptase. PCR was done with these primers and the amplified nucleotides were analyzed by a DNA sequencer. Only Cyp2b9r10 and 4a12 primers gave clear bands, although all primers gave clear bands from liver total RNA. Nucleotide sequences of these products amplified by PCR were identical with Cyp2b9 and 4a12. These findings indicate that Cyp2b9 and 4a12 were present in a macrophage cell line, RAW264.7, and the regulation of P450 by inducers and cytokine differed from that in liver. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; Macrophage; Lipopolysaccharide; Interferon-g
Abbreviations: P450 or CYP, cytochrome P450; NO, nitric oxide; NOS, nitric oxide synthase; LPS, lipopolysaccharide; IFNg , interferon-gamma; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PB, phenobarbital; CF, clofibrate; BNF, b-naphthoflavone; 3-MC, 3-methylchoranthrene; DMSO, dimethylsulfoxide; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; GAPDH, glycelaldehyde-3-phosphate dehydrogenase; RT–PCR, reverse transcriptase–polymerase chain reaction ) Corresponding author. Laboratory of Chemistry, Osaka City University Medical School, 1-4-54 Asahimachi, Abeno-ku, Osaka 545, Japan. Fax: q81-6-646-3922.
1. Introduction Nitric oxide Ž NO. is produced by nitric oxide synthase Ž NOS. w1x. NOS catalyzes the formation of NO from L-arginine in two steps w2x. The first step is an N-oxidation of L-arginine with consumption of NADPH and O 2 and formation of N v-hydroxy-Larginine, and the second step an oxidation of N v-hydroxy-L-arginine which also consumes NADPH and
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O 2 and leads to L-citrulline and NO. NOS is a natural fusion protein of NADPH-P450 reductase and cytochrome P450 and has similar characteristics with cytochrome P450 w3x. Recently, Boucher et al. w4x found that CYP3A isoform, a major P450 in liver, contributes to oxidation of N v-hydroxy-L-arginine and suggested that P450 participates in the synthesis of NO in liver. Macrophages produce large amounts of NO. Cytochrome P450 isoforms were detected in lymphocytes and polymorphonuclear leukocytes w5– 7x. The biological role of P450 in macrophage is not known and contribution of P450 to NO synthesis has not been demonstrated. The immune system is a direct target for toxic xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin ŽTCDD. w8x. Many of these chemicals require metabolic activation to reveal toxicity w9x. Previous studies have demonstrated that macrophage metabolizes benzoŽ a.pyrene w10x. Exposure of mice to benzoŽ a.pyrene resulted in an increase in the amounts of some benzoŽ a.pyrene metabolites generated by macrophages w11x. P450 in macrophages may have important roles in cell toxicity of xenobiotics and in the immune system. In this report, we investigated presence of P450 isoforms and induction of these P450s in a murine macrophage cell, RAW264.7 by chemical inducers. Furthermore, we investigated changes in the amount of P450s by treatment of RAW264.7 with lipopolysaccharide ŽLPS. and interferon-g ŽINF-g . and found that the level of Cyp4a12 was decreased by the treatment.
2. Materials and methods
2.2. Cell culture A murine macrophage cell line, RAW264.7, was obtained from the American Type Culture Collection ŽRockville, MD.. The cells were maintained by culturing in Ham’s F12 medium ŽDainippon Pharmaceutical, Osaka., supplemented with 50 U penicillinrml, 50 m grml streptomycin Ž Flow Laboratories, Irving Scotland., and 10% heat-inactivated fetal bovine serum ŽGibco, Grand Island, NY. , in a CO 2 incubator Ž5% CO 2 –95% humidified air. at 378C. The cells were seeded onto plastic plate dishes ŽFalcon, a1001, Deckton Dickinson. , and passaged twice a week. 2.3. Induction of inducible nitric oxide synthase ( iNOS ) in macrophages and treatment of macrophages with P450 inducers The cells were seeded at 4 = 10 5 cellsrml onto 60-mm petri dishes ŽFalcon, a1007. in 5 ml of fresh culture medium and then incubated at 378C overnight. The medium was replaced with 5 ml of fresh culture medium containing LPS Ž 100 ngrml. with IFN-g Ž10 Urml. or P450 inducers, PB Ž 0.4 mM. , CF Ž 0.1 mM., BNF Ž 0.01 mM. , 3-MC Ž0.01 mM. and DMSO Ž0.1%.. The cells were further incubated at 378C for 24 h for P450 inducers and 8 h for LPS activation w12x. The cells from 10 plates were collected by centrifugation and washed twice with PBS and homogenized in buffer A containing 0.25 M sucrose, 1 mM EDTA, and 1 mM PMSF in 10 mM HEPES– NaOH ŽpH 7.5.. The homogenates were centrifuged at 750 = g for 5 min at 48C and the supernatant was further centrifuged at 100,000 = g for 60 min at 48C. The pellet, microsomal fraction, was suspended in buffer B containing buffer A and 20% glycerol.
