Polycyclic Aromatic Hydrocarbons Inhibit Differentiation of Human Monocytes into Macrophages

This information is current as Julien van Grevenynghe, Sophie Rion, Eric Le Ferrec, Marc of September 23, 2021. Le Vee, Laurence Amiot, Renée Fauchet and Olivier Fardel J Immunol 2003; 170:2374-2381; ; doi: 10.4049/jimmunol.170.5.2374 http://www.jimmunol.org/content/170/5/2374 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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Polycyclic Aromatic Hydrocarbons Inhibit Differentiation of Human Monocytes into Macrophages1

Julien van Grevenynghe,2* Sophie Rion,2* Eric Le Ferrec,* Marc Le Vee,* Laurence Amiot,† Rene´e Fauchet,† and Olivier Fardel3*†

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are ubiquitous environmental carcinogenic contaminants exerting deleterious effects toward cells acting in the immune defense such as monocytic cells. To investigate the cellular basis involved, we have examined the consequences of PAH exposure on macrophagic differentiation of human blood monocytes. Treatment by BP markedly inhibited the formation of adherent macrophagic cells deriving from monocytes upon the action of either GM-CSF or M-CSF. Moreover, it reduced expression of macrophagic phenotypic markers such as CD71 and CD64 in GM-CSF-treated monocytic cells, without altering cell viability or inducing an apoptotic process. Exposure to BP also strongly altered functional properties characterizing macrophagic cells such as endocytosis, phagocytosis, LPS-triggered production of Downloaded from TNF-␣ and stimulation of allogeneic lymphocyte proliferation. Moreover, formation of adherent macrophagic cells was decreased in response to PAHs distinct from BP such as dimethylbenz(a)anthracene and 3-methylcholanthrene, which interact, like BP, with the arylhydrocarbon receptor (AhR) known to mediate many PAH effects. In contrast, benzo(e)pyrene, a PAH not activating AhR, had no effect. In addition, AhR was demonstrated to be present and functional in cultured monocytic cells, and the use of its antagonist ␣-naphtoflavone counteracted inhibitory effects of BP toward macrophagic differentiation. Overall, these data dem- onstrate that exposure to PAHs inhibits functional in vitro differentiation of blood monocytes into macrophages, likely through an http://www.jimmunol.org/ AhR-dependent mechanism. Such an effect may contribute to the immunotoxicity of these environmental carcinogens owing to the crucial role played by macrophages in the immune defense. The Journal of Immunology, 2003, 170: 2374–2381.

olycyclic aromatic hydrocarbons (PAHs)4 represent an im- and ultimately interaction with xenobiotic responsive elements portant class of widely distributed environmental contam- found in the 5Ј flanking regions of responsive genes (6). In this P inants. They are usually formed through the combustion of way, AhR is notably implicated in up-regulation of drug-metabo- fossil fuel and the burning of various substances and are found in lizing enzymes such as cytochromes P450 (CYPs), especially significant amounts in automobile exhaust, cigarette smoke, vari-

CYP1A1 (7), which metabolize PAHs into reactive intermediates by guest on September 23, 2021 ous foods, and industrial waste by-products (1). They can exert that are capable of interacting with DNA and most likely account major toxic effects, including development of cancers in various for mutagenic properties of these environmental contaminants (8). tissues, cardiovascular diseases, loss of fertility, and immunosup- Immunosuppression due to PAHs, which may indirectly contrib- pression (2Ð5). Many of these adverse effects are thought to be ute to their carcinogenic properties, also involves (at least in part) linked to the cytosolic arylhydrocarbon receptor (AhR), a ligand- AhR, because the AhR antagonist ␣-naphtoflavone counteracts dependent basic helix-loop-helix transcription factor to which some adverse effects of PAHs toward immune cells (4, 9). How- PAHs bind, thereby triggering translocation of the AhR into the ever, cellular and molecular mechanisms involved in PAH-related nucleus, association with the AhR nuclear translocator (ARNT), immunotoxicity remain incompletely understood, especially for hu- man cells, because most published studies have been performed on mice (9Ð11). Lymphocytes likely constitute important targets. In- *Faculte«de Pharmacie, Institut National de la Sante«et de la Recherche Me«dicale, deed, potent immunosuppressive PAHs such as benzo(a)pyrene Rennes, France; and †Faculte«deMe«decine, Laboratoire Universitaire d’He«matologie et de la Biologie des Cellules Sanguines, Rennes, France (BP) and dimethylbenz(a)anthracene (DMBA) inhibit murine T and B cell proliferation and alter T cell-related cytokine production and Received for publication July 10, 2002. Accepted for publication December 17, 2002. B cell-mediated Ab production (11Ð13). They also suppress mito- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance genesis of human T lymphocytes (14) and alter B cell lymphopoie- with 18 U.S.C. Section 1734 solely to indicate this fact. sis through triggering pre-B lymphocyte apoptosis (15). 1 This work was supported by grants from the Institut National de Recherche et de Besides lymphocytes, APCs such as macrophages and dendritic Se«curite«and the Ministe`re de l’Ame«nagement du Territoire et de l’Environnement. cells can also be affected by PAHs. Indeed, PAHs impair Ag pre- J.v.G. and S.R. are recipients of fellowships from the Ligue Nationale contre le Can- cer (Comite« des Coˆtes d’Armor) and the Fondation pour la Recherche Me«dicale, sentation by mouse macrophages (16), alter T cell-macrophage respectively. interaction (17), and suppress phagocytic activity of peritoneal 2 J.v.G. and S.R. contributed equally to this study. macrophages (18). BP also reduced esterase-positive macrophagic 3 Address correspondence and reprint requests to Dr. Olivier Fardel, Institut National cell population in mouse spleen (19). In fact, murine splenic mac- de la Sante«et de la Recherche Me«dicale Unité 456, Faculte«de Pharmacie, 2 avenue rophages, which have been demonstrated to metabolize PAHs du Pr. L. Bernard, 35043 Rennes, France. E-mail address: olivier.fardel@univ- rennes1.fr (20), are considered the cell types targeted by BP among the dif- 4 Abbreviations used in this paper: PAH, polycyclic aromatic hydrocarbon; AhR, ferent splenic cell populations and are responsible for PAH-related arylhydrocarbon receptor; ARNT, AhR nuclear translocator; CYP, cytochrome P450; suppression of splenic humoral immune response (21). Moreover, BP, benzo(a)pyrene; DMBA, dimethylbenz(a)anthracene; MC, 3-methylcholan- threne; BeP, benzo(e)pyrene; WST-1, 4-3-(4(iodophenyl)-2-(4-nitrophenyl)-2H-5tet- it is noteworthy that cigarette smoke condensates, whose major razole]-1,3-benzene disulfonate; MFI, mean fluorescence intensity. components consist of PAHs, markedly down-regulate functional

