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Disruption of human plasma cell differentiation by an environmental polycyclic aromatic hydrocarbon:… Allan, Lenka L; Sherr, David H Mar 24, 2010

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RESEARCH Open AccessDisruption of human plasma cell differentiationby an environmental polycyclic aromatichydrocarbon: a mechanistic immunotoxicologicalstudyLenka L Allan1,2, David H Sherr1*AbstractBackground: The AhR is a ligand-activated transcription factor that mediates immunosuppression induced byenvironmental PAH and HAH. Recently, a critical role for the AhR in development of T cells involved inautoimmunity (Th17 and Treg) has been demonstrated, supporting the hypothesis that the AhR plays a key role inimmune regulation both in the presence and absence of environmental ligands. Despite these results with T cellssystems, little is known of the role that the AhR plays in B cell development. We have demonstrated that B cellactivation with CD40 ligand, a stimulus that models adaptive immunity, induces AhR expression in primary humanB cells, suggesting that activation may increase human B cell sensitivity to AhR ligands and that the AhR may playa role in B cell development.Methods: To test these possibilities, we developed an in vitro system in which activated human B cells expressinghigh AhR levels are induced to differentiate into plasma cells. Consequently, the effects of benzo [a]pyrene, aprototypic environmental AhR ligand, on plasma cell differentiation could be investigated and this chemical couldbe exploited essentially as drug probe to implicate the role of the AhR in plasma cell development.Results: A previously unattainable level of B cell differentiation into plasma cells (up to 45% conversion) wasobserved. Benzo [a]pyrene significantly suppressed that differentiation. g-Irradiation after an initial proliferationphase induced by CD40 ligand and immediately prior to initiation of the differentiation phase blocked cell growthbut did not affect cell viability or plasma cell differentiation. B [a]P suppressed differentiation whether or not cellgrowth was inhibited by g-irradiation.Conclusions: 1) Extensive proliferation is not required during the differentiation phase per se for CD40L-activatedhuman B cells to undergo plasma cell differentiation, and 2) an environmental PAH blocks both proliferation anddifferentiation of AhR expressing B cells. The results uncover a new mechanism by which environmentallyubiquitous PAHs may negatively impact human B cell-mediated immunity.BackgroundPolycyclic aromatic hydrocarbons (PAHs) are ubiquitousenvironmental pollutants generated by the incompletecombustion of carbon sources. Numerous studies havedemonstrated that a variety of PAHs are carcinogenicand immunosuppressive. Indeed, carcinogenic PAHs,such as benzo [a]pyrene (B [a]P), suppress both humoral(B cell-mediated) and cellular (T cell-mediated) immuneresponses. Most of these adverse effects are mediated bythe aryl hydrocarbon receptor (AhR), a cytosolic recep-tor/transcription factor [1]. In some cases, lymphocyteimmunotoxicity may be mediated indirectly throughaccessory cells. For example, PAH-induced pre-B cellapoptosis is mediated by AhR-expressing stromal cellsin the bone-marrow microenvironment [2]. Directeffects of other PAHs on transformed B cells also havebeen reported [3]. Similarly, halogenated hydrocarbons* Correspondence: dsherr@bu.edu1Department of Environmental Health, Boston University School of PublicHealth, Boston, MA, 02118, USAAllan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15© 2010 Allan and Sherr; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.impair the ability of thymic stroma to support develop-ing T cell growth and/or differentiation [4-12].Many studies that evaluate the mechanisms by whichPAHs, or other related AhR ligands (e.g. halogenatedaromatic hydrocarbons/HAHs), mediate immunosup-pression have been performed in animal models. Thesestudies reveal that AhR ligands suppress immunity bytheir ability to compromise virtually every stage of lym-phocyte development, activation, and effector functionstudied. In this regard, PAHs and/or HAHs suppressmature B and T cell development in primary lymphoidorgans [4-12] as well as inhibit antibody production,alloantigen-specific mixed-lymphocyte reactions, T cellcytokine production, effector and memory T cell devel-opment, cytotoxic T cell responses, and host resistanceto infectious agents and transplantable tumors [7,13-19].Perhaps most intriguing are recent studies demon-strating that the AhR is intimately involved in the devel-opment of T cell subsets, e.g., regulatory T cells (Treg)and IL-17-secreting Th17 cells, and that activation ofthe AhR in vivo enhances autoimmunity, presumably byup-regulating Th17 development and/or down-regulat-ing Treg development [20-24]. This environmental che-mical alteration in development of T cell subsets helpsexplain the outcomes of pioneering studies of Kerkvlietet al in which AhR activation was shown to suppress Tcell-mediated