2.1. Chemicals
2.4. Immunoblotting of RAW264.7 microsomes
LPS from Escherichia coli was obtained from Sigma Ž St. Louis, MO. . 125 I-labeled protein A was synthesized as previously described w12x. Phenobarbital ŽPB., clofibrate Ž CF., b-naphthoflavone ŽBNF., 3-methylchoranthrene Ž3-MC., and dimethylsulfoxide ŽDMSO. were obtained from Wako ŽTokyo, Japan.. Recombinant INF-g was kindly supplied by Toray ŽTokyo, Japan..
Fifty micrograms aliquots of microsomes were subjected to SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membrane as described w13x. The proteins on the membrane were reacted with antibodies against CYP2A1, 2B1, and 4A2 and then 125 I-labeled protein A under conditions described previously w12x. Antibodies against rat CYP2A1, 2B1, and 4A2 were prepared
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with Japanese white rabbit Ž Bioteck, Saga, Japan. and were characterized previously w13x. The reacted bands on the membrane were detected with a BAS2000 bioimage analyzer Ž Fuji Film, Tokyo, Japan. . 2.5. RT–PCR and sequence analysis of RAW264.7 RNA Total RNA from RAW264.7 cells was isolated with ISOGEN ŽNippon Gene, Toyama, Japan. according to the manufacturer’s instructions. For reverse transcriptase–polymerase chain reaction, TAKARA RNA PCR Kit ŽTAKARA, Kyoto, Japan. was used including AMV RNA reverse transcriptase. This reaction was done in 20 m l containing 5 m g total RNA according to the manufacturer’s instructions. The mixture was preincubated for 10 min at 308C, then incubated for 30 min at 558C to convert the mRNA to cDNA, and for 5 min at 998C to inactivate the enzyme. PCR reactions were done in a 100-m l final volume consisting of 1 = Taq DNA polymerase buffer Ž50 mM KCl, 10 mM Tris–HCl, pH 9.0, Triton X-100, 1.25 mM MgCl 2 ., 0.2 mM of dNTP, 10 pmol each of forward and reverse primers, and 1 unit of Taq DNA polymerase Ž TAKARA, Kyoto, Japan.. The PCR was done for 30 cycles by sequence temperature, 1 min denaturation at 948C, 1 min annealing at 508C, 2.5 min extension at 728C. The forward and reverse primers for mouse P450s and GAPDH are summarized in Table 1. The primers were prepared from the published nucleotide sequences of Cyp2a4 w14x, 2a12 w15x, 2b9r10 w16x, 4a10 w17x, 4a12 w18x, and GAPDH w19x. PCR products were analyzed by electrophoresis with 2% agarose. If necessary, the band was cut out and the DNA extracted. The nucleotide sequence was analyzed with
Fig. 1. Immunoblotting of a macrophage-like cell line, RAW264.7, with anti-rat P450 antibodies. Macrophage microsomes Ž50 m g of protein. were applied to 7.5% polyacrylamide gel. The proteins were reacted with antibodies against P450 indicated at the right side of the membrane and then the resultant immune complex was reacted with 125 I-labeled protein A. Ab, antibody; PB, phenobarbital-treated; CF, chlofibrate-treated; BNF, b-naphthoflavone-treated; 3-MC, 3-methylcholanthrene-treated; DMSO, dimethylsulfoxide-treated; NT, non-treated.
an autosequencer Ž 377A, Perkin-Elmer. after fluorescent labeling.
3. Results 3.1. Effects of P450 inducers on RAW264.7 cells Three kinds of antibodies against rat CYP2A1, 2B1, and 4A2 were used. Cyp2a and 2b isoforms
Table 1 The primers used for PCR Cyp
Sense
Anti-sense
2a4 2a12 2b9r10 4a10 4a12 GAPDH
5X-TCT GTT GCT GAT GAA GTA CC-3X 5X-CCT GCT ACT AAT GAA GCA TC-3X 5X-CAG AGT TCC ATC ACC AGA AC-3X 5X-CTA ATG GTG CGT ACA GAT TG-3X 5X-TTG GCC ACA AAT CCT GAA CAT-3X 5X-CAG CCT CGT CCC GTA GAC AA-3X
5X-TTG TCA TCT AGG AAG TGC TT-3X 5X-TTG TGA TCC AGG AAG TGC TG-3X 5X-CTG TGG ATC ATG TAG AGC TG-3X 5X-AAT GGC CTG GAG GTA GGT CC-3X 5X-AAG ACC CTG GTG CAA ATC GA-3X 5X-GTA GTT GAG GTC AAT GAA GGG-3X
The primers were prepared from published nucleotide sequences of Cyp2a4 w14x, 2a12 w15x, 2b9r10 w16x, 4a10 w17x, 4a12 w18x, and GAPDH w19x. Primers for Cyp2b9r10 recognized both Cyp2b9 and 2b10.