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 2375 capacities of macrophages (22). More specialized APCs, i.e., densed or fragmented chromatin, were counted. Results were expressed as Langerhans cells, have also been found to be altered after topical percentages of the total cell number. application of DMBA (23). Interestingly, PAHs have been recently demonstrated to strongly impair functional differentiation and mat- Flow cytometric immunolabeling assays uration of human monocytes into dendritic cells (24). This indi- Phenotypic analysis of monocytic cells was performed using flow cyto- cates that differentiation pathways of monocytes may be compro- metric direct or indirect immunofluorescence as previously described (24). mised by PAHs. Owing to the fact that macrophages are likely Cells were first incubated for 30 min in PBS with 5% human AB serum at 4¡C to avoid any nonspecific mAb binding. Several mAbs were then used major targets of PAHs as reported above, it is tempting to specu- for direct or indirect immunofluorescence labeling: PE-conjugated mouse late that PAHs may affect not only dendritic cell differentiation, but mAbs against CD11b and CD40; FITC-conjugated Abs directed against also macrophage generation from monocytes. To test such a hy- CD14, CD16, CD86, CD80, HLA class I, HLA class II, CD71, CD64, Ј pothesis, in the present study we have examined the effects of PAH CD11a, CD11c, CD29, CD36, CD49e, and F(ab )2; and unconjugated mAb against CD49f. Isotypic control labeling was performed in parallel. Monoclo- exposure on functional differentiation of peripheral blood mono- nal Abs were purchased from Immunotech (Marseille, France), except for Abs cytes into macrophages upon the action of cytokines such as GM- Ј against HLA class I, CD36, and F(ab )2, which were obtained from Sigma- CSF and M-CSF. Our data indicate that PAHs inhibit generation of Aldrich, Ortho Diagnostics (Raritan, NJ), and Silenus (Melbourne, Australia), functional macrophagic cells from human monocytes likely respectively. Thereafter, cells were analyzed with a FACSCalibur flow through, at least in part, an AhR-related mechanism. Such an al- cytometer (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences). Results were expressed as mean fluorescence intensity (MFI) tered macrophagic differentiation may significantly contribute to calculated as follows: mean fluorescence (mAb of interest) Ϫ mean PAH-related immunotoxicity, owing to the crucial role played by fluorescence (control mAb). macrophages in the immune response, and supports the conclusion Downloaded from that monocyte differentiation pathways represent major cellular Cytokine measurements events affected by PAHs. Levels of TNF-␣ in the supernatants of LPS-treated monocytic cultures were quantified using an ELISA kit obtained from BD Biosciences. Anal- Materials and Methods yses were conducted according to the manufacturer’s instructions. Chemicals and reagents Endocytosis assay BP, DMBA, 3-methylcholanthrene (MC), benzo(e)pyrene (BeP), ␣-naph- http://www.jimmunol.org/ Cultured monocytic cells were incubated with 1 mg/ml FITC-dextran for toflavone, LPS, PMA, and FITC-dextran (Mr, 40,000 kDa) were provided by Sigma-Aldrich (St. Louis, MO). Dihydrorhodamine 123 and Hoechst 60 min at 37¡C. Cellular uptake of FITC-dextran was then monitored by 33342 were obtained from Molecular Probes (Eugene, OR). Chemicals flow cytometry. A negative control was performed in parallel by incubating were commonly used as stock solutions in DMSO. Final concentrations of cells with FITC-dextran at 4¡C instead of 37¡C. solvent in culture medium did not exceed 0.2% (v/v); control cultures received the same dose of solvent as for treated counterparts. [3H]Thymi- Phagocytosis assay dine (sp. act., 5 Ci/mmol) was obtained from Amersham (Les Ulis, France). ␮ Fluorescent latex microspheres were purchased from Polysciences (War- Cultured monocytic cells were incubated with 15 loffluorescent latex rington, PA). The tetrazolium salt 4-[3-(4(iodophenyl)-2-(4-nitrophenyl)- microspheres for 30 min at 37¡C. Cellular phagocytosis of latex beads was then monitored by flow cytometry. A negative control was performed in 2H-5tetrazole]-1,3-benzene disulfonate (WST-1) was provided by Roche by guest on September 23, 2021 Diagnostics (Meylan, France). Human GM-CSF (sp. act., 1.2 ϫ 108 U/mg) parallel by incubating cells with latex beads at 4¡C instead of 37¡C. was provided by Schering Plough (Lyon, France), whereas M-CSF (sp. act., 1 ϫ 108 U/mg) was obtained from Promocell (Heildelberg, Germany). Mixed leukocyte reaction Preparation and culture of human monocytes Ability of cultured monocytic cells to stimulate allogeneic T cell prolifer- ation was determined using MLR as previously described (24). Briefly, PBMCs were obtained from blood buffy coats of healthy donors through 0.3 ϫ 105 LPS-pretreated monocytic cells were cultured with 1.5 ϫ 105 Ficoll gradient centrifugation and from cytapheresis products of nonpatho- allogeneic T cells in round-bottom 96-well microplates. After 5 days of logical peripheral blood. Monocytes were then prepared by a 2-h adhesion culture, cells were pulsed with 1 ␮Ci of [3H]thymidine for 18 h. Incorpo- step, which routinely obtained Ͼ90% of adherent CD14-positive cells as ration of the radionucleide into DNA was further measured by scintillation assessed by immunostaining. These monocytic cells were next cultured in counting. Basal thymidine incorporation into monocytic cells was deter- RPMI 1640 medium supplemented with 2 mM glutamine, antibiotics, and mined in parallel from wells containing monocytic cells alone and was 10% FCS in the presence of 800 U/ml GM-CSF or 50 U/ml M-CSF to get substracted from thymidine counts observed in T cell-monocytic cell co- macrophages as previously reported (25). cultures. Results were expressed as radioactivity (cpm) per well.

