Protein-1, and Antioxidant Response Elements Stress-Activated Protein

Macrophage Activation by Polycyclic Aromatic Hydrocarbons: Evidence for the Involvement of Stress-Activated Protein Kinases, Activator Protein-1, and Antioxidant Response Elements This information is current as of October 1, 2021. David Ng, Niels Kokot, Timothy Hiura, Mary Faris, Andrew Saxon and Andre Nel J Immunol 1998; 161:942-951; ; http://www.jimmunol.org/content/161/2/942 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Macrophage Activation by Polycyclic Aromatic Hydrocarbons: Evidence for the Involvement of Stress-Activated Protein Kinases, Activator Protein-1, and Antioxidant Response Elements1

David Ng, Niels Kokot, Timothy Hiura, Mary Faris, Andrew Saxon, and Andre Nel2

Polycyclic aromatic hydrocarbons (PAH) contained in fossil fuel combustion particles enhance the allergic response to common environmental Ags. A key question is: what are molecular pathways in the immune system by which PAH and conversion products drive allergic inflammation? Circumstantial evidence suggests that macrophages are involved in PAH-induced responses. We demonstrate that a representative PAH, ␤-napthoflavone (BNF), and a representative quinone metabolite, tert-butylhydroxyquinone (tBHQ), induce Jun kinase and p38 mitogen-activated protein kinase activities in parallel with the generation of activator Downloaded from protein-1 (AP-1) mobility shift complexes in THP-1 and RAW264.7 macrophage cell lines. Activation of mitogen-activated protein kinases was dependent on generation of oxidative stress, and could be inhibited by N-acetylcysteine. Another genetic response pathway linked to PAH is the antioxidant response element (ARE), which regulates expression of detoxifying enzymes. BNF and tBHQ activated a human ARE (hARE) reporter gene in RAW264.7 cells. Interestingly, bacterial lipopolysaccharide also induced hARE/chloramphenicol acetyltransferase activity. While the hARE core, GTGACTCAGC, contains a consensus AP-1 sequence (underlined), AP-1 was not required for hARE activation. This suggests that PAH and their conversion products operate via http://www.jimmunol.org/ ARE-specific transcription factors in the immune system. BNF and tBHQ did, however, induce AP-1 binding to the hARE, while constitutively active Jun kinase interfered in hARE/chloramphenicol acetyltransferase activation. This suggests that AP-1 proteins negatively regulate the hARE. These data establish important activation pathways for PAH in the immune system and provide us with targets to modulate the effect of environmental pollutants on allergic inflammation. The Journal of Immunology, 1998, 161: 942–951.

ir pollution is an important public health issue, and a vious studies have directly addressed the effects of such combus-

number of pollutants, including suspended particles, car- tion particles and their associated polycyclic aromatic hydrocar- by guest on October 1, 2021 A bon monoxide, lead, nitrogen dioxide, sulfur dioxide, bons (PAH) on the allergic response (6–11). In particular, DEP and ozone, are being monitored by the U.S. Environmental Pro- have been shown to enhance IgE production in humans and ani- tection Agency (1). While ozone and sulfur dioxide have been mals in response to challenge with environmental or experimental studied in some detail, particulate matter is receiving increased allergens (6–11). A key question has become: what are the cellular attention due to accumulating evidence that particles of 10 ␮mor targets and molecular pathways in the immune system by which less (PM10) can exacerbate respiratory disease, particularly PAH and their conversion products drive allergic inﬂammation? asthma (2–4). An important component of PM10 is fossil fuel Our nasal challenge studies have shown that DEP alters IgE pro- combustion products, e.g., diesel exhaust particles (DEP)3 (5). Pre- duction both qualitatively and quantitatively. However, the direct effects on B cells occur primarily in cells already committed to IgE production, suggesting involvement of another cell type in the ob- Division of Clinical Immunology and Allergy, Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095 served in vivo outcomes (8, 11). Similarly, while DEP plus aller- Received for publication December 1, 1997. Accepted for publication March gen challenge increased the in vivo production of Th2 cytokines in 16, 1998. our nasal challenge studies (9), our preliminary data have failed to The costs of publication of this article were defrayed in part by the payment of page show a direct DEP or PAH effect on Th2 cytokine production, charges. This article must therefore be hereby marked advertisement in accordance including activation of the IL-4 and IL-5 promoters in T lympho- with 18 U.S.C. Section 1734 solely to indicate this fact. cytes (not shown). This suggests that a major target for PAH in the 1 This work was supported by U.S. Public Health Service Grant AI-34567 (University of California, Los Angeles, Asthma, Allergy, and Immunologic Disease Center mucosal immune system is a nonlymphoid cell type. funded by National Institute of Allergy and Infectious Diseases and National Institute Macrophages are a PAH target in the respiratory tract for in- on Environmental Health Sciences). haled PAH. It has been demonstrated that a variety of xenobiotics, 2 Address correspondence and reprint requests to Dr. Andre Nel, University of Cal- including PAH, polychlorinated biphenyls, and halogenated aro- ifornia, Los Angeles, School of Medicine, Department of Medicine, CIA, 52-175 CHS, 10833 Le Conte Ave., Los Angeles, CA 90095. E-mail address: matic hydrocarbons, exerts effects on macrophages, including pul- [email protected] monary alveolar macrophages (10, 12–19). These effects include 3 Abbreviations used in this paper: DEP, diesel exhaust particles; Ahr, aromatic hy- induction of oxidative burst activity (10, 12), increased expression drocarbon receptor; AP-1, activator protein-1; ARE, antioxidant response element; of MHC-II gene products (13), induction of cytochrome P4501A1 ATF2, activating transcription factor 2; BNF, ␤-napthoﬂavone; CAT, chloramphenicol acetyltransferase; comp, complex; CYP1A1, cytochrome P4501A1; GST, gluta- (CYP1A1) activity (12, 16, 18), and conversion of benzo(a)pyrene thione S-transferase; hARE, human antioxidant response element; JNK, c-Jun N- terminal kinase; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NQO1, nicotinamide-adenine dinucleotide phosphate (NADPH):quinone oxidoreduc- oxygen species; tBHQ, tert-butylhydroxyquinone; TCDD, 2,3,7,8-tetrachlorodibenzo- tase; PAH, polycyclic aromatic hydrocarbon; RE, response element; ROS, reactive p-dioxin; XRE, xenobiotic response element.