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3.2. Changes in the leÕels of P450 by actiÕation of RAW264.7 by LPS and IFN-g LPS and INF-g strongly induce iNOS in macrophages. RAW264.7 cells were treated with LPS and INF-g . Changes in the protein levels of Cyp2a, 2b and 4a were investigated Ž Fig. 2. . Cyp2a and 2b levels did not change but Cyp4a levels decreased significantly. Fig. 2. Changes in the levels of P450 by treatment with LPS and INF-g . Microsomes were isolated from non-treated RAW264.7 cells or those treated with LPS and INF-g . Levels of P450 were assayed by immunoblotting. The results are expressed as relative values with the control levels set at 100%. The values are expressed as mean"SD of four different preparation of microsomes. )Significantly different from the non-treated; p- 0.05.
were first isolated from mouse liver or kidney w14–16x and Cyp4a is a major form in extrahepatic tissue such as kidney w17,18x. The band at similar mobility with a purified rat P450 was detected with each antibody, suggesting the presence of Cyp2a, 2b, and 4a in RAW264.7 microsomes ŽFig. 1.. PB decreased Cyp2b but other inducers including CF, BNF, 3-MC, and DMSO did not affect the levels of P450 in RAW264.7 cells.
Fig. 3. RT–PCR of P450 mRNA in RAW264.7 cells and mouse liver. Total RNA was isolated from RAW264.7 without treatment ŽNT., those treated with LPS and INF-g ŽLPSqINF., and mouse liver. RNA Ž5 m g for cells and 0.5 m g for liver. was converted to cDNA with reverse transcriptase and amplified by PCR with the primers shown in Table 1.
3.3. Detection of P450 mRNA in RAW264.7 To confirm the presence of P450 in RAW264.7, RT–PCR with total RNA from the cells was done using the primers indicated in Table 1. The primers of GAPDH, Cyp2b9r10, and Cyp4a12 gave clear bands ŽFig. 3.. Other sets of primers did not give
Fig. 4. Nucleotide sequences of fragments amplified from RAW264.7 mRNA by RT–PCR. The lower lines are results in this study. The numbers indicated in the left side are positions from published data w16,18x. The nucleotides underlined were used for PCR primers, which were also used for sequence analysis.
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bands of the sizes predicted from the cDNA sequences although they gave clear bands when total RNA from mouse liver was used Ždata not shown.. The bands of RT–PCR products from RAW 264.7 RNA were cut out from the gels and the DNA extracted. The nucleotide sequence was analyzed. The sequences of the bands from Cyp2b9r10 and Cyp4a12 were identical with mouse Cyp2b9 and Cyp4a12 cDNA sequences although substitution of some nucleotides in Cyp4a12 was seen Ž Fig. 4. . The primers from Cyp2a4 and 2a12 gave no band although an anti-rat CYP2A1 antibody gave a clear band, suggesting the presence of another subfamily of Cyp2a isoform in RAW264.7 cells. The results from RT–PCR qualitatively suggested that treatment with LPS and INF-g decreased Cyp4a12 mRNA levels as well as protein levels.
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Although liver has Kupffer cells which are of macrophage-lineage, the major constituent of the liver cells is the parenchymal cells that are known to be an abundant source of P450 and responsible for the drug metabolism. This might explain the difference in the distribution of P450 as well as the response of P450 isoforms when certain drugs or biological response modifiers like LPS and INF-g are administered to liver or macrophages. Based on the variety of responses of macrophages to the pathological and pharmacological reagents, studies on P450 species in macrophages, especially in the process of macrophage activation, will lead to new findings on the role of P450 in regulation of macrophage functions.
References 4. Discussion In this study, we found that Cyp2b9 and 4a12 were present in a murine macrophage cell line, RAW264.7. The presence of CYP1A1 in macrophages was already reported and macrophages have aryl hydrocarbon hydroxylation activity which is responsible for CYP1A1 activity w5,11x. TCDD which suppresses the immune system is a strong inducer of CYP1A1 w8,20x. The P450 in macrophages may have an important role in immunosuppression of chemicals such as TCDD. The biological role of Cyp2b9 and 4a12 in RAW264.7 is not clear. CYP2B forms metabolize many drugs efficiently in liver w21x. CYP4A forms metabolize fatty acids and prostaglandins in kidney w22x. Macrophages oxidize low density lipoprotein w23x. CYP4A in macrophages may oxidize lipoprotein. PB is a strong inducer of Cyp2b forms in liver w5x but it did not induce Cyp2b9 protein in RAW264.7. CF is an inducer of Cyp4a forms w24x but it did not induce Cyp4a12 protein. LPS or INF-g usually suppresses the expression of P450, although suppression levels of P450 are different w25–27x. Regulation of P450s in macrophage is different from that in liver. The response to LPS and INF-g was also different between liver and macrophage-like cell line, RAW264.7.
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