Cellular viability determination Respiratory burst activity detection Cellular viability was determined by microscopic analysis of cellular ex- Respiratory burst activity detection was performed using the reactive ox- clusion of trypan blue dye and flow cytometric analysis of cellular pro- ygen intermediate-sensitive probe dihydrorhodamine 123 (26). Monocytic pidium iodide staining as previously reported (24). cells were first prestimulated for 45 min with 100 ng/ml PMA, a chemical commonly used to trigger oxidative burst activity in phagocytic cells (27). Cellular adhesion assay After washing, cells were incubated with 5 ␮g/ml dihydrorhodamine 123 Adherent fraction of cultured monocytic cells was analyzed using the for 45 min at 37¡C. Cellular fluorescence due to rhodamine 123, formed WST-1 assay in 96-well microplates. Briefly, after careful removal of non- upon the action of reactive oxygen intermediates such as hydrogen perox- adherent cells by gentle aspiration, adherent cells were washed and then ide, was then measured by flow cytometry. Data were expressed as fluo- incubated with 10 ␮l of WST-1. The yellow formazan product formed by rescence arbitrary units. viable adherent cells was then quantified by detection of its absorbance at 450 nm using a Titertek Multiskan spectrophotometer (Flow Laboratories, RNA isolation and RT-PCR analysis Puteaux, France). Total RNA was isolated from cells using the guanidinium thiocyanate/ Detection of apoptosis cesium chloride method of Chirgwin et al. (28). RT-PCR analysis of CYP1A1, AhR, ARNT, and ␤-actin mRNA expression was then performed Microscopic detection of apoptotic cells was performed after Hoechst as previously reported (24). The primers used for CYP1A1, AhR, and 33342 labeling of nuclei as previously reported (26). Briefly, cells were ARNT detection were exactly those used by Baron et al. (29) and Roberts first stained with Hoechst 33342 (0.5 ␮g/ml) in PBS at room temperature et al. (30). ␤-Actin detection was performed as a loading control with the for 15 min in the dark. Thereafter, they were observed by fluorescence following primers: sense, 5Ј-GCCCAGAGCAAGAGAG-3Ј, and anti- microscopy using a fluorescence Olympus BX60 microscope (Olympus, sense, 5Ј-GGCATCTCTTGCTCG-3Ј. PCR products were separated on Melville, NY). Cells with an apoptotic nucleus, i.e., a nucleus with con- 1.2% agarose gels and stained with ethidium bromide. 2376 ALTERED MACROPHAGIC DIFFERENTIATION BY PAHs

Statistical analysis Data were analyzed with the nonparametric Wilcoxon’s test. The level of significance was p Ͻ 0.05. Results PAHs alter the formation of adherent macrophagic cells in GM-CSF-treated monocyte cultures Blood monocytes cultured for 6 days in the presence of GM-CSF developed into adherent macrophagic cells displaying a “fried- egg”-like morphology, i.e., round cells with a large nucleus cen- tered in the cytoplasm, as previously reported (25) (Fig. 1). In the presence of 10 ␮M BP throughout the culture, the formation of adherent macrophagic cells was markedly reduced, and most of the cells remained as nonadherent cells in the culture medium and exhibited a smaller size than their untreated counterparts (Fig. 1). However, BP treatment did not result in alteration of cell viability as assessed by determination of trypan blue exclusion (Fig. 2A) and by measurement of the percentages of propidium iodide- Downloaded from stained cells (Fig. 2B). In addition, the proportion of apoptotic cells remained low, i.e., below 10%, in both BP-treated and untreated monocytic cultures as assessed by Hoechst 33342 labeling of ap- optotic nuclei (Fig. 2C). The decrease in the formation of adherent macrophagic cells in response to BP was further quantified using the WST-1 assay. As shown in Fig. 3A,10␮M BP reduced adherent monocytic cell num- http://www.jimmunol.org/ ber to ϳ25Ð30% of that found in untreated cultures. Such a decrease was similarly observed in BP-exposed monocytic cells maintained in culture for 4, 6, or 10 days. Shorter exposure to BP, i.e., Ͻ 4 days, was not tested since a 4- to 6-day culture period is usually required for getting macrophages from monocytes in response to GM-CSF (25). The action of BP was dose dependent: concentrations such as 10 and FIGURE 2. Effect of BP treatment on cellular trypan blue exclusion (A), 1 ␮M maximally reduced adherent macrophagic cell number, propidium iodide staining (B), and Hoescht 33342 labeling (C) in mono- ␮