of rat and murine phase II enzyme promoters (Fig. 1) (36, 53, 54). One hypothesis is that AP-1 proteins play a role in the activation of the ARE (36, 42, 43, 53, 54). This notion is contentious, however, as some studies have shown that the ARE can be activated independent of AP-1 protein(s) (39–41, 45). Whatever the relationship between ARE and AP-1 proteins, the molecular events leading to the activation of these RE are sensitive biochemical tools to study immune cellular activation by PAH. One of the regulatory pathways for AP-1 proteins are the MAPK cascades that affect transcriptional activation as well as expression of AP-1 FIGURE 1. Base pair sequence of the ARE in different gene promoters, proteins (55). We are particularly interested in the c-Jun N-termi- including reagents used in this work for studying the ARE. The extended nal kinases (JNK), also known as stress-activated protein kinases,

ARE in the human NQO1, rat NQO1, and the mouse GST-Ya promoters are and the p38 MAPK cascade because both pathways play a role in shown at the top. The ARE core with an overlapping AP-1 or AP-1-like cellular responses to environmental stress, including exposure to sequence, as well as the upstream AP-1-like elements are shown (under- toxic drugs and chemicals, LPS, inﬂammatory cytokines, oxidative lined). The hARE sequence, provided by Dr. Anil Jaiswal in the hARE- stress, or hyperosmolarity (55–58). tk-CAT vector, is shown below together with mutant versions of this re- There have been no systematic studies in macrophages of acti- porter gene, designated core mutant (cm) or AP-1 mutant (mAP1). Double- vation of the ARE or the AP-1 pathway by PAH. We sought to stranded oligonucleotides used in gel-shift studies are shown below.

determine whether a representative PAH, ␤-naphtoﬂavone (BNF), Downloaded from and a representative quinone derivative, tert-butylhydroxyquinone (tBHQ), can induce MAPK activation in the macrophage cell lines, to redox-active quinones and other DNA adducts (16, 17, 19). THP-1 and RAW264.7 (29–31, 43). In addition, we investigated Macrophages, including pulmonary alveolar macrophages, play an the effects of these chemicals on ARE activation. Our data show important role in allergic inﬂammation through their effects on Ag that BNF and tBHQ induce JNK and p38 MAPK activation in

presentation, expression of costimulatory molecules that activate T parallel with the generation of AP-1 electrophoretic mobility shift http://www.jimmunol.org/ cells, and production of cytokines and chemokines that enhance complexes. While these chemicals induced AP-1 interactions with IgE production (20–27). Recently, Prell and Kerkvliet have shown the hARE, hARE reporter gene activity could be activated inde- that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) inhib- pendent of AP-1 protein binding. These data indicate that ARE and its CD86 expression on Mac1ϩ cells, and suggested that a major AP-1 response elements may play important roles in macrophage target for dioxin in the immune system is APCs, including activation by PAH. macrophages (28). While metabolic pathways for PAH are well described in hepatocytes, not much is known about PAH pathways for cellular ac- Materials and Methods tivation in immune cells, including macrophages. The best-char- Reagents by guest on October 1, 2021 acterized PAH metabolic pathway involves PAH interaction with RPMI 1640 was purchased from Irvine Scientific (Santa Ana, CA). the aromatic hydrocarbon receptor (AhR), which translocates to DMEM, penicillin-streptomycin, and L-glutamine were purchased from the nucleus and transcriptionally activates genes that express the Life Technologies (Baltimore, MD). The polyclonal rabbit IgG anti-JNK2 Ab and polyclonal rabbit IgG anti-phospho-p38 Ab was purchased from xenobiotic response element (XRE) (29–32). Genes that utilize an New England Biolabs (Beverly, MA). A p38 MAPK Ab that recognizes XRE include CYP1A1, which, in turn, is responsible for the con- nonphosphorylated epitopes and that can be used for immunoblotting and version of PAH to oxidatively labile metabolites that damage cel- immunoprecipitation was bought from Santa Cruz Biotechnology (Santa lular DNA, proteins, and lipids (29–34). Some PAH metabolites, Cruz, CA). The anti-phosphotyrosine Ab, 4G10, was purchased from Up- e.g., quinone derivatives, participate in additional pathways such date Biotechnology (Lake Placid, NY), while anti-pan-Fos and anti-pan- Jun antisera were from Santa Cruz Biotechnology. tBHQ was purchased as 1-electron reductions that yield reactive oxygen species (ROS) from Aldrich (Milwaukee, WA). BNF, LPS (O55:B5 serotype), phenan- (19, 35–37). In addition to the role of the XRE, more recent studies threne, and N-acetylcysteine (NAC) were purchased from Sigma (St. in hepatocytes have focused on the role of PAH and their quinone Louis, MO). Silica gel TLC plates were purchased from VWR Scientific derivatives on cellular activation via the generation of oxidative (San Francisco, CA). Wild-type and hARE core mutant reporter plasmids (hARE-tk-CAT and hAREcm-CAT, respectively) were obtained from Dr. stress (35–38). An important genetic response element (RE) that is Anil Jaiswal (Fox Chase Cancer Center, Philadelphia, PA) (Fig. 1) (38, 42). affected by oxidative stress is the antioxidant or electrophile re- The cDNAs for DA-MEKK1 (MEKK⌬) and DN-MEKK1 (MEKK ⌬K432 sponse element (ARE or EpRE) (35–45). The ARE, with the core M) were a gift from Dr. G. Johnson (National Jewish Center for Immu- sequence GTGACNNNCA (39–41), has been linked to the ex- nology and Research, Denver, CO) (59). [14C]Chloramphenicol was from ␥ 32 ␣ 32 pression of genes that encode phase II drug metabolizing enzymes, Amersham (Arlington Heights, IL), while [ - P]ATP and [ - P]dCTP were from NEN (Boston, MA). e.g., glutathione S-transferase (GST) and nicotinamide-adenine dinucleotide phosphate (NADPH):quinone oxidoreductase Cell culture and stimulation (NQO1) (Fig. 1). A major effect of phase II enzymes is to protect cells against toxic effects of xenobiotics and their oxidatively labile THP-1 cells were obtained from American Type Culture Collection (Ma- products (45–49). Recently, it has been shown that ARE elements nassas, VA). The cells were cultured at 37°C in a 5% CO2 atmosphere in are found in the promoters of other critical cellular genes, includ- RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% glutamine. RAW264.7 cells were generously provided by Dr. Steven ing the IL-6 gene (50). Smale (University of California, Los Angeles). These cells were cultured in Chemicals that induce phase II enzymes also induce the expres- DMEM containing 10% FBS, 1% penicillin-streptomycin, and 1% sion of AP-1 proteins (36, 43, 44, 51–54). Moreover, within the glutamine. human ARE (hARE) core in the NQO promoter, GTGACT BNF, tBHQ, and phenanthrene were dissolved in DMSO, while TCDD 1 was dissolved in ethanol. Before use, the chemicals were made up in cul- CAGC, is a consensus AP-1 response element (underlined) in ad- ture media at the indicated concentrations, keeping the final carrier con- dition to an AP-1 site immediately upstream of the hARE core centration at 0.1%. LPS was dissolved in PBS before adding it to the (Fig. 1) (36, 40, 43). AP-1-like sequences also appear in the ARE culture medium. 944 MACROPHAGE ACTIVATION BY POLYCYCLIC AROMATIC HYDROCARBONS

