Bone Marrow Stromal-B Cell Interactions in Polycyclic Aromatic Hydrocarbon-Induced Pro/Pre-B Cell Apoptosis

Lenka L. Allan*,{dagger}, Koren K. Mann{ddagger}, Raymond A. Matulka§, Heui-Young Ryu*, Jennifer J. Schlezinger* and David H. Sherr*,1

* Department of Environmental Health, {dagger} Department of Microbiology, Boston University Schools of Medicine and Public Health, Boston, Massachusetts; {ddagger} Lady Davis Institute for Medical Research, Montreal, Canada; and § The Burdock Group, Vero Beach, Florida

Received July 24, 2003; accepted September 5, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Environmental polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons are immunotoxic in a variety of systems. In a model system of B lymphopoiesis, PAH exposure rapidly induces apoptosis in CD43- pre-B and CD43+ pro/pre-B cells. Apoptosis induction by 7,12-dimethylbenzo[a]anthracene (DMBA) is dependent upon AhR+ bone marrow stromal cells and likely involves DMBA metabolism within the stromal cell. However, it is not known if PAH-treated stromal cells release free metabolites or soluble factors that may directly induce B cell death or if the effector death signal is delivered by stromal cell-B cell contact. Here, we demonstrate that supernatants from DMBA-treated bone marrow stromal cells contain an activity capable of inducing apoptosis in pro/pre-B cells cocultured with stromal cells. This activity (1) is not produced when stromal cells are cotreated with DMBA and {alpha}-naphthoflavone ({alpha}-NF), an aryl hydrocarbon receptor (AhR) and cytochrome P-450 inhibitor, (2) is >= 50 kDa, (3) is trypsin and heat sensitive, and (4) is dependent on AhR+ stromal cells, which in turn deliver the effector death signal to pro/pre-B cells. The results (1) argue against a role for a soluble, stromal cell-derived cytokine as the effector of PAH-induced pro/pre-B cell death, (2) exclude the possibility of a free metabolite acting directly on AhR- pro/pre-B cell targets, and (3) suggest the elaboration by stromal cells of a relatively stable, DMBA metabolite-protein complex capable of acting on other stromal cells at some distance. Collectively, these studies suggest that, while stromal cell products, e.g., metabolite-protein complexes, may affect the function of distant stromal cells, the effector death signal delivered by stromal cells to bone marrow B cells is mediated by cell–cell contact.

Key Words: polycyclic aromatic hydrocarbon; B cell apoptosis; stromal cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons (HAH) are both carcinogenic and immunosuppressive. For example, the prototypic PAHs benzo[a]pyrene (B[a]P) and 7,12 dimethlybenz[a]anthracene (DMBA) induce a variety of tumors in animals (Rigdon and Neal, 1969Go) and compromise the immune system by suppressing cytokine production, B and T cell mitogen responses, tumor-specific CD8+ T cell induction, and B cell antibody production (Burchiel et al., 1992Go, 1993Go; Davilla et al., 1996Go; Thurmond et al., 1988Go; White et al., 1985Go; Wojdani et al., 1984Go).

The apparent correlation between PAH carcinogenicity and immunotoxicity suggests that overlapping signaling pathways may mediate these divergent biologic outcomes. Two elements in the pathway to malignant transformation are the aryl hydrocarbon receptor (AhR), which transforms into a transcription factor when bound by ligand, and PAH metabolites, production of which is facilitated by AhR-regulated cytochrome P-450 monooxygenases (Christou et al., 1987Go; Uno et al., 2001Go). There is a considerable body of evidence supporting the hypothesis that the AhR influences PAH-mediated immunosuppression (Dertinger et al., 2001Go; Kerkvliet, 1995Go; Laiosa et al., 2002Go; Lawrence et al., 1996Go; Mann et al., 1999Go; Near et al., 1999Go; Staples et al., 1998Go; Thurmond et al., 2000Go; Vorderstrasse and Kerkvliet, 2001Go; Yamaguchi et al., 1997aGo,bGo), as well as PAH-induced malignant transformation (Poland et al., 1974Go; Safe and Krishan, 1995Go). Similarly, PAH metabolites are both highly carcinogenic and immunosuppressive. Indeed, some PAH metabolites suppress antigen-specific T and B cell responses or compromise lymphocyte development at doses lower than those required to induce immunosuppression with the respective parent compounds (Davilla et al., 1996Go; Kawabata and White, 1987Go; Ladics et al., 1991Go; Mann et al., 1999Go; Thurmond et al., 1988Go).