whereas the addition of 0.5 M BP resulted in only a partial effect, cyte cultures. Blood monocytes were cultured with GM-CSF in the absence by guest on September 23, 2021 and lower concentrations such as 0.1 and 0.01 ␮M had no effect (Fig. or presence of 10 ␮M BP for 6 days. Cellular viability was then determined 3B). Besides BP, other PAHs used at 10 ␮M, such as DMBA and by analysis of cellular trypan blue exclusion (A)orbyflow cytometric MC, also decreased the formation of adherent macrophagic cells in measurement of propidium iodide-stained cells (B), whereas apoptotic cells GM-CSF-treated monocytic cultures (Fig. 3C). In contrast, 10 ␮M were determined through examination of Hoescht 33342-labeled nuclei BeP failed to alter the adherent macrophagic population (Fig. 3C). (C). Data shown are expressed as percent of trypan blue-negative cells comparatively to total cells (A), as flow cytometric graphs indicating the PAHs alter expression of phenotypic surface markers in proportion of propidium iodide-positive cells (B), and as percent of apo- Ϯ GM-CSF-treated monocytic cell cultures ptotic cells (C). They are the means SD of at least five independent experiments (A and C) or are representative of three independent experi- To investigate the effects of PAHs on expression of surface mark- ments (B). ers, we first analyzed levels of CD71 in PAH-treated and untreated monocytic cells cultured in the presence of GM-CSF for 6 days. Besides CD71, other phenotypic surface markers such as the Indeed, CD71, which corresponds to the transferrin receptor, is costimulation molecules CD80 and CD86, the Fc␥RI receptor well known as a macrophagic differentiation marker (31, 32). In CD64, the integrins CD11a, CD11c, CD29, CD49e, and CD49f, agreement with this point, monocytic cells, which initially did not and HLA class I molecules were significantly down-regulated in express CD71, exhibited a marked expression of this surface BP-treated monocytic cells when compared with their untreated marker after 6 days of culture, i.e., when they had acquired a mac- counterparts (Table I). Expression of the LPS surface receptor rophagic phenotype (Fig. 4). In the presence of PAHs such as BP CD14 was also reduced in BP-exposed cells; however, such a de- and DMBA, however, CD71 expression remained very low (Fig. crease did not statistically reach a significant level, which may 4). In contrast, CD71 levels were unaffected by BeP (Fig. 4). reflect the fact that heterogeneous and variable levels of expression of this surface molecule were observed in control untreated mono- cytic cells. Several surface markers were not found to be signifi- cantly altered by BP treatment: the integrin CD11b, the costimu- lation molecule CD40, the scavenger receptor CD36, and HLA class II molecules (Table I).

PAHs alter functional properties of GM-CSF-treated monocytic FIGURE 1. BP treatment inhibits the GM-CSF-triggered formation of cells adherent human macrophagic cells. Blood monocytes were cultured with GM-CSF in the absence or presence of 10 ␮M BP for 6 days. Photographs We next analyzed the consequences of PAH exposure during GM- of cultured cells were then taken by phase-contrast microscopy. Original CSF-triggered monocytic cell differentiation toward several mac- magnification, ϫ60. rophagic functions. Exposure to 10 ␮M BP of cultured monocytic The Journal of Immunology 2377 Downloaded from

FIGURE 4. Effects of PAHs on CD71 up-regulation in GM-CSF-treated monocytic cells. Monocytes were cultured with GM-CSF for 6 days in the absence or presence of 10 ␮M BP, 10 ␮M DMBA, or 10 ␮M BeP. Parental and cultured monocytes were stained with mAb directed against CD71 http://www.jimmunol.org/ (filled histograms) or with isotypic control (open histograms). The data shown are representative of at least three independent experiments.

cally differ between BP-exposed and unexposed cells (Fig. 7). In addition, the cellular fluorescence values reflecting basal levels of reactive oxygen intermediates in the absence of PMA were similar in both BP-treated and untreated monocytic cells. by guest on September 23, 2021 PAHs inhibit M-CSF-triggered formation of adherent macrophagic cells from monocytes FIGURE 3. PAH exposure decreases the number of adherent cells in To determine whether the inhibitory effects of PAHs such as BP GM-CSF-treated monocyte cultures. Monocytes were cultured with GM- observed in GM-CSF-treated monocytic cultures may also occur CSF in the absence or presence of 10 ␮M BP for 4, 6, or 10 days (A)orof various BP concentrations (from 0.01 to 10 ␮M) for 6 days (B)orof during GM-CSF-unrelated macrophagic differentiation, we have ␮ various PAHs (BP, DMBA, MC, and BeP) used at 10 ␮M for 6 days (C). examined the action of 10 M BP on M-CSF-mediated macroph- Numbers of adherent monocytic cells were then determined using the agic differentiation. As shown in Fig. 8A and in agreement with WST-1 assay as described in Materials and Methods. Data are expressed as percent of adherent cell counts found in BP-untreated cultures and are the means Ϯ SD of five independent experiments performed in quadripli- Table I. Phenotypic analysis of cultured monocytic cells either p Ͻ 0.05 when compared with BP-untreated cells. untreated or exposed to BP a ,ء .cate