Transfections 1). For cold competition, 100-fold excess ununlabeled probe was incubated for 15 min at room temperature with the above mixture before addition of RAW264.7 cells were washed once in PBS and resuspended in DMEM the labeled probe. For Ab supershift, 0.5 ␮g anti-pan-Fos or anti-pan-Jun ϫ 6 ␮ with 20% FCS at a final concentration of 7 10 cells/200 l. These cells antisera were added to the binding reaction for 15 min before addition ␮ ␮ were incubated in a 0.4-cm cuvette together with 20 g plasmid and 30 l of the labeled probe. Shift complexes were electrophoresed on a 6% PBS at room temperature for 10 min. Cells were transfected at 260 V and polyacrylamide-glycerol gel, and the dried gel was exposed to ␮ 975 F in a Bio-Rad (Richmond, CA) Gene Pulser. The transfected cells autoradiographic film. were rested at room temperature for 10 min, washed once in PBS, and placed in six-well plates in complete medium (see above). Results Construction of a hARE-CAT construct with mutation of the tBHQ and BNF activate the JNK cascade in an antioxidant- AP-1 site sensitive manner in human and murine macrophage cell lines The hARE promoter was excised from the hARE-tk-CAT vector (pBLCAT tBHQ is mechanistically representative of the quinone products backbone) with XbaI, followed by BamHI digestion to provide an over- that form when PAH are metabolized by cytochrome P4501A1 Ј hanging site on the 3 end. A hARE promoter with a mutagenized AP-1 site (CYP1A1) (46). Although tBHQ is classified as a phenolic anti- (Fig. 1) was ligated into the double digested plasmid. The correct orien- tation and presence of the mutagenized sequence were confirmed by DNA oxidant, its breakdown actually generates ROS, which leads to sequence analysis. The resulting plasmid was designated hARE(mAP1). cellular activation and generation of nuclear responses in hepatocytes (35, 51). One type of nuclear response is AP-1 protein ex- CAT assay pression, which affects AP-1 response elements (36, 43, 44, 46, RAW264.7 cells transfected with hARE-tk-CAT or mutant versions 51–54). We were interested to determine whether tBHQ could ac- thereof (Fig. 1) were allowed to rest for 24 h before stimulation with 50 tivate JNK, since this cascade regulates the expression and tran- Downloaded from ␮ ␮ ␮ M BNF, 50 M tBHQ, and 10 g/ml LPS for an additional 24 h. The scriptional activation of AP-1 proteins (55). Treatment of the hu- cells were harvested and washed once in PBS. Cell pellets were resuspended in 100 ␮l1ϫ reporter lysis buffer (Promega, Madison, WI) and man macrophage cell line, THP-1, and the murine macrophage cell incubated at 4°C for 30 min while shaking. The lysates were freeze thawed line, RAW264.7, with tBHQ induced JNK activation, as deter- once on dry ice and spun at 14,000 ϫ g for 10 min. A total of 50 ␮gof mined by in vitro kinase assay (Fig. 2, A and B). JNK activation protein per stimulation was heat inactivated at 65°C for 4 min. Each sample commenced within 30 min of adding the chemical, peaked within was then coincubated with 25 ␮g N-butyryl-CoA (Promega) and 0.25 ␮Ci