Of particular concern for environmental exposures is the extreme sensitivity of the developing immune system to PAHs and HAHs (Holladay and Smith, 1995Go; Lai et al., 2000Go; Urso and Johnson, 1988Go). In a series of studies using an in vitro model of B lymphopoiesis, our laboratory demonstrated that PAHs (B[a]P and DMBA) rapidly induce apoptosis in primary pre-B cells or in a nontransformed CD43+ pro/pre-B cell line (BU-11) at doses lower than those required for cell transformation (i.e., >= 10-8 M) (Hardin et al., 1992Go; Mann et al., 1999Go, 2001Go; Near et al., 1999Go; Quadri et al., 2000Go; Yamaguchi et al., 1997aGo,bGo). However, PAH toxicity in this system is not due to direct effects of PAH on the B cells, which do not express the AhR, but rather is mediated indirectly through PAH-treated AhR+ bone marrow stromal cells. The ability of common environmental pollutants such as B[a]P to effect changes in bone marrow stromal cell activity has implications for multiple hematopoietic cell types, since the growth and/or development of all eight hematopoietic lineages is dependent on these stromal elements.

Induction of the stromal cell-derived, pro/pre-B cell-directed death signal with relatively low PAH doses (i.e., 10-6–10-8 M) is blocked by AhR inhibitors (Quadri et al., 2000Go; Yamaguchi et al., 1997bGo) and requires that the stromal cells express a functional AhR (Mann et al., 1999Go; Near et al., 1999Go). Furthermore, there appears to be some role for PAH metabolism in the induction of the death signal, in that poorly metabolized AhR ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related PCBs do not induce apoptosis while a DMBA metabolite, DMBA-3,4-dihydrodiol, is a potent inducer of apoptosis. Furthermore, a role for a stromal cell monooxygenase, CYP1B1, in induction of apoptosis in the transformed pre-B cell line, 70Z3, has been demonstrated (Heidel et al., 1999Go, 2000Go). Again, these results suggest a similarity between PAH carcinogenicity and immunotoxicity.

The nature of the death signal that PAH-treated stromal cells deliver to pre- or pro/pre-B cells is not known. Work in our laboratory suggests that it is not mediated by Fas ligand, TNF-{alpha}, TNF-ß, lymphotoxin ß, TGF-ß1, ß2, or ß3, glucocorticoids, or other obvious inducers of lymphocyte apoptosis (data not shown). It has been suggested by studies performed in a related system that at least some of the toxicity is mediated by direct effects of PAH metabolites on B cells (Heidel et al., 1999Go). However, no data are available that directly support this hypothesis.

In the present study, the potential for DMBA-treated stromal cells to deliver a death signal to pro/pre-B cells via a soluble apoptosis-inducing activity was evaluated. Of particular interest was the possibility that stromal cells could generate metabolites that directly activate an early B cell death pathway.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines.
Cell lines where maintained at 37°C in 10% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (BioWhittaker, Walkersville, MD) supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, and 5 x 10-5 M 2-mercaptoethanol (Life Technologies, Grand Island, NY). Pro/pre-B cell lines were obtained by harvesting stromal cell-adherent B cells from primary Whitlock-Witte cultures (Whitlock et al., 1984Go; Yamaguchi et al., 1997aGo) and transferring them to monolayers of a cloned bone marrow stromal cell line, BMS2 (Pietrangeli et al., 1988Go), kindly provided by Dr. P. Kincade. Cells of one such line, termed BU-11, uniformly express the late pro-B/early pre-B cell marker CD43 and contain a rearranged immunoglobulin heavy chain gene (Yamaguchi et al., 1997bGo).