Surface Marker Ϫ BP ϩ BP

cells was found to markedly suppress cellular endocytosis of CD11a (n ϭ 13) 56.8 Ϯ 31.6 24.7 Ϯ 9.2b FITC-dextran when compared with untreated cells (Fig. 5A). Sim- CD11b (n ϭ 7) 631.8 Ϯ 229.4 577.8 Ϯ 194.6 ilarly, cellular phagocytosis of fluorescent microspheres was CD11c (n ϭ 9) 284.3 Ϯ 121.4 148.2 Ϯ 81.5b down-regulated in response to BP (Fig. 5B). Addition of BP during CD14 (n ϭ 11) 53.9 Ϯ 73.6 18 Ϯ 19.2 CD29 (n ϭ 7) 117.9 Ϯ 36.5 36.7 Ϯ 10.7b macrophagic differentiation also markedly diminished LPS-medi- ϭ Ϯ Ϯ ␣ CD36 (n 6) 30.3 16.8 17.4 7.1 ated secretion of TNF- by monocytic cells as demonstrated by CD40 (n ϭ 7) 129.7 Ϯ 90.7 111.7 Ϯ 61.6 ELISA measurements of TNF-␣ levels in culture supernatants CD49e (n ϭ 9) 35.2 Ϯ 14.5 9.7 Ϯ 7.4b (Fig. 6A). This indicates that LPS-stimulated release of TNF-␣ was CD49f (n ϭ 7) 49.2 Ϯ 21.5 20.1 Ϯ 17.2b ϭ Ϯ Ϯ b likely impaired in PAH-treated cells. BP exposure also strongly CD64 (n 11) 56.7 31.2 2.7 2.5 CD71 (n ϭ 27) 94.3 Ϯ 46.8 12 Ϯ 16b inhibited the ability of cultured monocytic cells to induce prolif- CD80 (n ϭ 14) 46 Ϯ 29.3 10.5 Ϯ 9.8b eration of allogeneic T lymphocytes. Indeed, unlike their untreated CD86 (n ϭ 9) 47.2 Ϯ 16.1 26.6 Ϯ 12.8b counterparts, BP-exposed monocytic cells failed to stimulate DNA HLA class I (n ϭ 10) 660.2 Ϯ 234.6 456 Ϯ 121.7b synthesis in allogeneic T lymphocytes (Fig. 6B). However, BP did HLA class II (n ϭ 9) 126.2 Ϯ 54.3 159.8 Ϯ 79.2 not affect respiratory burst activity. Indeed, exposure to PMA trig- a Monocytes were cultured with GM-CSF for 6 days in the absence or presence of gered production of reactive oxygen intermediates in both un- 10 ␮M BP. Phenotype analysis was then performed as described in Materials and Methods. Results are expressed as MFI and are the means Ϯ SD of n independent treated and BP-treated monocytic cells, and the levels of reactive experiments. oxygen intermediates found after PMA treatment did not statisti- b p Ͻ 0.05 when compared with BP-untreated cells. 2378 ALTERED MACROPHAGIC DIFFERENTIATION BY PAHs Downloaded from http://www.jimmunol.org/