tumor immunity or allograft rejection[25,26]. These results support our working hypothesisthat the AhR plays a key role in immune regulation andthat environmental AhR ligands can significantly com-promise immunity by altering AhR-dependent lympho-cyte development and/or function.Despite these exciting results with T cells systems, lit-tle is known of the role that the AhR plays in B celldevelopment in general and in human cells in particu-lar. For example, while now classic studies demon-strated that low doses of TCDD (10-9 M) suppressantibody secretion by immmortalized murine B celllines [27,28], little is known of the ability of AhRligands to affect human B cell differentiation intoplasma cells, a critical event in the ultimate productionof protective antibodies. The studies that have been per-formed in this area relied primarily on transformed celllines or mixed lymphocyte populations with which it isdifficult to pinpoint the cell subset directly affected by agiven AhR ligand.To begin to bridge this gap, we developed an in vitromodel to demonstrate that activation of primary humanB cells with CpG or CD40L, stimuli that mimic innateor adaptive B cell responses respectively, dramaticallyincreases AhR expression [29]. These results suggestthat B cell stimulation following pathogen exposure mayincrease B cell sensitivity to environmental AhR ligandsthrough AhR up-regulation. Since CD40L-stimulated, Bcells undergo rapid proliferation in germinal centers fol-lowing antigenic stimulation and then differentiate intoplasma cells, it seemed plausible that AhR ligands couldinterfere with the growth of what is presumed to be Bcells expressing high AhR levels ("AhRhigh“) and/or theirdifferentiation into plasma cells. The former possibilitywas confirmed by the demonstration that B [a]P inhibitsproliferation of CD40L-activated primary human B cells[29]. To test the latter possibility, we defined conditionsunder which purified human B cells could be induced toundergo differentiation in vitro and then evaluated theeffects of B [a]P, a prototypical environmental PAH andAhR ligand, on that process. The results demonstratethat, while proliferation may be required for B cells toreach a state at which they are capable of differentiatinginto plasma cells, ongoing B cell proliferation is notrequired during the differentiation process itself.Furthermore, results suggest that an environmentalPAH acts directly on AhRhigh human B cells to suppresstheir differentiation into plasma cells, and that differentclasses of AhR ligands differentially affect biologicoutcomes.MethodsChemicalsB [a]P (Sigma, St. Louis, MO) was dissolved in dimethyl-sulfoxide (DMSO) (Sigma). Cells were dosed from a1000× stock so that the final concentration was 0.1%.Cell cultureCD40L-transfected L cells (American Type Tissue Cul-ture Collection, VA) were maintained at 37°C in 10%CO2 in RPMI supplemented with 10% FBS, 2 mM L-glutamine, 5 μg/ml Plasmocin (Invivogen, San Diego,CA) and hypoxanthine thymidine (HT). Unless other-wise indicated, all culture reagents were obtained fromCellgro (Mediatech, Herndon, VA).B cell preparationPeripheral blood mononuclear cells (PBMC) were pre-pared from anonymous individual blood donors (NewYork Biologics, Inc., New Jersey, NY) by centrifugationof 50-100 ml whole blood through Ficoll (AmershamBiosciences, Uppsala, Sweden) as previously described[29]. PBMCs were depleted of T cells by sheep redblood cell (ICN Biomedicals, Aurora, OH) rosetting anda second centrifugation through Ficoll. PBMCs werestained with FITC-labeled CD20-specific antibody (BDPharMingen (Chicago, IL) and purified by fluorescence-activated cell sorting (MoFlo, Dako Cytomation) basedon forward and side scatter parameters and CD20expression. Approximately 107 B cells (>99% CD20+)were recovered per donor from approximately 108PBMCs.Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 2 of 12Plasma cell generation (two step cell culture)In the first stage of plasma cell generation, purified Bcells were plated at approximately 3 × 106 cells/well in6-well plates on irradiated CD40L-transfected L cells inB cell media (Iscove’s medium; Invitrogen, Carlsbad,CA) supplemented with 5% human AB serum (MP Bio-medicals), 50 μg/ml transferrin (Invitrogen), 0.5%human serum albumin (Aventis Behring, Kanakakee,IL), 5 μg/ml insulin (Sigma), and 25 μg/ml Plasmocinplus the following cytokines: rIL-2 (50 U/ml), rIL-4 (50ng/ml), rIL-10 (50 ng/ml) and rIL-12 (2 ng/ml). After 4days of culture (step 2), B cells were harvested, washedand seeded in a single well (24 well plate) at 4 × 105/mlmedia containing rIL-2 (50 U/ml), rIL-6 (25 ng/ml), rIL-10 (50 ng/ml), rIL-12 (2 ng/ml) and rIFN-a (100 U/ml)in the absence of CD40L-transfected L cells. All cyto-kines were obtained from Research Diagnostics Inc.