AhR+/- mice with a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). To generate primary bone marrow stromal cells from wild-type and AhR-/- littermates, AhR+/- heterozygote breeding pairs were established and offspring analyzed for AhR genotype by AhR-specific PCR analysis of tail DNA. Primary bone marrow stromal cell cultures were obtained from femurs of AhR+ and AhR- male mice as previously described (Whitlock et al., 1984Go). Briefly, bone marrow plugs were flushed from the femurs of 4- to 6-week-old mice and RBC-depleted bone marrow cells were cultured in RPMI-1640 supplemented with 10% FBS, L-glutamine, 2-mercaptoethanol, and penicillin/streptomycin (Gibco/BRL) at an approximate cell density of 2–4 x106/ml. Nonadherent cells were removed 7 days later by vigorous washing. Adherent cells were treated with 0.05% trypsin–0.53 mM EDTA (Gibco/BRL), harvested, washed, and transferred to fresh tissue culture flasks containing a 1:1 ratio of fresh media and conditioned media.

Hepa-1c1c7 (Hepa-1) cells, kindly provided by Dr. James Whitlock (Stanford University), were maintained in DMEM supplemented with 5% FBS, 2 mM L-glutamine, and 5 x10-5 M ß-mercaptoethanol. Hepa-1 cells were passed three times per week at a 1:10 dilution and never were grown to more than 80% confluency. For the AhR-mediated CYP1A1 induction assay, Hepa-1 cells were plated at a cell density of 105 cells/ml and treated the next day with vehicle (dimethylsulfoxide, DMSO), 10-5–10-8 M DMBA, or conditioned media (supernatants) from vehicle- or 10-5 M DMBA-treated BMS2 cells. Eighteen hours later, the monolayers were washed with phosphate-buffered saline (PBS) (Gibco/BRL). Cells were detached with 0.05% trypsin–0.53 mM EDTA, harvested, and stored at -80°C until use.

Production of stromal cell conditioned supernatant.
BMS2 cells (0.6 x105/ml) were plated in 10 ml DMEM as described above and treated the next day with vehicle (0.01% DMSO final concentration) or 10-5–10-8 M DMBA (Sigma/Aldrich Chemical Co., Milwaukee WI) dissolved in DMSO. Eighteen h later all media were removed. Plate-adherent BMS2 monolayers were washed extensively with four changes of PBS, and fresh media were added (a 1:1 ratio of RPMI-1640 and DMEM supplemented with 5% FBS, L-glutamine, and 2-mercaptoethanol). After 24 h, conditioned supernatants were removed and sterile filtered through a 0.22 µm syringe filter (Fisherbrand, Fisher Scientific, Pittsburgh, PA).

Dialysis/ ultrafiltration.
Supernatants from vehicle- or BMS2-treated cells were dialyzed using either Spectra/Por 6 (MWCO 1,000) or Spectra/Por CE membranes against a 10-fold excess of 1:1 RPMI/DMEM media for 18 h with one change of media (Spectrum Laboratories, Inc., Dominguez, CA). Alternatively, supernatants were concentrated using a Centricon-10 microconcentrator (10 kDa membrane exclusion) (Amicon Corp., Danvers, MA), a Biomax PB 50,000 NMWL ultrafiltration membrane (50 kDa membrane exclusion) (Millipore Co., Bedford, MA) or an Amicon Ultrafiltration cell (50 kDa membrane exclusion) to one-tenth the starting volume. For some experiments, concentrated supernatants were heat inactivated by incubating in a 60°C water bath for one h, incubated for one h at 37°C as a control, or digested with an equal volume of 0.25% trypsin for one h at 37°C. Following the treatment, concentrates were reconstituted with media to the original volumes, and retentates and filtrates were added to BU-11/BMS2 cultures.