FIGURE 5. BP treatment decreases endocytic (A) and phagocytic (B) activity of cultured monocytic cells. Monocytes were cultured with GM- CSF for 6 days in the absence or presence of 10 ␮M BP. Cells were then incubated with FITC-dextran (A)orfluorescent latex microbeads (B)at4¡C FIGURE 6. BP treatment inhibits TNF-␣ secretion (A) and stimulation or 37¡C. Cellular uptakes of FITC-dextran and microbeads were then de- of allogeneic lymphocyte DNA synthesis (B) by monocytic cells. Mono- termined by flow cytometry. The data shown are representative of five cytes were cultured with GM-CSF for 6 days in the absence or presence of by guest on September 23, 2021 independent experiments. 10 ␮M BP. A, TNF-␣ secretion in culture medium, triggered by a 2-day stimulation by LPS, was then measured by ELISA. Data are the means Ϯ SD of TNF-␣ values from seven independent experiments. B, Capacity of LPS-pretreated monocytes to stimulate DNA synthesis of allogeneic T previous reports (25), blood monocytes maintained for 6 days in lymphocytes in MLR was determined as described in Materials and Meth- the presence of M-CSF developed into adherent macrophagic cells ods. Data are expressed as cpm per well and are the means Ϯ SD of five Ͻ ء displaying an elongated or stellate morphology. In the presence of independent experiments performed in triplicate. , p 0.05 when com- 10 ␮M BP throughout the culture, the formation of adherent mac- pared with BP-untreated counterparts. rophagic cells was markedly diminished, and most of the cells remained as nonadherent cells in the culture medium (Fig. 8A). This loss of adherent macrophagic cells was fully illustrated using the WST-1 adhesion assay, because adherent monocytic cell counts in rophagic cells when compared with untreated counterparts BP-treated cells were found to represent 20.8 Ϯ 15.1% of the values (n ϭ 7, p Ͻ 0.05). detected in untreated counterparts (n ϭ 7, p Ͻ 0.05, when compared ␣ with untreated cells). In addition, BP exposure was found to down- The AhR inhibitor -naphtoflavone counteracts PAH inhibitory regulate surface levels of the macrophagic markers CD71, CD16, and effects toward macrophagic differentiation from monocytes CD64 in M-CSF-treated monocytic cells, whereas expression of We finally investigated the putative involvement of AhR in PAH- CD14 remained unchanged (Fig. 8B). It also reduced cellular endo- mediated inhibitory effects toward macrophagic differentiation us- cytosis of FITC-dextran (data not shown). ing the AhR antagonist ␣-naphtoflavone (9, 33). We first verified that AhR was present and functional in our monocytic cell cul- PAHs exert inhibitory effects toward differentiated macrophages tures. As shown in Fig. 9A, mRNAs of AhR and of its cofactor To analyze PAH effects on differentiated macrophages, blood ARNT were detected in cultured monocytic cells as well as in monocytes were first cultured with GM-CSF for 6 days and the human hepatocytes used here as positive cellular controls (6). macrophages obtained were then maintained in the absence or Moreover, treatment of monocytic cells with 10 ␮M BP was dem- presence of 10 ␮M BP for 4 days. Treatment by the PAH led to a onstrated to markedly up-regulate CYP1A1 expression (Fig. 9B), loss of adherent macrophagic cells; indeed, the WST-1 adhesion indicating that AhR was most likely fully functional in our mono- assay indicated that adherent monocytic cell counts in BP-exposed cyte culture system because this receptor is well-known to play a macrophages corresponded to 48 Ϯ 19.7% of the values found in key role in PAH regulation of CYP1A1 (7). untreated cells (n ϭ 10, p Ͻ 0.05, when compared with untreated Cultured monocytes were then treated with BP or cotreated with cells). In addition, levels of CD71 and CD64 expression were BP and ␣-naphtoflavone throughout the GM-CSF-triggered mac- reduced by 2.7- and 3.8-fold, respectively, in BP-treated mac- rophagic differentiation process. Microscopic examination of The Journal of Immunology 2379

FIGURE 7. Effect of BP on PMA-triggered burst respiratory activity in cultured monocytic cells. Monocytes were cultured with GM-CSF for 6 days in the absence or presence of 10 ␮M BP. PMA-triggered production of reactive oxygen intermediates was then determined by flow cytometry using Downloaded from the dihydrorhodamine 123 probe. Cellular levels of reactive oxygen inter- mediates are expressed in fluorescence arbitrary units and are the means Ϯ SD p Ͻ 0.05 when compared with PMA- FIGURE 9. AhR, ARNT, and CYP1A1 expression in cultured monocytic ,ء .of five independent experiments untreated counterparts. cells. A, AhR, ARNT, and ␤-actin mRNA levels were analyzed in GM-CSF- treated monocytic cells and primary human hepatocytes by RT-PCR analysis. B, CYP1A1 expression and ␤-actin mRNA levels in untreated and BP-treated 6-day cultures revealed that ␣-naphtoflavone was capable of mark- monocytic cells were determined using RT-PCR analysis. The data shown are http://www.jimmunol.org/ edly counteracting the inhibitory effects of BP toward the forma- representative of three independent experiments. tion of adherent macrophagic cells (data not shown). In agreement with these morphological observations, data from WST-1 adhesion the down-regulation of surface levels of the macrophagic marker assays (Fig. 10A) indicated that adherent macrophagic cell num- CD71 occurring in response to BP (Fig. 10B). bers were similar whether the cells were untreated, exposed to ␣-naphtoflavone alone, or cotreated with the combination of ␣-naphtoflavone and BP. In contrast, exposure to BP markedly Discussion diminished the adherent monocytic cell fraction as already re- Many PAHs are well recognized as potent immunosuppressive by guest on September 23, 2021 ported above. Moreover, addition of ␣-naphtoflavone suppressed agents (9Ð12). Although lymphocytes have been shown to be tar- gets (5), there is growing evidence that monocyte-derived cells such as myeloid dendritic cells and macrophages are also directly implicated in PAH immunotoxicity. Indeed, PAHs strongly impair functional differentiation and maturation of human monocytes into myeloid dendritic cells (24), and various experimental results in- dicate that functional capacities of macrophages are altered by PAHs (16Ð19). Our present data fully support the view that mono- cytic cells are important targets for PAHs, because we demon- strated that these chemicals inhibit GM-CSF- and M-CSF-medi- ated generation of functional macrophagic cells from human monocytes. Thus, we have found through microscopic examina- tion of cultures and the use of an adhesion cell count assay that exposure to PAHs such as BP, MC, and DMBA during GM-CSF- triggered macrophagic differentiation decreased formation of ad- herent monocytic cells and prevented acquisition of characteristic morphological features of macrophages. BP was also demonstrated to exert a similar action in M-CSF-treated monocyte cultures. This loss of adhesion ability might be linked to altered expression of some integrins such as CD29, CD49e, and CD49f triggered by BP treatment in macrophagic cells. Interestingly, the effect of BP on cell adhesion was dose dependent and the active BP concentra- tions, i.e., 0.5Ð10 ␮M, were in the range of those known to affect FIGURE 8. BP treatment alters M-CSF-triggered formation of adherent PAH-sensitive cells such as oocytes, lymphocytes, and vascular macrophagic cells. Blood monocytes were cultured with M-CSF in the smooth cells (4, 5, 34). BP effects persisted at least up to day 10 of absence or presence of 10 ␮M BP for 6 days. A, Photographs of cultured culture, suggesting that BP continuously inhibited macrophagic cells were then taken by phase-contrast microscopy (original magnifica- tion, ϫ40). B, CD14, CD16, CD64, and CD71 expressions were deter- differentiation from monocytes rather than simply delaying it. mined by flow cytometric immunofluorescence as described in Materials PAH exposure was associated with decreased expression of sur- and Methods; data are expressed as the percentage of MFI values found in face phenotypic markers usually up-regulated in macrophagic monocytic cells not exposed to BP and are the means Ϯ SD of six inde- cells, especially CD71. Functional macrophagic properties such as p Ͻ 0.05 when compared with BP-untreated cells. endocytosis, phagocytosis, cytokine production, and APC activity ,ء .pendent experiments 2380 ALTERED MACROPHAGIC DIFFERENTIATION BY PAHs