[14C]chloramphenicol in a 250 mM Tris/HCl buffer for2hat37°C. The 60 min, and returned to near baseline in about 4 h (Fig. 2, A and http://www.jimmunol.org/ reaction was stopped with 500 ␮l ethyl acetate and centrifuged at 14,000 ϫ B). The magnitude of the response was more robust in THP-1 than g for 30 s. The top organic layer was removed, dried, and resuspended in in RAW264.7 cells (Fig. 2, A and B). Compared with a potent JNK 15 ␮l ethyl acetate and spotted on silica TLC plates. The TLC plates were stimulus, e.g., LPS (63), tBHQ induced a response of almost sim- placed in a TLC tank that was equilibrated previously with 145 ml chlo- ilar magnitude in THP-1 cells, but elicited only 50% of the LPS roform and 5 ml methanol. The TLC plates were dried and exposed to radiographic film or a PhosphorImager screen (Molecular Dynamics, response in RAW264.7 cells (Fig. 2, A and B). Sunnyvale, CA). BNF is representative of the bifunctional PAHs, compounds that induce the expression of CYP1A1 via the XRE, and induce ex- MAPK assays pression of phase II enzymes (e.g., GST) via the ARE (46–48). 6 Following stimulation of 3 ϫ 10 cells for the indicated time period, JNK The induction of phase II enzyme activity requires that BNF be by guest on October 1, 2021 ␮ kinase activity was assessed as previously described (60). Briefly, 100 g converted to redox active quinones by CYP1A1 (46–48). Treat- cellular protein was coincubated with GST-c-Jun(1–79), immobilized on glutathione beads. After washing of the beads, the phosphorylation reaction ment of THP-1 cells with BNF induced a small but definite in- was initiated by the addition of [␥-32P]ATP at 20°C for 15 min (60). The crease in JNK activity, which maximally amounted to 12% of the p38 MAPK assay followed the same procedure, except that the kinase was LPS-induced response (Fig. 2C). The response to BNF in purified by an anti-p38 MAPK Ab, with GST-ATF2 acting as substrate RAW264.7 cells was much stronger and achieved 31% of the LPS- (61). Phosphorylated substrates were resolved on 10% SDS polyacryl- induced response (see left-hand panel, Fig. 3A). Compared with amide gels that were dried and autoradiographed. the brief activation of JNK by tBHQ (Fig. 2, A and B), BNF- JNK2 immunoprecipitation and Western blotting induced JNK activation was discernible for up to 22 h after stim- Aliquots of 7 ϫ 106 THP-1 cells were stimulated with 50 ␮M BNF and 50 ulation (Fig. 2C). Taken together, these data indicate that tBHQ ␮M tBHQ for the indicated time periods. Cells were lysed in 200 ␮l lysis and BNF can be added to the growing list of compounds and envi- buffer containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mM ronmental stress stimuli that activate the JNK cascade (56–58). NaCl, 1 mM EDTA, 10 mM NaF, 2 mM PMSF, 10 ␮g/ml leupeptin, 2 ␮ We investigated the role of ROS and protein phosphorylation in U/ml aprotinin, and 1 mM Na3VO4. A total of 200 g of lysate protein was incubated with 0.5 ␮g anti-JNK2 Ab for1hat4°C. After absorption to JNK activation. Guyton et al. have shown previously that MAPK protein A-Sepharose beads for 1 h, the beads were washed and boiled in activity, including JNK activity, can be induced by H2O2 (64). We SDS sample buffer. Proteins were resolved on a 10% polyacrylamide gel, determined what effect the antioxidant, NAC, had on BNF- and transferred to an Immobilon-P membrane, and overlaid with anti- tBHQ-induced JNK activation (Fig. 3, A and B). Exposure to 20 phosphotyrosine (4G10) Ab, as previously described (62). mM NAC abrogated subsequent BNF- and tBHQ-induced JNK Electrophoretic mobility shift assays (EMSA) activation (Fig. 3, A and B), while decreasing the LPS response by A total of 1 ϫ 107 RAW264.7 cells was stimulated with 50 ␮M BNF or 50 50% (Fig. 3A). During the performance of these experiments, we ␮M tBHQ for the indicated time period. Nuclear extraction was performed, noted that NAC lowered resting JNK activity, suggesting that con- as previously described, with a few modifications (40). Briefly, cells were tinuous production of ROS may drive basal JNK activity in mac- pelleted and resuspended in lysis buffer containing 0.1% Nonidet P-40, 50 rophages. We also determined the effects of tBHQ and BNF stim- mM Tris-HCl, pH 8, 10 mM NaCl, and 5 mM MgCl for 1 min. The 2 ulation upon tyrosine phosphorylation of JNK (Fig. 3C). JNK process was repeated with the same lysis buffer containing 0.5% Nonidet P-40 for 1 min. Nuclear proteins were eluted in an extraction buffer con- activity is induced by phosphorylation of threonine and tyrosine taining 500 mM NaCl, 20 mM HEPES, pH 7.9, 1 mM EDTA, and 20% residues in the TPY allosteric effector site of that kinase (55–57). glycerol. For DNA-protein binding, 10 ␮g of nuclear protein was incubated While resting cells lacked tyrosine phosphorylation of the 55-kDa 5 32 together with 10 cpm P-labeled probe in the presence of a binding buffer JNK isoform (Fig. 3C, lane 2), BNF and tBHQ induced phosphor- and 3 ␮g poly(dI/dC) for 20 min. Probes used in this study include a: 1) AP-1 consensus oligonucleotide, 5Ј-GATCCGTGACTCAGCGCG-3Ј;2) ylation of p55 JNK (lanes 3 and 4). Equal amounts of the 55-kDa human ARE oligonucleotide (Fig. 1); and 3) a mutant hARE oligonucle- JNK isoform were being immunoprecipitated, as confirmed by anti- otide in which 3 bp in the AP-1 consensus site have been changed (see Fig. JNK immunoblotting (not shown). The Journal of Immunology 945 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. The activation of JNK by tBHQ and BNF is dependent on generation of oxidative stress and induction of JNK phosphorylation. Cells were incubated in 20 mM NAC for 16 h before treatment with 50 ␮M BNF for the indicated time periods, or 50 ␮M tBHQ for 1 h. The positive control was RAW264.7 cells treated with 10 ␮g/ml LPS for 1 h. JNK activity was determined as described in Figure 2. Antiphosphotyrosine immunoblotting of JNK2 immunoprecipitates was performed as described in Materials and Methods. A, Depicts the effect of NAC on induction of JNK activity by BNF in RAW264.7 cells. The autoradiogram of the phosphorylated substrate is shown at the top, and 32P incorporation is depicted below. B, Shows the effects of NAC on JNK activation by tBHQ in THP-1 cells. C, Shows antiphosphotyrosine overlay of the JNK2 immunoprecipitate. This FIGURE 2. tBHQ and BNF activate the JNK cascade in macrophage JNK isoform migrates at 55 kDa. NIS ϭ nonimmune serum (used as a cell lines. Aliquots of 3 ϫ 106 human THP-1 and murine RAW264.7 cells control immunoprecipitate using lysates from BNF-treated cells). were treated with 50 ␮M tBHQ or 50 ␮M BNF for the indicated time periods. As a positive control, cells were treated with 10 ␮g/ml LPS for 1 h. Cells were lysed, and 100 ␮g cellular protein was incubated with GST-c- tBHQ and BNF induce p38 MAPK activity in an antioxidant- Jun(1–79), immobilized on glutathione beads. After kinase capture, the sensitive fashion in THP-1 and RAW264.7 cells beads were washed and the phosphorylation reaction was initiated by adding [␥-32P]ATP (60). The autoradiogram shows the phosphorylated sub- In addition to JNK, a second MAPK, p38 MAPK, plays a role in strate, while the bar graph shows 32P incorporation in cpm. A, Shows JNK macrophage activation by LPS and other forms of acute cellular activation by tBHQ in THP-1 cells. B, Shows JNK activation by tBHQ in stress (58). To determine whether tBHQ and BNF induce p38 RAW264.7 cells. C shows JNK activation by BNF in THP-1 cells. MAPK activation in THP-1 and RAW264.7 cells, we used an in 946 MACROPHAGE ACTIVATION BY POLYCYCLIC AROMATIC HYDROCARBONS Downloaded from