(RDI, Flanders, NJ). The cell recovery after the second 4day culture was over 2 × 106 cells/well, representingapproximately a 6-fold increase in cell numbers duringthe second culture.Cells were treated with vehicle (DMSO) or 10-6 M B[a]P dissolved in DMSO on the first day of cultureand/or on day 4 at the start of the second culture step.For all studies, cells were washed extensively after thefirst culture and the same number of viable cells,4 × 105, was added to each well in the second (differen-tiation) culture. In studies evaluating the requirementfor cell proliferation during plasma cell differentiation,activated B cells were g-irradiated (700 Rads) on day 4prior to the initiation of the second culture. Whereindicated, 30 μM ZVAD-fmk (Biomol International,Plymouth Meeting, PA) was added during the secondculture step to maximize cell viability. Approximately4 × 105 cells/ml were recovered in vehicle-treated wellsfollowing cell irradiation with or without ZVAD-fmksupport.Surface phenotype and viability of cultured B andplasma cellsCells were harvested on day 8 of culture and viabilitywas determined by trypan blue and propidium iodideexclusion by light microscopy and flow cytometry,respectively. Phenotypic analyses of B and plasma cellswere performed using FITC-conjugated anti-CD20, PE-conjugated anti-CD38 antibody (BD PharMingen (Chi-cago, IL) or fluorochrome-labeled isotype controls. Non-specific mAb binding was blocked by incubating cellsfor 10 min in PBS containing 5% FBS, 1% sodium azideand 0.01 mg/ml normal mouse IgG (Caltag Laboratories,Burlingame, CA). Cells then were labeled with mAb orisotype matched control antibodies according to themanufacturer’s instructions. Following one wash, cellswere fixed in PBS containing 3.7% paraformaldehydeand analyzed in a Becton Dickinson FACScan flow cyt-ometer using CellQuest software (BD Biosciences).Quadrants were set using dot plots obtained with iso-type controls.Giemsa stainFollowing the second culture, sorted CD20lo/CD38hicells were resuspended in PBS (70,000 cells/100 μl) andcytocentrifuged (Thermo Shandon, Thermo ElectronCorporation, Pittsburg, PA) for 5 minutes at 400 rpmonto pre-cleaned Colorfrost/Plus microscope slides(Fisher Scientific). Slides were immediately fixed with90% ethanol for 1 minute and allowed to dry beforestaining for 30 minutes with 1:4 Giemsa stain/deionizedwater solution (Sigma). Excess stain was removed fromslides by dipping in water, followed by dipping in 0.01%acetic acid. Slides were dehydrated by dipping into 95%ethanol and 100% ethanol, placed into xylene andmounted with Permount (Fisher Scientific) and a cover-slip. Images were digitally captured using DIC optics(Nikon).Proliferation assaysCD40L-activated B cells were plated at a density of 105cells/well into 96-well plates for 20 hours. [3H]-thymi-dine (1 μCi/well) was added and plates were incubatedfor an additional 18 hours. Cells were harvested onto fil-ter strips using a cell harvester (Brandel, Gaithersbug,MD) and radionucleotide incorporation was measuredusing a liquid scintillation counter (Wallac, Turku, Fin-land). For each donor, B cell treatments were performedin triplicate. The means of the triplicate radioactivitycounts per minute (cpm) from each donor were used toobtain an average for each indicated data point.Apoptosis assayPrimary cultured B cells were harvested and washedonce with cold PBS containing 5% FBS and 0.01 Msodium azide (Sigma). For propidium iodide (PI) stain-ing, cells were resuspended in 0.15 ml hypotonic buffercontaining 50 μg/ml PI (Sigma), 0.1% sodium citrateand 0.1% Triton X-100 and analyzed by flow cytometryas we previously described [1,29-31]. Cells undergoingDNA fragmentation (i.e. apoptosis) have a weaker PIfluorescence than those in the typical G0/G1 stages ofcell cycle [31].Statistical AnalysesThe Student’s paired t-test and one-factor ANOVA wereused to analyze the data using Statview (SAS Institute,Cary, NC). For ANOVA, the Dunnett’s or the Tukey/Kramer multiple comparisons tests were used to com-pare experimental and vehicle-treated groups or to com-pare all possible combinations of groups respectively.Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 3 of 12ResultsGeneration of plasma cells using two-step cell culturesIn order to address whether environmental AhR ligandsaffect the differentiation of human B cells into plasmacells, it was necessary first to develop an in vitro differen-tiation system in which significant numbers of humanplasma cells could be reliably generated from activated,AhRhigh B cells. To this end, modifications were made topreviously published protocols in which activation ofhuman B cells was shown to significantly up-regulateAhR expression [32,33]. In the first phase of the protocol,CD20+ human B cells, purified from PBMC by fluores-cence-activated cell sorting, were activated by growth onmonolayers of human CD40L-transfected L cells for 4days in the presence of rIL-2, rIL-4, rIL-10 and rIL-12.Robust B cell proliferation was observed during thefirst culture period with B cell numbers increasingapproximately 8-fold [29]. Significant up-regulation ofAhR expression occurred within 24 hours of CD40Lactivation and remained high for several weeks [1,29].After this first culture, the cells expressed high levels ofCD20 and low levels of the plasma cell/activated lym-phocyte marker CD38 (Figure 1A, center dot plot).Activated CD20hi/CD38lo B cells then were culturedfor an additional 4 days with rIL-2, rIL-10, rIL-12, rIL-6,and rIFN-a but without rIL-4 or CD40L-transfectedcells. Although stimulation through CD40 ceased afterthe first culture of our system, B cell continued to divideduring a second culture period. In control cultures fromindividual experiments using cells from five donors, thenumber of cells harvested at the end of the eight dayculture period (2.6 × 106 ± 0.1 cells/well; Figure 2D,first histogram) was approximately 6-fold higher thanthe number plated on day four, demonstrating a signifi-cant level of proliferation during the second culture.