to BP treatment. The observation that levels of surface phenotypic markers such as CD11b, CD36, CD40, CD14, and HLA class II molecules were not significantly altered in response to BP also argues against a major PAH toxicity. Moreover, this conclusion is supported by the preservation of PMA-triggered burst respiratory activity in BP-treated monocytic cells, indicating that some of the monocytic functions were not affected by PAHs. In addition, BP treatment did not elicit a major oxidative stress in cultured mono- cytes because similar cellular basal formation of reactive oxygen intermediates was found in both untreated and BP-treated mono- cytic cells. A major oxidative toxic insult, which has already been shown to occur in response to PAHs in cell types such as vascular smooth muscle cells (34), was consequently not likely operating in our cell culture system. It is also noteworthy that BP exposure did not result in a marked apoptosis induction because apoptotic cell number in response to BP did not overrun 10% of total monocytic cell population as assessed by Hoechst 33342 labeling of apoptotic nuclei. Similarly, BP and the halogenated arylhydrocarbon 2,3,7,8-

tetrachlorodibenzo-p-dioxin failed to induce apoptosis in cultured Downloaded from monocytic RAW 264.7 and U937 cells (35, 36), whereas PAHs are well-known to induce apoptosis in murine pre-B cells (15). Such data suggest that apoptotic effects of PAHs depend on cell types. The cellular and molecular mechanisms underlying the inhibi- tory action of PAHs toward macrophagic differentiation remain to