FIGURE 5. tBHQ and BNF induce p38 MAPK activity through the generation of oxidative stress and induction of p38 phosphorylation. A,107 THP-1 cells were treated with 10 ␮g/ml LPS, 50 ␮M BNF, 50 ␮M tBHQ, http://www.jimmunol.org/ and 10 nM PMA for 1 h. Cellular lysates were resolved on 10% SDS- PAGE, and proteins were transferred to an Immobilon-P membrane. The blot was overlaid with 0.1 ␮g/ml of an anti-phosphopeptide Ab that recognizes activated p38 MAPK. The increase in p38 phosphorylation reﬂects its activation. These differences are not the result of different amounts of p38 MAPK protein, as immunoblotting with an Ab to the whole protein showed equal staining intensity in control and treated cells. In a duplicate blot, overlay of cellular lysates with an anti-p38 MAPK antiserum, which recognizes the whole protein, showed equal amounts of kinase protein in

control and treated cells. In B, RAW264.7 cells were exposed to 20 mM by guest on October 1, 2021 NAC for 16 h before treatment with 50 ␮M BNF, 50 ␮M tHBQ, or 10 ␮g/ml LPS for 1 h. p38 MAPK activity was determined as described in FIGURE 4. tBHQ and BNF induce p38 MAPK activity in THP-1 and Figure 4. RAW264.7 cells. Aliquots of 3 ϫ 106 cells were treated with 50 ␮M tBHQ or 50 ␮M BNF for the indicated time period. For a positive control, we ␮ ␮ used stimulation with 10 g/ml LPS for 1 h. A total of 100 g/ml of RAW264.7 cells to 20 mM NAC abrogated BNF- and tBHQ-in- cellular lysate was immunoprecipitated with anti-p38 Ab (Santa Cruz Bio- duced p38 MAPK activity, while decreasing the LPS response by technology). After washing, the lysates were incubated together with 5 ␮g ␥ 32 43% (Fig. 5B). Similar to what was seen for JNK, NAC lowered GST-ATF2 and [ - P]ATP for 25 min at room temperature. The substrate was resolved by 10% SDS-PAGE. The autoradiogram of the phosphory- basal p38 MAPK activity, suggesting that ROS may sustain basal lated substrate is shown at the top, and 32P incorporation is shown below. p38 MAPK activity in macrophages (Fig. 5B). A, Shows p38 MAPK activation during tHBQ treatment. B, Shows p38 MAPK activation during BNF treatment. BNF and tBHQ induce AP-1 electrophoretic mobility shift complexes in RAW264.7 cells Based on the effects of MAPK cascades on the expression and vitro immune complex kinase assay to measure the activity of p38 transcriptional activation of AP-1 proteins, we sought to determine MAPK (Fig. 4, A and B). The magnitude of the response to tBHQ whether tBHQ and BNF could induce AP-1 mobility shift com- and BNF amounted to 9 and 20%, respectively, in THP-1, and 45 plexes in macrophages. RAW264.7 cells were treated with 50 ␮M and 31%, respectively, in RAW264.7 cells (Fig. 4, A and B). tBHQ or BNF, as indicated in Figure 6, and nuclear extracts were While details about the pathway by which these chemicals en- incubated together with a labeled AP-1 consensus oligonucleotide. gage the p38 MAPK cascade are unknown, p38 MAPK itself is BNF induced a discernible increase in AP-1 shift complexes activated by a dual speciﬁcity MAPK kinase, MKK6 (65). MKK6 within 2 h; this effect was sustained during the entire observation activates p38 MAPK by phosphorylating threonine and tyrosine period of 14 h (Fig. 6, lanes 2–5). The addition of a 100-fold residues in the TGY allosteric effector site of the latter kinase (58, excess unlabeled AP-1 probe abrogated protein binding to the la- 65). The phosphorylation of p38 MAPK can be studied by anti- beled probe (Fig. 6, lane 6). Prior treatment of nuclear extracts phosphopeptide Abs that recognize the phosphorylated but not the with anti-pan-Fos or anti-pan-Jun antisera decreased the abun- native species. Overlay of whole cell lysates with anti- dance of the shift complexes, demonstrating that these complexes phosphopeptide antiserum showed increased p38 phosphorylation contain proteins from both major AP-1 protein families (Fig. 6, in THP-1 cells resulting from treatment with LPS, BNF, tBHQ, lanes 7 and 8). Similar results were obtained with nuclear extracts and PMA (Fig. 5A). We examined the role of oxidative stress in from tBHQ-treated cells, except that the relative abundance of the p38 MAPK activation, and found that prior exposure of shift complexes declined within 6 h (Fig. 6, lanes 9–17). These The Journal of Immunology 947 Downloaded from