(Note that the number of cells entering the secondculture was always adjusted to a constant number,i.e., 4.0 × 105 cells/well). Following this second cultureCD40-B cells(day 4)PBMC(day 0)Differentiated PC(day 8)CD20C D 3 8GiemsaCD38ABFigure 1 Differentiation of peripheral human B cells into plasma cells. CD20hi B cells were purified by fluorescence-activated cell sortingand activated for 4 days on CD40L-expressing cells in the presence of rIL-2, rIL-4, rIL-10, and rIL-12. Activated B cells then were adjusted to 4.0 ×105 cells/ml and cultured for an additional 4 days without CD40L cells but with rIL-2, rIL-6, rIL-10, rIL-12 and rIFN-a. A) The phenotype of freshPBMCs prior to sorting, and of B lineage cells obtained after the first and second cultures was determined by flow cytometry following stainingwith CD20- and CD38-specific or isotype control monoclonal antibodies. Quadrants were set using isotype control antibodies. CD20high/CD38lowstarting B cells and CD20lo/CD38hi plasma cells are indicated by the octagons in the leftmost and rightmost dot plots respectively. B) CD20lo/CD38hi cells generated after the second culture were sorted by flow cytometry, cytocentrifuged onto glass slides, and treated with Giemsa stainto visualize plasma cell morphology.Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 4 of 12step (culture day 8), 31 ± 17% (mean ± SE; n = 4donors) of the cells expressed the phenotypic character-istics of plasma cells, i.e. low CD20 and high CD38expression (Figure 1A, right dot plot).Although many studies validate this phenotype as ahallmark of human antibody secreting plasma cells[32,33], CD20lo/CD38hi cells induced in our two stepculture were sorted by flow cytometry to >95% purityand stained with Giemsa to visualize cell morphologyand to formally confirm their identity as plasma cells.As expected, these cells displayed morphologic charac-teristics of plasma cells, namely a basophilic cytoplasm,an accentric nucleus, and a pale Golgi zone (Figure 1B).It was concluded, therefore, that the 2-step culture wassuitable for analysis of plasma cell development in thepresence of environmental chemicals.B [a]P suppresses production of plasma cellsTo determine the effect of B [a]P exposure on the pro-duction of plasma cells, B cells were treated with vehicleCD20C D 3 840.3% 42.1%VEH/VEH VEH/B[a]PB[a]P/VEH B[a]P/ B[a]P46.0% 29.8%A.CD38B.C.D. Cells/Well (x 10 6 )***CD20lo/CD38hiCells/Well (x 10 6)VEH/VEHVEH/B[a]PB[a]P/VEHB[a]P/ B[a]P0.*CD20lo/CD38hi (%)0102030405060708090100Figure 2 B [a]P suppresses plasma cell production. Purified CD20+ B cells from 4 donors were individually cultured in two steps as in Figure1 to generate plasma cells. Cultures were treated with vehicle or 10-6 M B [a]P during the first (B [a]P/VEH), the second (VEH/B [a]P), or both (B[a]P/B [a]P) culture periods. A) Dot plots of cells obtained from a representative donor stained with CD20- and CD38-specific or isotype controlantibodies are shown. Quadrants were set using isotype control antibodies. The percentages of CD20lo/CD38hi plasma cells are indicated in theupper left quadrants. B) The percentages of CD20lo/CD38hi plasma cells obtained after the second culture period when cells were exposed tovehicle or B [a]P during the first, second, or both cultures are presented as means + SE. C) The total number of CD20lo/CD38hi plasma cellsobtained after both cultures (day 8) are presented as means + SE. D) The total number of cells obtained after both cultures are presented asmean + SE. An asterisk (*) indicates a significant decrease relative to vehicle-treated cells (p < 0.05; Dunnett’s).Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 5 of 12or 10-6 M B [a]P during the first and/or second culturestep and the number of CD20lo/CD38hi plasma cells wasassessed. Although individual experiments occasionallysuggested a decrease in the percentage of CD20lo/CD38hi plasma cells after treatment with B [a]P duringboth cultures (e.g., Figure 2A, lower right dot plot), nosignificant differences were observed overall in the per-centage of plasma cells generated in cultures exposed toB [a]P during any point of the cultures (Figure 2B).Treatment with B [a]P during either the first or the sec-ond culture tended to decrease the absolute number ofplasma cells recovered, although statistical significance(p < 0.05) was not reached unless B [a]P was present inboth cultures (Figure 2C). The apparent decrease inplasma cell yield following B [a]P exposure coincidedwith a significant decrease in the total number of cellsin cultures treated during the first, second, or both cul-tures (Figure 2D). B [a]P did not induce significantlevels of overt death or apoptosis, as assayed by trypanblue exclusion and propidium iodide staining and flowcytometry, under any of the conditions shown in Figure2 (~7% apoptotic in both vehicle and B [a]P-treated cul-tures). These experiments indicate that B [a]P signifi-cantly reduces the total number of B cells andcompromises the production of plasma cells in thismodel. However, they do not distinguish between B [a]P-mediated effects on B cell proliferation during the sec-ond, differentiation culture, from proliferation-indepen-dent effects on B cell differentiation into plasma cells.B [a]P suppresses plasma cell differentiation duringconditions of minimal proliferationIt has been proposed that differentiation of B cells intoplasma cells requires cell proliferation, at least during aninitial stage of activation during which activated B cellsbecome differentiation-competent [34-39]. As notedabove, CD40L-activated B cells proliferate during boththe initial activation culture and the subsequent differ-entiation culture. Previously, we demonstrated that B [a]P inhibits proliferation of CD40L-activated B cells[34-39]. Consequently, the reduced recovery of plasmacells in B [a]P-treated cultures could reflect a reductionin cell proliferation required to generate differentiation-competent cells during the first or second culture and/or inhibition of differentiation itself in the second cul-ture. To develop a model in which the effects of B [a]Pon B cell proliferation could be dissociated from itsputative effects on differentiation, purified human Bcells were activated for the first four days with CD40ligand with cytokines and then g-irradiated (700 Rads)to block further cell proliferation.As expected, little or no growth was observed in theirradiated B cells, as indicated by the minimal level of[3H]-thymidine incorporation during an 18 hour periodfollowing irradiation (Figure 3A). When irradiated Bcells were re-cultured under conditions that induceplasma cell differentiation, 41.8 ± 3.2% (mean ± SE, n =4 donors) of the cells differentiated into CD20lo/CD38hiplasma cells (e.g. Figure 3B). This level of plasma celldifferentiation was comparable to that seen in non-irra-diated cultures (Figure 2). These results demonstrateFigure 3 g-Irradiation inhibits cell growth but has no effect onB cell differentiation into plasma cells. Purified CD20+ B cellswere activated for 4 days as described in Figure 1. Activated B cellswere then g-irradiated (700 Rads) and cultured without CD40L-transfected cells but with IL-2, IL-6, IL-10, IL-12 and IFN-a. A) [3H]-thymidine was added to cultures of irradiated and control cells at21 hours and [3H]-thymidine incorporation assayed 18 hours later.Data from 3 donors are presented as mean cpm ± SE. An asteriskindicates a significant decrease in [3H]-thymidine incorporation ascompared with corresponding non-irradiated controls (p < 0.05;paired t-test). B) Cells cultured as in “A” above were phenotyped forCD20 and CD38 expression by flow cytometry 4 days afterirradiation and culture under plasma cell differentiation conditions.Dot plots obtained with cells from a representative donor areshown. The percentage of CD20lo/CD38hi plasma cells is indicatedin the upper left quadrant.Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 6 of 12that inhibition of cell division during the second stageculture has no discernable effect on differentiation, aresult consistent with the hypothesis that, after an initialproliferative phase, differentiation can occur in theabsence of continual cell proliferation [33,40-43].Given these results, it was possible to determine theputative effects of B [a]P exposure on plasma cell differ-entiation during the second culture under conditions ofminimal cellular proliferation. Treatment of activated,irradiated human B cells with B [a]P only during the dif-ferentiation phase (i.e., the second culture) significantlyreduced the percentage (Figure 4A and 4B: VEH/B [a]P)and the absolute number (not shown) of CD20lo/CD38hiplasma cells recovered. These results strongly suggestthat B [a]P inhibits the process of plasma cell differen-tiation independent of its ability to suppress prolifera-tion of activated B cells.Similarly, treatment with B [a]P in the first culturestep, during which extensive proliferation takes place,resulted in a significant reduction in the number (notshown) and percentage of plasma cells recovered (Figure4A and 4B: B [a]P/VEH). B [a]P treatment during bothcultures (B [a]P/B [a]P) resulted in the greatest decreasein the percentage of plasma cells recovered, a resultconsistent with an additive effect of B [a]P on the gen-eration/proliferation of differentiation-competent B cellsin the first culture and on their ability to differentiateinto plasma cells in the second culture.Irradiated cells