Induction and quantitation of apoptosis.
Apoptosis induction and quantitation was performed as described previously (Ryu et al., 2003Go). Briefly, BMS2 cells were plated at a density of 0.4 x105/ml in 24-well culture dishes (Costar, Corning Inc., NY) and allowed to adhere overnight. In experiments where primary stromal cells were used in place of BMS2 cells, AhR+ or AhR- stromal cells were seeded at a density of 105 cells/ml and allowed to adhere for 48 h. Subsequently, BU-11 cells were added to stromal cells at a density of 0.5 x105/ml and allowed to settle for 6–8 h before treating individual wells with vehicle (0.01% DMSO), 10-5–10-8 M DMBA dissolved in DMSO, or conditioned supernatants from vehicle- or DMBA-treated BMS2 cells. In experiments where BU-11 were cultured in the absence of the supporting BMS2 cells for less than 48 h, the media was supplemented with recombinant interleukin-7 (rIL-7) to maintain BU-11 viability. When adding BMS2 supernatants, > 95% the media was removed from BU-11/BMS2 cultures and replaced with an equal volume of conditioned supernatant from vehicle- or DMBA-treated BMS2 cells. Forty-eight h later, BU-11 cells were harvested and washed once with cold PBS containing 5% FBS and 1% sodium azide. Cells then were resuspended in 0.3 ml of hypotonic buffer consisting of 50 µg/ml propidium iodide (P.I.) (Sigma Chemical Co., St. Louis, MO), 1% sodium citrate, and 0.1% Triton X-100 and analyzed in a Becton Dickinson FACScan flow cytometer. Cells undergoing DNA fragmentation and apoptosis are weaker in P.I. fluorescence than those in the G0/G1 phase of cell cycle (Mann et al., 1999Go, 2001Go).

Semiquantitative RT-PCR.
Total RNA was prepared from Hepa-1 cell pellets using the Qiagen RNeasy kit (Qiagen Inc. Valencia, CA). Five µg of total RNA was combined with 50 ng of random hexamer primer and water in a volume of 11 µl and annealed at 70°C for 10 min. The primed RNA was chilled on ice and reverse transcribed using 160 units of SuperScript II reverse transcriptase in PCR buffer (5 mM MgCl2, 20 mM DTT, and 2 mM dNTPs) at 42°C for 50 min followed by 70°C for 10 min. After chilling on ice, 1.6 units of RNase H were added, and the mixture was incubated for 20 min at 37°C. All enzymes were obtained from Life Technologies/Invitrogen Corp. (Carlsbad, CA). The PCR was conducted using 2 µl of cDNA in 10 x PCR buffer, 1.5 mM MgCl2, 0.2 µM dNTP mix, 5 units Taq polymerase and 0.2 µM CYP1A1-specific primers (sense 5'-TCTGGAGACCTTCCGGCATT-3'/antisense 5'-CCGATGCACTTTCGCTTGCC-3') or ß-actin-specific primers (sense 5'-GTCGTCGACAACGGCTCCGGCATGTG-3'/antisense 5'-CATTGTAGAAGGTGTGGTGCCAGATC-3'). Twenty-two cycles of amplification were performed in a programmed thermocycler (Barnstead/Thermolyne, Dubuque, IA), and 5 µl of each product was separated on a 1% agarose gel and visualized with ethidium bromide. Images were captured by digital photography (Kodak Transilluminator and Kodak DC290 Digital Camera). Relative band intensities were determined with the Kodak Digital Sciences ID program. CYP1A1-band intensity was normalized to the corresponding sample ß-actin band intensity.

Statistics.
Statistical analyses were performed with Statview (SAS Institute, Cary, NC). The Student’s t-test and one-factor ANOVAs were used to analyze the data. For the ANOVAs, the Dunnett’s or Tukey/Kramer multiple comparisons tests were used to determine significant differences.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conditioned Supernatants from DMBA-Treated Stromal Cells Induce Pro/Pre-B Cell Apoptosis in BU-11/BMS2 Cocultures
Previous studies demonstrated that AhR negative pro/pre-B cells (BU-11) maintained on AhR+ primary or cloned bone marrow-derived stromal cells undergo apoptosis when cultures are exposed to PAH such as DMBA or B[a]P (Mann et al., 1999Go, 2001Go; Near et al., 1999Go; Quadri et al., 2000Go; Yamaguchi et al., 1997aGo,bGo). BU-11 cells grown in the presence of rIL-7 but in the absence of stromal cells are resistant to DMBA (Yamaguchi et al., 1997aGo,bGo). Furthermore, addition of rIL-7 to cocultures of BU-11 and BMS2 cells does not protect BU-11 cells from DMBA-induced apoptosis (Yamaguchi et al., 1997bGo). These data demonstrate that pro/pre-B cell apoptosis is not due to a loss of stromal cell function (e.g., loss of IL-7 secretion) but rather is mediated by the active production of a stromal cell-dependent death signal.