FIGURE 6. tBHQ and BNF induce AP-1 mobility shift complexes. A total of 107 RAW264.7 cells was stimulated with 50 ␮M BNF or 50 ␮M tHBQ for the indicated time period. The positive control was stimulation with 10 nM PMA for 1 h. Nuclear extraction and incubation of nuclear proteins with the 32P-labeled consensus AP-1 probe (Fig. 1) were described http://www.jimmunol.org/ in Materials and Methods. Cold competition was performed with a 100- ␮ fold excess unlabeled probe. For Ab supershift, 0.5 g anti-pan-Fos or FIGURE 7. tBHQ and BNF induce AP-1 binding to a labeled ARE anti-pan-Jun antiserum was added for 15 min before addition of the labeled probe. A total of 107 RAW264.7 cells was stimulated with 50 ␮M BNF or probe. 50 ␮M tBHQ for 2 h. Nuclear extraction was performed as described in Materials and Methods, and electrophoretic mobility shift assay (EMSA) was conducted with 32P-labeled wild-type or mutant ARE probe (Fig. 1). results are in agreement with the differences observed in the ki- The hARE(mAP1) probe contains a change of 3 bp in the AP-1 sequence, netics of JNK activation (Fig. 2). Taken together, our data indicate which does not affect the hARE core (Fig. 1). Cold competition was per-

that BNF and tBHQ induce heterodimeric Fos/Jun complexes in formed with a 100-fold excess probe. Ab supershift was performed with by guest on October 1, 2021 macrophage cell lines. This agrees with the ability of these chemicals anti-pan-Jun antiserum, as described in Figure 6. to induce AP-1 shift complexes in hepatocytes (36, 44, 52–54).

tBHQ and BNF induce AP-1 binding to a labeled ARE hARE-speciﬁc transcription factor(s) (39–41). We therefore consensus oligonucleotide: effects of site-directed mutation in changed the TCA sequence in the hARE (GTGACTCAGC) to the overlapping AP-1 site GTGACGCTGCA), disrupting the AP-1 consensus (GTGACT

The hARE sequence in the NQO1 promoter (GTGACTCAGC) CAGC), but not the hARE core sequence. Coincubation of this contains a consensus AP-1 sequence (underlined) (38, 40, 43). labeled probe, designated hARE(mAP1) (Fig. 1), with nuclear ex- Utilizing the labeled hARE oligonucleotide shown in Figure 1, tracts from RAW264.7 cells, resulted in a single shift complex that together with nuclear extracts from tBHQ- or BNF-treated hepa- comigrated with comp 3 of the consensus hARE probe (lanes tocytes, results in at least two shift complexes (39–41). To deter- 7–11). While cold competition with hARE(mAP1) blocked comp mine whether RAW264.7 cells contain hARE-binding factors, we 3 formation, a molar excess of a consensus AP-1 probe had no used the same labeled hARE probe together with nuclear extracts effect (lanes 10 and 11). Taken together, these data indicate that from BNF- or tBHQ-treated RAW264.7 cells. BNF treatment re- tBHQ and BNF induce AP-1 binding to the hARE. These results sulted in the occurrence of a new shift complex, designated comp are in agreement with data in hepatocytes, which indicate that 1, in addition to two shift complexes (comp 2 and 3) seen in un- while the slower migrating complex contains PAH- and tBHQ- stimulated cells (Fig. 7, lanes 1 and 2). BNF also induced an inducible AP-1 proteins, the faster migrating complex represents a increase in the abundance of comp 2 (Fig. 7, lanes 1 and 2). While noninducible, AP-1-independent complex (39–41). Nguyen and all three complexes were effectively inhibited by a molar excess of Pickett suggested that the latter complex represents a hARE-spe- unlabeled hARE (lane 4), an unlabeled AP-1 consensus oligonu- ciﬁc transcription factor (39–41). cleotide competed for comp 2 only, suggesting that this complex contains AP-1 proteins (lane 5). Supershift analysis showed that BNF, tBHQ, and LPS induce hARE reporter gene activity comp 2 could be inhibited by an anti-pan-Jun Ab (lane 6). tBHQ- ARE activation in hepatocytes is dependent on the generation of treated cells contained two complexes (comp 2 and 3), one of ROS (35, 36). Since macrophages generate ROS in response to which (comp 2) was induced by tBHQ (lane 3). xenobiotics (10, 12) as well as ligation of various membrane re- In light of AP-1 protein interactions with the hARE, we tested ceptors, we sought to determine whether these stimuli induce whether an alteration of the AP-1 sequence affected the observed hARE activation in RAW264.7 cells. RAW264.7 cells were cho- mobility shift complexes. This was possible because the so-called sen for their ease of gene transfection compared with THP-1 cells. hARE core sequence, GTGACTCAGC, contains three degenerate We used a copy of the hARE linked to a CAT reporter (hARE- bases (TCA) that are nonessential for binding of the putative tk-CAT; Fig. 1) to study ARE activation during treatment with 948 MACROPHAGE ACTIVATION BY POLYCYCLIC AROMATIC HYDROCARBONS

FIGURE 8. BNF, tBHQ, and LPS induce hARE-CAT reporter activity: effects of mutageniz- ing the hARE core or AP-1 sequences. A total of 7 ϫ 106 RAW264.7 cells in 200 ␮l complete medium was incubated with 20 ␮g of the hARE-tk- CAT or hARE-cm-CAT plasmid for 10 min. The cells were transfected at 260 V and 975 ␮Fina Bio-Rad Gene Pulser. The cells were allowed to rest for 24 h before stimulation with 50 ␮M BNF, 50 ␮M tHBQ, or 10 ␮g/ml LPS for an additional 24 h. Cells were lysed, and CAT assays were performed as described in Materials and Methods. CMV-CAT transfection (lane 10) was used as a positive control. CAT-enz (lane 11) is a puriﬁed Downloaded from CAT enzyme used to standardize the assay. In A, the wild-type hARE-CAT and the hARE core mutant (cm) are compared. hAREcm contains changes in the hARE core as well as the AP-1 sequence shown in Figure 1. In B, the wild-type hARE-CAT and AP-1 mutant are compared. hARE(mAP1)-CAT http://www.jimmunol.org/ contains a 3-bp change that disrupts the consensus AP-1, but not the hARE core sequence (Fig. 1). by guest on October 1, 2021