treated with vehicle or B [a]P appearedas healthy as non-irradiated cells at the end of cultureas assessed by trypan blue and propidium iodide stain-ing (~7% apoptotic). Nevertheless, it seemed formallypossible that the apparent block by B [a]P of B cell dif-ferentiation could in part reflect preferential death ofshort-lived plasma cells [36] upon B [a]P exposure. Toaddress this possibility, a separate series of experimentswas performed in which a relatively high dose (30 μM)of a potent pan-caspase inhibitor, ZVAD-fmk [31], wasadded to the second culture to minimize B/plasma cellapoptosis. We have shown that >15 μM ZVAD-fmk orother caspase inhibitor efficiently blocks PAH-inducedapoptosis of bone marrow B cell cells in vitro [31].Apoptosis was measured by permeabilizing cells andstaining DNA with propidium iodide, as described pre-viously [31]. A relatively small population of cells (4.6 +0.89%) was undergoing apoptosis following vehicle treat-ment (Table 1). No significant differences in the percen-tage of apoptotic cells were observed when cells weretreated with B [a]P (Table 1). Nevertheless, B [a]P,added either at the beginning of the first culture periodor during the differentiation stage, significantlydecreased the percentage of CD20lo/CD38hi plasma cellsrecovered (Figure 5A, B). These results are equivalent tothose obtained with irradiated cells in the absence ofZVAD-fmk (Figure 4). Therefore, it is concluded thatthe decrease in plasma cell recovery seen after B [a]Ptreatment of the second, differentiation culture resultsprimarily from a block in plasma cell differentiation andnot from inhibition of growth or from overt toxicitymanifest as preferential plasma cell death.DiscussionThe adverse effects of PAH exposure on B cell-depen-dent, immune responses have been well documented[44-46]. While many studies performed in animal mod-els demonstrated reduced immunoglobulin levels follow-ing in vivo or in vitro treatment with PAHs such as B[a]P or DMBA, the exact cellular mechanisms, e.g., inhi-bition of antigen presentation, reduction in T helperactivity, or inhibition of antibody secretion, was gener-ally not investigated and indeed could not be evaluatedin the context of mixed lymphocyte populations. A sig-nificant advance came with the demonstration that anAhR ligand suppresses LPS-induced antibody secretionin a transformed murine B cell line in vitro [47,48].While these studies demonstrated a mechanism throughwhich an activated AhR could suppress immunoglobulingene transcription, they did not address the effect ofAhR ligands on the process of differentiation per sefrom activated B cell to plasma cell. The present studyis the first to address specifically this issue and toextend studies to nontransformed, highly enriched pri-mary human cells.In addition, the current studies were motivated by ourfinding that human B cells, activated through CD40L asa surrogate for antigen-stimulated CD40L+ T helpercells, up-regulate AhR expression within 24 hours byapproximately 5-fold [29]. In addition to suggesting thepossibility that the AhR plays an important role in Bcell function, these results led us to investigate whetheractivated B cells undergo normal levels of proliferationand differentiation in the presence of AhR ligands.Treatment of purified human B cells with B [a]P dur-ing either the first or second stage of culture resulted ina significant decrease in the total number of cells recov-ered (Figure 2C and 2D). This result alone indicates thatsuppression of humoral immunity may be mediated bydirect effects of PAHs on activated AhRhigh B cells. Themaximal decrease in the number of plasma cellsobserved after B [a]P exposure during the entire cultureperiod suggests an additive effect resulting from B [a]Pinhibition of both B cell proliferation in the first cultureand differentiation in the second.In order to distinguish between the effects of B [a]P onB cell proliferation from additional effects on differentia-tion, we developed a system in which plasma cell differ-entiation could be studied under conditions of minimalcell proliferation. To this end, cells were g-irradiatedAllan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 7 of 12Figure 4 B [a]P suppresses plasma cell differentiation under conditions of minimal proliferation. Purified B cells were activated for 4 daysas described in Figure 1. B cells then were g-irradiated (700 Rads) and re-cultured for an additional 4 days under plasma cell differentiationconditions. Cells were treated with vehicle or 10-6 M B [a]P during the first and/or second culture periods. A) Cells were phenotyped for CD20and CD38 expression by flow cytometry. Dot plots obtained with cells from a representative donor (4 total) are shown. The percentages ofCD20lo/CD38hi plasma cells are indicated in the upper left quadrants. B) Data from cells obtained from four donors and treated as in “A” arepresented as mean percentage of CD20lo/CD38hi cells + SE. An asterisk (*) indicates a significant decrease relative to vehicle-treated cells(p < 0.05; Dunnett’s).Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 8 of 12prior to the differentiation (second) culture. Prior to thisstage, activated B cells do not express any markers char-acteristic of plasma cells. Radiation (700 R) significantlyreduced proliferation without inducing apoptosis, asmeasured by propidium iodide staining or overt death asmeasured by trypan blue exclusion. Notably, the greaterthan 90% decrease in cell proliferation during the criticalstage of differentiation, i.e., the second culture, had noeffect on plasma cell differentiation. This importantresult extends previous reports in which it was concludedthat extensive proliferation of B cells is required prior todifferentiation into plasma cells [34-36,39]. In fact, thedata presented herein indicate that, while CD40L+ T cell-dependent proliferation of B cells prior to a differentia-tion signal may be required to generate “differentiation-competent” cells, little or no cell division is required dur-ing the differentiation phase itself [40-43,49].To evaluate the effects of B [a]P on differentiation perse, B cells were irradiated at the beginning of the secondculture to inhibit proliferation and then exposed to vehi-cle or B [a]P. B [a]P reduced the number (not shown)and percentage (Figure 4) of CD20lo/CD38hi plasma cellssignificantly, thereby demonstrating for the first time thatthis prototypic PAH directly inhibits differentiation of Bcells into plasma cells. It also was interesting to note thattreatment of B cells during the first culture only, andprior to irradiation and differentiation, also decreased thepercentage of plasma cells recovered despite the fact thatcells were washed extensively to remove B [a]P and thatcell numbers were adjusted to a standard level (4 × 105cells/well) for the second culture. These results are con-sistent with the hypothesis that, by suppressing cell divi-sion, B [a]P exposure reduces the number of B cells thatare competent to undergo differentiation, thereby nega-tively impacting the production of plasma cells.While the mechanism underlying the block in B celldifferentiation into plasma cells is not known, severalpossibilities exist. Pax5 is a transcription factor that pro-motes transcription of B cell lineage genes whilerepressing J-chain, IgH and XBP-1 genes, expression ofwhich is necessary for plasma cell differentiation [49].Notably, the murine and human Pax5 promoters containthree AhR response elements (AhREs), affording a directtranscriptional mechanism through which the AhRcould up-regulate the expression of Pax5 and suppressgenes required for plasma cell differentiation. In supportof this possibility, at least one AhR ligand, TCDD, pre-vents an LPS-mediated decrease in Pax5 expression andsuppresses antibody secretion in a transformed murineB cell line, CH12.LX [50].A second mechanism through which AhR activationmay alter the expression of genes required for plasmacell differentiation is through the modulation of Pax5transcriptional activity. The Pax5 DNA recognitionsequence (G/ANNCANTGNNGCGT/GG/AACC/GA/G)contains a consensus AhR ‘core’ binding site (bold) [51].In fact, AhR found in nuclear extracts prepared fromtwo human B cell lines recognizes a consensus Pax5binding site in the CD19 promoter [52]. Therefore, it ispossible that B [a]P augments repression of genesrequired for plasma cell differentiation, e.g. XBP-1, byvirtue of their expression of Pax5/AhR binding sites.The ability of B [a]P to suppresses plasma cell differen-tiation in the system described herein could reflect theability of B cells to metabolize B [a]P. In this vein, we haveshown that CYP1A1-dependent B [a]P metabolism into B[a]P-7,8 dihydrodiol-9,10-epoxide is required for suppres-sion of CD40L-activated B cell proliferation [1]. Further-more, B [a]P can be metabolized by cellular peroxidases toform B [a]P-quinones. These metabolites are convertedinto reactive oxygen species that induce oxidative stress.Cellular redox potential controls the DNA binding activityof several transcription factors, including NF-B, AP-1,and p53 [53-57]. In addition, oxidative stress in human Bcells induces expression of a multifunctional enzymeknown as APE/Ref-1 (or HAP-1)[58]. APE/Ref-1 modu-lates the DNA binding activity of transcription factorssuch as Pax5 by reducing cysteine residues [59-62]. Whileoxidized Pax5 is unable to bind DNA, reduced Pax5 bindsDNA regulatory sites with high affinity [58,61,62]. Thus,oxidative stress induced by B [a]P-quinones or B [a]P-epoxides may activate APE/Ref-1, which in turn promotesPax5 transcriptional activity suppressing XBP-1 expressionand plasma cell differentiation.Regardless of the mechanism(s) through which B [a]Pinhibits plasma cell differentiation, the data presentedhere demonstrate a second level, in addition to inhibi-tion of B cell growth [1], through which this prototypic,environmental PAH is likely to suppress B cellresponses. Collectively, the data support the hypothesisthat up-regulation of AhR expression in activatedhuman B cells renders them particularly susceptible toimmunosuppressive PAHs.Table 1 B [a]P does not