be clarified. The AhR, already known to mediate many immuno- http://www.jimmunol.org/ suppressive effects of PAHs (5, 15), is most likely involved be- cause 1) this receptor and its cofactor ARNT are present in cul- tured monocytes in agreement with a previous report (37) and are fully functional as assessed by RT-PCR analyses of CYP1A1 ex- FIGURE 10. Effect of ␣-naphtoflavone on BP-mediated decrease of ad- pression, 2) among PAHs such as BP, DMBA, MC, and BeP, only herent cell number (A) and on BP-triggered CD71 down-regulation (B)in the latter, which does not interact with AhR in contrast with the cultured monocytic cell cultures. GM-CSF-cultured monocytes were un- others (38), failed to down-regulate the formation of adherent mac- treated, exposed to 1 ␮M BP alone or 1 ␮M ␣-naphtoflavone alone, or rophagic cells, and 3) the AhR antagonist ␣-naphtoflavone coun- cotreated with 1 ␮M BP and 1 ␮M ␣-naphtoflavone. A, Numbers of ad- teracted BP action on the macrophagic differentiation pathway. by guest on September 23, 2021 herent monocytic cells were then determined using the WST-1 assay as Therefore, through interacting with AhR in monocytic cells, PAHs described in Materials and Methods. Data are expressed as percent of might activate unidentified factors that hamper macrophage for- adherent cell counts found in untreated monocytic cultures and are the mation or, alternatively, might down-regulate factors required for means Ϯ SD of five independent experiments performed in quadriplicate. B, Cells were stained with mAb directed against CD71 (filled histograms) macrophagic differentiation. Beyond PAH effects, the putative or with isotypic control (open histograms). The data shown are represen- physiological role of AhR in monocytic cells may be worth con- tative of five independent experiments. sidering because one might hypothesize that endogenous Ahr li- gands such as bilirubin (39) would negatively regulate macro- phagic differentiation. More generally, our data fully support the were also impaired in PAH-exposed monocytic cells. It is idea that AhR may play a role in differentiation processes as re- noteworthy that inhibitory effects of PAHs toward macrophage cently suggested (40, 41). Further studies are certainly required to generation were observed in both GM-CSF- and M-CSF-exposed investigate this point. monocyte cultures. This makes unlikely a specific interaction of Owing to the major role played by macrophages in innate and PAHs with GM-CSF- or M-CSF-restricted transduction pathways acquired immune defense, inhibition of their differentiation from and rather supports a general inhibitory effect of PAHs toward monocytes in response to PAHs might significantly contribute to monocyte differentiation pathways, whatever the cellular or mo- the potent immunosuppressive properties of these environmental lecular factor initially triggering this differentiation process. The contaminants. This conclusion agrees with numerous data obtained fact that PAHs also inhibit formation of functional dendritic cells from PAH-treated mice, pointing out an implication of macro- from blood monocytes upon the action of GM-CSF and IL-4 (24) phagic cells in PAH immunotoxicity (16Ð19, 21). It is also sup- fully supports this conclusion. In addition, it is noteworthy that BP ported by the phenotypic pattern of BP-treated monocytic cells, was demonstrated to exert inhibitory effects toward macrophages especially the down-regulation of costimulation molecules such as previously differentiated upon the action of GM-CSF, thus result- CD80 and CD86 involved in Ag presentation to lymphocytes, the ing in decreased cell adhesion and down-regulation of the mac- decreased expression of Fc␥ receptors such as CD16 and CD64 rophagic markers CD71 and CD64. This suggests that PAHs can acting in Ab-dependent phagocytosis and cytotoxicity, and the di- affect both the generation of macrophages and the properties of minished levels of integrins such as CD11a, CD11c, CD29, differentiated macrophages. These two effects may add up, leading CD49e, and CD49f responsible for cell-cell interactions, cell ad- to a major impairment of the monocytic/macrophagic cell lineage hesion, and migration. Moreover, BP-related alteration of endocy- in response to PAHs. tosis, phagocytosis, and allogeneic T cell stimulation likely im- Inhibition of macrophage generation in PAH-exposed monocyte pairs other macrophagic functions such as Ag processing and cultures was not associated with a nonspecific toxicity due to presentation and apoptotic cell clearance, whereas abolition of PAHs because we did not find any loss in cell viability in response TNF-␣ production may compromise the development of local or The Journal of Immunology 2381 systemic inflammation. Interestingly, monocytes, whose differen- 19. Blanton, R. H., M. Lyte, M. J. Myers, and P. H. Bick. 1986. Immunosuppression tiation pathways toward dendritic cells (24) or macrophages ap- by polyaromatic hydrocarbons in mice and murine cells. Cancer Res. 46:2735. 20. Ladics, G. S., T. T. Kawabata, A. E. Munson, and K. L. White, Jr. 1992. Me- pear as key targets for PAHs, are also included in the main cell tabolism of benzo(a)pyrene by murine splenic cell types. Toxicol. Appl. Phar- types activating PAHs into carcinogenic metabolites (42). Taken macol. 116:248. 21. Ladics, G. S., T. T. Kawabata, A. E. Munson, and K. L. White, Jr. 1992. Eval- together, these data highlight the probable crucial role of mono- uation of murine splenic cell type metabolism of benzo(a) pyrene and function- cytes in global pathogenesis of PAH toxicity. In addition, it is ality in vitro following repeated in vivo exposure to benzo(a)pyrene. Toxicol. noteworthy that the lowest concentrations of BP active in vitro on Appl. Pharmacol. 116:258. ␮ 22. Braun, K. M., T. Cornish, A. Valm, J. Cundiff, J. L. Pauly, and S. Fan. 1998. monocytes, i.e., 0.5Ð1 M (125Ð250 ng/g), are close to those Immunotoxicology of cigarette smoke condensates: suppression of macrophage found in charcoal-broiled foods (up to 50 ng/g) (43) or in the responsiveness to interferon ␥. Toxicol. Appl. Pharmacol. 149:136. mainstream smoke of a cigarette (20Ð40 ng) (44) that, moreover, 23. Ragg, S. J., G. W. Dandie, G. M. Woods, and H. K. Muller. 1995. Abrogation of afferent lymph dendritic cell function after cutaneously applied chemical carcin- contain additional toxic PAHs (45). These data are consistent with ogens. Cell. Immunol. 162:80. the idea that humans may be exposed to PAH concentrations af- 24. Laupeze, B., L. Amiot, L. Sparfel, E. Le Ferrec, R. Fauchet, and O. Fardel. 2002. Polycyclic aromatic hydrocarbons affect functional differentiation and maturation fecting monocytic cells. of human monocyte-derived dendritic cells. J. Immunol. 168:2652. In summary, our data indicate that exposure to PAHs strongly im- 25. Young, D. A., L. D. Lowe, and S. C. Clarck. 1990. Comparison of the effects pairs differentiation of human monocytes into macrophages. This in- of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture: anal- hibitory effect, which likely involves, at least partly, activation of the ysis of macrophage antibody-dependent cellular cytotoxicity. J. Immunol. 145: AhR, may contribute to the potent immunotoxicity of PAHs. 607. 26. Lecureur, V., D. Lagadic-Gossmann, and O. Fardel. 2002. Potassium antimonyl tartrate induces reactive oxygen species-related apoptosis in human myeloid leu- Acknowledgments kemic HL60 cells. Int. J. Oncol. 20:1071. Downloaded from 27. Siddiqi, M., Z. C. Garcia, D. S. Stein, T. N. Denny, and Z. Spolarics. 2000. We thank Dr. C. Leberre for providing us with blood buffy coats, Dr. B. Relationship between oxidative burst activity and CD11b expression in neutro- Drenou for helpful facilities for flow cytometric immunolabeling experi- phils and monocytes from healthy individuals: effects of race and gender. Cy- ments, and Drs. A. Guillouzo and D. Lagadic for critical reading of the tometry 46:243. manuscript. 28. Chirgwin, J. M., E. A. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribo- nuclease. Biochemistry 18:5294.

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