BNF, tBHQ, and LPS (38). Constitutively active CMV-CAT Constitutive activation of the JNK cascade by DA-MEKK1 served as a positive control, while a hARE mutant, hAREcm- interferes in expression of hARE-CAT activity CAT, which contains an altered ARE core sequence, served as The data in Figures 6 through 8 suggest that BNF and tBHQ negative control (Fig. 1). Treatment with BNF and tBHQ in- may regulate both hARE and AP-1 response elements. While duced a 3- and 6.9-fold increase, respectively, in hARE-CAT hARE-specific transcription factors remain to be identified, pre- activity in the intact reporter gene (Fig. 8A, lanes 2–4). The vious studies have shown that the hARE response pathway is magnitude of these responses is comparable with hARE-CAT regulated by ROS (35, 36). Since ROS play a role in PAH- responses in hepatocytes (38). Interestingly, LPS induced a 6.7- induced JNK activation in macrophages (Figs. 2 and 3), and the fold increase in CAT activity, implying that an LPS-binding hARE contains an overlapping AP-1 sequence (Fig. 1), we receptor such as the CD14 receptor (66) may activate a genetic sought to determine whether the JNK cascade regulates hARE- response element that has classically been linked to chemical CAT activity. We used a dominant active (DA) Jun kinase ki- effects. No CAT activity was expressed in hAREcm-CAT-trans- nase kinase, MEKK⌬, to activate the JNK cascade RAW264.7 fected cells (Fig. 8A, lanes 6–9). cells (59). First, we determined whether DA-MEKK1 was ex- A critical question raised by the data in Figure 7 is whether the pressed in transfected cells by using anti-MEKK1 immunoblot- AP-1 sequence in the unmodified hARE affects the transcriptional ting (Fig. 9A). Compared with mock-transfected cells or cells activity of this reporter. For that reason, we subcloned the transfected with an empty vector, MEKK⌬-transfected cells ex- hARE(mAP1) sequence, which disrupts the consensus AP-1, but not pressed the dominant active MAPKKK (Fig. 9A). Moreover, we the hARE, into the tk-CAT reporter (Fig. 1). This mutant reporter was confirmed that JNK activity was increased in DA-MEKK1 com- transfected into RAW264.7 cells in parallel with the unmodified pared with untreated or empty plasmid-transfected cells (Fig. hARE-CAT construct, and cells were stimulated with the same stim- 9B). Subsequently, we cotransfected hARE-CAT with 1) an uli depicted in Figure 8A (Fig. 8B). Compared with the hARE core empty vector, 2) DA-MEKK1, or 3) dominant negative or ki- mutant, the hARE(mAP1) mutant showed definitive expression of nase inactive (KI) MEKK1 [pSR␣-MEKK⌬(K432 M)] (59). CAT activity (Fig. 8B, lanes 6–9). These data show that although the Compared with BNF-, tBHQ-, or LPS-inducible reporter gene hARE sequence overlaps with the AP-1 site, an intact AP-1 site is not activity in empty vector (Fig. 9C, lanes 1–4) or KI-MEKK1- necessary for generation of hARE-CAT activity. These data support expressing cells (lanes 9–12), the dominant active MEKK1- the existence of AP-1-independent, ARE-specific transcription factors transfected cells showed neither basal nor inducible CAT ac- in macrophages (39–41, 45). tivity (lanes 5–8). These data indicate that although the hARE The Journal of Immunology 949 Downloaded from