induce apoptosis duringdifferentiation of irradiated B cells to plasma cellsTreatment % Apoptotic CellsVehicle/Vehicle 4.6 ± 0.9Vehicle/B [a]P 6.5 ± 1.1B [a]P/Vehicle 6.4 ± 0.8Cells were activated with CD40L + rIL-2, rIL-4, rIL-10, and rIL-12 with vehicle or10-6 M B [a]P for 4 days as described in Materials and Methods. Cells wereharvested, washed, irradiated (700 Rads) and re-plated at a concentration of4 × 105/ml in media containing rIL-2, rIL-6, rIL-10, rIL-12, and rIFN-a in theabsence of CD40L-transfected L cells and in the presence of vehicle or B [a]P.The percentage of cells undergoing apoptosis on day 8 of culture wasquantified by propidium iodide staining of permeabilized cells followed byflow cytometric analyses. Data are derived from samples shown in Figure 4and are presented as means ± SE of cells falling into the sub G0/G1 peak aswe previously described [30,31].Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 9 of 12Figure 5 Preferential plasma cell apoptosis does not account for a decrease in the percentage of plasma cells in B [a]P-treatedcultures. Purified B cells were activated for 4 days as described in Figure 1. B cells then were g-irradiated and re-cultured in the presence of thepan-caspase inhibitor ZVAD-fmk (30 μM) for an additional 4 days. Cells were treated with vehicle or 10-6 M B [a]P during the first and/or secondculture periods. A) Cells were phenotyped for CD20 and CD38 expression by flow cytometry. Dot plots obtained with cells from a representativedonor (4 total) are shown. The percentages of CD20lo/CD38hi plasma cells are indicated in the upper left quadrants. B) The percentages ofplasma cells (4 donors) obtained 4 days after irradiation (day 8) and after treatment with vehicle or 10-6 M B [a]P in the presence or absence of30 μM ZVAD-fmk are presented as means ± SE. An asterisk indicates a significant decrease in the percentage of plasma cells relative to control,vehicle-treated cells (p < 0.05; Dunnett’s test).Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 10 of 12ConclusionsUsing a novel human B cell differentiation system it waspossible to evaluate the ability of environmental AhRligands to directly suppress human B cell differentiationinto plasma cells in the absence of other lymphoid cellpopulations. From these studies it is concluded that atleast some AhR ligands are capable of suppressing thedifferentiation of activated human B cells into plasmacells without inducing cell death. Inhibition of differen-tiation with a prototypic PAH was seen even in theabsence of B cell proliferation or significant levels of celldeath, suggesting that a specific AhR-regulated signalingpathway(s) is impacted by PAH exposure. Taken withour previous studies showing up-regulation of AhRexpression and activity in activated human B cells [29],these studies support the hypothesis that activated Bcells, which express high levels of the AhR, are particu-larly susceptible to PAH exposure and that the AhRmay play an important role in differentiation from acti-vated B cell to plasma cell. These results suggest amechanistic underpinning for epidemiological studies inwhich exposure to environmental chemicals, includingmetabolizable AhR ligands, results in reduced antibodyresponses [63].AbbreviationsAhR: Aryl Hydrocarbon Receptor; B [a]P: Benzo [a]pyrene; HAH: HalogenatedAromatic Hydrocarbon(s); PAH: Polycyclic Aromatic Hydrocarbon(s).AcknowledgementsThe authors thank Dr. Douglas Allan for assistance with DIC optics. This workwas supported by NIEHS Grants P42 ES07381, RO1 ES06086 and PO1ES11624.Author details1Department of Environmental Health, Boston University School of PublicHealth, Boston, MA, 02118, USA. 2Department of Pathology and LaboratoryMedicine, University of British Columbia, Vancouver, British Columbia,Canada.Authors’ contributionsLA and DS jointly conceived of the project, compiled and analyzed the data,and co-wrote the manuscript. LA developed the plasma cell differentiationassay and performed the experiments, data from which are presentedherein. All authors have read and given final approval of the version to bepublished.Competing interestsThe authors declare that they have no competing interests.Received: 2 September 2009 Accepted: 24 March 2010Published: 24 March 2010References1. Allan LL, Schlezinger JJ, Shansab M, Sherr DH: CYP1A1 in polycyclicaromatic hydrocarbon-induced B lymphocyte growth suppression.Biochem Biophys Res Commun 2006, 342:227-235.2. 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Heilmann C, Grandjean P, Weihe P, Nielsen F, Budtz-Jorgensen E: Reducedantibody responses to vaccinations in children exposed topolychlorinated biphenyls. PLoS Med 2006, 3:e311.doi:10.1186/1476-069X-9-15Cite this article as: Allan and Sherr: Disruption of human plasma celldifferentiation by an environmental polycyclic aromatic hydrocarbon: amechanistic immunotoxicological study. Environmental Health 2010 9:15.Allan and Sherr Environmental Health 2010, 9:15http://www.ehjournal.net/content/9/1/15Page 12 of 12


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