To determine if BU-11 cell apoptosis is mediated by an activity released into culture media, BMS2 cells were treated with vehicle (0.01% DMSO) or 10-5–10-7 M DMBA for 24 h. Media were removed and cells washed extensively prior to addition of fresh media. Conditioned supernatants were harvested 24 h later, sterile filtered, and added to BU-11/BMS2 cultures at various ratios of conditioned to fresh media. BU-11 cells were harvested 48 h later, treated with propidium iodide, and assayed for the percentage of cells undergoing apoptosis as described previously (Mann et al., 1999Go, 2001Go; Near et al., 1999Go; Quadri et al., 2000Go). As a positive control, separate BU-11/BMS2 cocultures were treated with DMBA directly and assayed 48 h later for apoptosis.

Consistent with previous studies, addition of 10- 5–10-7 M DMBA directly to BU-11/BMS2 cocultures induced a significant (p < 0.05) level of BU-11 cell apoptosis, as visualized by an increase in the percentage of cells exhibiting a sub G0/G1 staining pattern (Fig. 1AGo, left histograms). While control supernatants from vehicle-treated BMS2 cells had no effect in BU-11/BMS2 cultures, conditioned supernatants from BMS2 cells treated with 10-5 or 10-6 M DMBA induced a level of BU-11 cell apoptosis approximately the same as that induced by adding DMBA directly to cocultures (Fig. 1AGo, right histograms). Similar results were obtained when BU-11 cells were cultured on primary bone marrow stromal cells (below).



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 1. Conditioned supernatants from DMBA-treated BMS2 cells induce apoptosis in BU-11 pro/pre-B cells maintained on stromal cell monolayers. BU-11 cells grown on BMS2 monolayers were treated with vehicle, DMBA, or supernatants from vehicle- or DMBA-treated BMS2 cells. Forty-eight h later, BU-11 cells were harvested, and the percentage of apoptotic cells was assessed by P.I. staining and flow cytometry. (A) BU-11/BMS2 cocultures were treated with vehicle, 10-5–10-7 M DMBA ("Control"), supernatants from vehicle-treated BMS2 cells, or supernatants from BMS2 cells treated with 10-5–10-7 M DMBA ("Supe"). Data from three experiments are expressed as mean ± SE. An asterisk denotes statistical significance relative to corresponding vehicle controls (p < 0.05; Dunnett’s test). (B) BU-11/BMS2 cocultures were treated with supernatants from vehicle- or 10-5 M DMBA-treated BMS2 cells. The supernatants were added neat or diluted 3:1, 1:1, 1:3, or 1:10 in fresh media prior to addition to BU-11/BMS2 apoptosis assay cocultures. Data from three experiments are expressed as the mean ± SE. An asterisk denotes statistical significance relative to corresponding vehicle controls (p < 0.02).

 
More than 30% of the BU-11 cells underwent apoptosis when conditioned supernatants from DMBA-treated BMS2 stromal cells were diluted as much as 1:3 (Fig. 1BGo p, < 0.01). A lower, but statistically significant level of apoptosis was seen when these supernatants were diluted as much as 1:10 (p < 0.02). These results indicate that DMBA-treated stromal cells elaborate a soluble factor(s) capable of inducing pro/pre-B cell death in BU-11/BMS2 cocultures.