FIGURE 9. Constitutive activation of the JNK cascade by DA-MEKK1 interferes in expression of hARE-CAT activity. RAW264.7 cells were transfected with 20 ␮g of the CMV-MEKK⌬ (DA-MEKK1), 20 ␮g of an empty plasmid (pCDNA 1.1), or 20 ␮gofpSR␣-MEKK⌬ (K432 M) (DN-MEKK1) http://www.jimmunol.org/ in the absence or presence of 20 ␮g hARE-tk-CAT vector, as described in Figure 8. A, Depicts immunoblotting of cellular extracts with an anti-MEKK1 antiserum. This shows expression of the dominant active MEKK1 kinase in MEKK⌬-transfected cells. B, Autoradiogram showing GST-c-Jun phosphorylation in an vitro JNK assay. This assay was performed as described in Figure 2. LPS stimulation was used as a positive control. C, Shows a reporter gene assay in hARE-CAT-transfected cells in the presence of empty vector, DA-MEKK1, or DN-MEKK1. interacts with AP-1 proteins, the JNK pathway that regulates entry into the cytosol, polycyclic aromatic and halogenated hydro- AP-1 proteins exerts an inhibitory effect on hARE transcrip- carbons are bound by the 95-kDa ligand-binding subunit of the by guest on October 1, 2021 tional activity. To show that the inhibitory effect of DA- AhR, which translocates to the nucleus, where it associates with MEKK1 on the hARE is mediated via the internal AP-1 se- the aryl hydrocarbon receptor nuclear translocator protein (29– quence, we cotransfected MEKK1 with the hARE(mAP1) 32). The AhR/aryl hydrocarbon receptor nuclear translocator com- reporter and did not find interference in the tBHQ- or BNF- plex acts as a transcriptional activator of genes that express the inducible hARE responses (not shown). Taken together, this XRE, leading to the expression of CYP1A1 and other phase I suggests that while BNF and tBHQ induce two distinct bio- drug-metabolizing enzymes. The XRE pathway is functional in chemical events in macrophages, i.e., JNK and hARE activa- macrophages and other immune cells, and has been best studied in tion, the former exerts a negative regulatory effect on the hARE. the context of immunosuppression by polycyclic aromatic hydrocarbons and dioxin (28, 67–69). Recent findings of impaired lym- Discussion phocyte development in AhR knockout mice also suggest that In this study, we show that a bifunctional polycyclic aromatic hy- physiologic AhR ligands exist that play a role in the function of the drocarbon, BNF (48), and a redox active quinone, tBHQ (37), immune system (70). activate the c-Jun and p38 MAPK cascades in parallel with gen- Our studies focused on the activation of two additional path- eration of AP-1 electrophoretic mobility shift complexes (Figs. ways that have been linked to PAH stimulation, namely the AP-1 2–6). The induction of these responses in human and murine mac- (Fig. 6) and the ARE response pathways (Figs. 7–9). PAH-induced rophage cell lines was dependent on the generation of oxidative ARE activation is dependent on prior XRE activation and expres- stress (Figs. 3 and 5). An additional response pathway in macro- sion of CYP1A1 activity. CYP1A1 is able to convert PAH to redox phages is activation of the antioxidant response element indepen- active compounds such as quinones (46, 53). One hypothesis is dent of the effect of chemicals on AP-1 proteins (Figs. 7–9). At the that ARE activation depends on the generation of oxidative stress level of the human ARE, JNK activation suppressed hARE re- by quinone derivatives (35, 36). PAH that activate XRE and ARE porter gene activity (Fig. 9). These data establish two important response elements induce both phase I (cytochromes P450) and and novel biochemical activation pathways for PAH and their de- phase II (detoxifying) enzymes, and are known as bifunctional rivatives in the immune system, and provide us with potential tar- inducers (46–48). In contrast, compounds such as quinones and gets by which to interfere in the effects of environmental pollutants other phenolic antioxidants that induce phase II enzyme expression on allergic inflammation. without affecting the activity of phase I enzymes are known as Our understanding of the molecular mechanisms by which PAH monofunctional agents (46–48). Our data show that BNF and and related xenobiotics impact the immune system is rudimentary tBHQ induce ARE reporter gene activity (Figs. 8 and 9), suggest- compared with the understanding of the metabolic pathways that ing that the phase II enzyme detoxification pathway is important in mediate the effects of such chemicals in hepatocytes. Best charac- macrophages. In addition, ARE consensus sequences have been terized is the pathway that is mediated by the AhR (29–32). After found in the promoters of the IL-6, P450 aromatase, ferritin-L, 950 MACROPHAGE ACTIVATION BY POLYCYCLIC AROMATIC HYDROCARBONS collagenase, and tyrosinase genes, and the ARE pathway may hARE activation in macrophages showed that mutation of the AP-1 therefore also be involved in these cellular responses (50). Future consensus sequence at a site that does not overlap with the hARE core studies will address which macrophage genes are activated via this does not affect ARE activation (Fig. 8B). However, mutation of bases route by PAH. It is interesting that bacterial LPS induced ARE- in the hARE core interfered in basal and inducible ARE activity (Fig. CAT activity (Fig. 8A), as this suggests that a LPS-binding recep- 8B). Gel-shift assays also showed that, while the wild-type hARE tor may act as an ARE inducer. Although there are no reports yields at least two shift complexes, a constitutive shift complex re- linking membrane receptors to the ARE pathway, it is possible that mained when the AP-1 sequence was disrupted (Fig. 7). This complex the CD14 and other macrophage receptors that induce ROS may may represent the ARE-specific transcription factor postulated by induce cellular activation via an ARE (66). We are in the process Pickett et al. (41). Our study does not rule out a role for AP-1 proteins of exploring the effects of those receptors on the ARE response in the extended ARE region, because our oligonucleotide probes and pathway. reporter gene constructs did not include a complete copy of the up- The activation of AP-1 response elements can contribute to a stream AP-1-like element (Fig. 1) (36). wide range of biologic responses. PAH-induced NQO1 or gluta- Taken together, our data show that a PAH and a representative thione S-transferase (GST-Ya) gene expression in hepatoma cells quinone product induce oxidative stress in macrophages. Oxidative is associated with an increase in AP-1 activity (36, 44, 52–54). stress leads to the activation of stress-activated protein kinases that tBHQ and BNF induce the expression of c-Jun, Jun B, Jun D, Fra1, are involved in AP-1-mediated gene responses. Another response and Fra2 (36, 43, 44, 52–54). In addition, Ainbinder et al. (54) pathway linked to PAH in macrophages is activation of the anti- showed that BNF and tBHQ induce GST-Ya gene expression (Fig. oxidant RE, which also contributes to gene expression. These ac-

1) by activating a signaling pathway that involves AP-1 proteins, tivation responses by PAH in inhaled particulates are most likely Downloaded from Ras, and protein tyrosine kinases. Other groups have reported that an important contributor to airway inﬂammation. PAH can induce Ha-Ras, c-myc, and protein tyrosine kinase activities (71–73). We focused on MAPK activation, since these cas- References cades are functionally related to Ras and AP-1 proteins. While we 1. Folinsbee, L. J. 1993. Human health effects of air pollution. Environ. Health failed to obtain extracellular signal-regulated kinase (ERK) acti- Perspect. 100:45. 2. Pope, C. A., D. W. Dockery, and J. Schwartz. 1995. Review of epidemiological

vation (not shown), both JNK and p38 MAPK activities were in- http://www.jimmunol.org/ evidence of health effects of particulate air pollution. Inhal. Toxicol. 7:1. creased in human and murine macrophage cell lines upon exposure 3. Schwartz, J., and D. W. Dockery. 1992. Increased mortality in Philadelphia as- to BNF and tBHQ (Figs. 2–4). The kinetics of activation was sociated with daily air pollution concentrations. Am. Rev. Respir. Dis. 145:600. faster and the intensity of the kinase responses more robust with 4. Peterson, B., and A. Saxon. 1996. Global increases in allergic respiratory disease: the possible role of diesel exhaust particles. Ann. Allergy Asthma Immunol. 77:263. tBHQ compared with BNF treatment (Figs. 2 and 4). This may be 5. McClellan, R. O. 1987. Health effects of exposure to diesel exhaust particles. due to direct engagement of the stress kinase pathway by tBHQ, Annu. Rev. Pharmacol. Toxicol. 27:279. 6. Takafuji, S., S. Suzuki, K. Koizumi, K. Tadokoro, T. Miyamoto, R. Ikemori, and while BNF may require conversion to quinone products before H. Tokiwa. 1986. Diesel-exhaust particulate inoculated by the intranasal route acting as a stress stimulus. Our results are in agreement with the have an adjuvant activity for IgE production in mice. J. Allergy Clin. Immunol. ability of butylated hydroxyanisole, a tBHQ homologue, to acti- 77:616. 7. Takafuji, S., S. Suzuki, M. Muranaka, and T. Miyamoto. 1989. Influence of vate the JNK cascade in Jurkat T cells (74). MAPK promote AP-1 environmental factors on IgE production: IgE, mast cells and the allergic re- by guest on October 1, 2021 activation by two independent but complementary actions. The sponse. Ciba Found. Symp. 147:188. first is transcriptional activation of AP-1 proteins, e.g., phosphor- 8. Diaz-Sanchez, D., A. R. Dotson, H. Takenaka, and A. Saxon. 1994. 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