{alpha}-Naphthoflavone ({alpha}-NF) Blocks Elaboration of Stromal Cell-Derived, Apoptosis-Inducing Activity
BU-11 cell apoptosis induced by addition of DMBA directly to BU-11/BMS2 cocultures is blocked by {alpha}-naphthoflavone ({alpha}-NF), an AhR and cytochrome P-4501A1 inhibitor (Mann et al., 1999Go; Quadri et al., 2000Go; Yamaguchi et al., 1997bGo). To determine if {alpha}-NF similarly blocks release of the death-inducing activity, BMS2 cells were pretreated with vehicle or 10-6 M {alpha}-NF. Vehicle or 10-5–10-6 M DMBA was added 1 h later, and cultures were incubated for 18 h. Cells were then washed extensively and incubated for 24 h in fresh media, and conditioned supernatants were collected and tested for apoptosis-inducing activity as in Figure 1Go. As seen when added directly to DMBA-treated cocultures (Mann et al., 1999Go; Quadri et al., 2000Go; Yamaguchi et al., 1997bGo), {alpha}-NF completely blocked the induction of the soluble death-inducing activity from DMBA-treated BMS2 cells (Fig. 2Go). These results suggest a role for either the AhR or PAH-metabolizing enzymes in production of an apoptosis-inducing activity.



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 2. An AhR and cytochrome P-4501A1 inhibitor ({alpha}-NF) blocks production of the DMBA-induced, stromal cell-derived apoptosis activity. BMS2 cells were pre-treated with vehicle or 10-6 M {alpha}-naphthoflavone ({alpha}-NF) for 1 h. Vehicle or 10-5–10-6 M DMBA was added and cultures were incubated for eighteen h. Plate adherent BMS2 cells were washed extensively, and fresh media was applied. Conditioned supernatants from these cultures were harvested 24 h later and added to BU-11/BMS2 cocultures. Forty-eight h later, BU-11 cells were harvested, and the percentage of apoptotic cells was assessed as in Figure 1Go. Data from four experiments are presented as the mean ± SE. An asterisk indicates a significant decrease in the percentage of apoptotic cells (p < 0.02; Student’s t test).

 
Characterization of Apoptosis-Inducing Activity
To begin to characterize the stromal cell-derived apoptosis-inducing activity, conditioned supernatants were prepared from vehicle- or DMBA-treated BMS2 cells and then added directly to BU-11/BMS2 cocultures or dialyzed for 18 h in 1-kDa exclusion dialysis membranes prior to addition to cocultures. As in previous experiments, supernatants from DMBA-treated stromal cells contained significant levels of apoptosis-inducing activity (Fig. 3AGo, left histograms). These supernatants retained this activity when dialyzed overnight (Fig. 3AGo, right histograms), indicating that the apoptosis-inducing activity is relatively stable and is larger than 1 kDa. This result is not consistent with a role for the carryover of free DMBA or a free DMBA metabolite to the BU-11/BMS2 cocultures. Indeed, maintenance of DMBA in media for 18 h ablated its ability to induce pro/pre-B cell apoptosis (data not shown).



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 3. DMBA-induced, stromal cell-derived apoptosis activity is retained by dialysis and is >= 50 kDa. Supernatants from vehicle- or 10-5 M DMBA-treated BMS2 cells were left untreated ("Untreated Supe"), dialyzed against fresh media overnight using a 1-kDa molecular cutoff dialysis membrane ("Dialyzed Supe"), or concentrated to one-tenth the original volume using a 50-kDa cutoff ultrafiltration membrane. Dialyzed supernatants, filtrates, or retentates restored to the original volume of the supernatant were added to BU-11/BMS2 cocultures, and BU-11 cell apoptosis was assayed forty-eight h later as in Figure 1Go. (A) Data from three experiments are expressed as the mean ± SE. An asterisk indicates a statistically significant level of apoptosis compared with corresponding vehicle controls (p < 0.01; Student’s t test). (B) Data from a separate series of three experiments are expressed as the mean ± SE. An asterisk indicates a significant level of apoptosis (p < 0.01).

 
To estimate the size of the factor(s) responsible for this activity, supernatants from vehicle- or DMBA-treated BMS2 cells were concentrated with one of two commercial preparations of 50-kDa exclusion filters. The supernatant retentate was diluted back to the original volume of the conditioned supernatant, and both the filtrate and retentate were tested for apoptosis-inducing activity in BU-11/BMS2 cocultures. Significant apoptosis-inducing activity was present in the retentate, while the filtrate had no such activity (Fig. 3BGo). Similar results were obtained with 5-kDa and 10-kDa exclusion filters (not shown). Therefore, the death-inducing activity is >= 50 kDa.

To determine if the apoptosis-inducing activity is associated with protein, conditioned media from vehicle- or DMBA-treated BMS2 cells were concentrated using a 50-kDa exclusion filter and treated with or without trypsin for 1 h at 37°C. All supernatants were then diluted back to the original volume of conditioned media and added to BU-11/BMS2 cocultures. Incubation of supernatant from DMBA-treated BMS2 cells at 37°C for 1 h had no effect on its ability to induce apoptosis in BU-11 cells (Fig. 4Go, left histograms). In contrast, treatment of supernatant from DMBA-treated BMS2 cells with trypsin for 1 h at 37°C completely eliminated its ability to induce BU-11 cell apoptosis (Fig. 4Go, middle histograms), indicating a role for a protein component in the apoptosis-inducing activity. Maintenance of the supernatant for 1 h at 60°C similarly ablated apoptosis-inducing activity (Fig. 4Go, right histograms), a result consistent with a role for a heat-labile protein.



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 4. DMBA-induced, stromal cell-derived death activity is sensitive to proteolytic digestion and heat inactivation. Conditioned supernatants from vehicle- or 10-5 M DMBA-treated BMS2 cells were concentrated using a 50K NMWCO Amicon ultrafiltration membrane as in Figure 3Go, incubated for one h at 37°C in the presence or absence of trypsin or heat inactivated at 60°C for one h. Concentrated supernatants (heated or trypsin digested) were diluted to the original volume of conditioned media and added to BU-11/BMS2 cocultures, and apoptosis was assayed forty-eight h later as in Figure 1Go. Data obtained with untreated or 37°C-treated supernatants from DMBA-treated stromal cells were indistinguishable and were pooled here for simplicity. Data from three to six experiments are expressed as the mean ± SE. An asterisk denotes a statistically significance decrease in apoptosis relative to untreated supernatant controls (p < 0.05; Tukey/Kramer).

 
Stromal Cell-Derived, Apoptosis-Inducing Activity Requires AhR+ Stromal Cells
To determine if the apoptosis-inducing activity could act directly on pro/pre-B cells, supernatants from vehicle- or DMBA-treated BMS2 cells were added to BU-11 cells grown in rIL-7 in the absence of stromal cells. While supernatant from DMBA-treated cells induced significant apoptosis when added to BU-11/BMS2 cocultures containing rIL-7, it failed to affect BU-11 cell viability when added to BU-11 cells in the absence of stromal elements (Fig. 5Go). This result indicates that, like DMBA-2,3-dihydrodiol (Mann et al., 1999Go), the activity elaborated by DMBA-treated stromal cells does not directly affect pro/pre-B cells, but that it mediates its activity indirectly through stromal elements. Furthermore, the data demonstrate that an activity elaborated by one stromal cell may affect other stromal cells at a distance.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 5. Stromal cell-derived, apoptosis-inducing activity does not directly target pro/pre-B cells. Supernatants from vehicle- or 10-5–10-7 M DMBA-treated BMS2 cells were added to BU-11 cells cocultured with BMS2 cells or cultured in the absence of BMS2 cells but in the presence of rIL-7. Forty-eight h later, BU-11 cells were harvested, and the percentage of apoptotic cells assayed as in Figure 1Go. Data from twelve experiments are expressed as the mean ± SE. An asterisk indicates a statistically significance level of apoptosis relative to vehicle controls (p < 0.01; Student’s t test).

 
To determine if production of the supernatant activity bypasses the previously described requirement for AhR in stromal cells, supernatants from vehicle- or DMBA-treated BMS2 cells were added to cocultures of BU-11 cells and primary bone marrow stromal cells derived from wild-type AhR+/+ or littermate AhR-/- mice. BU-11 cell apoptosis was assayed 48 h later. Supernatants from DMBA-treated BMS2 cells readily induced apoptosis in BU-11 cells cultured on primary AhR+/+ bone marrow stromal cells (Fig. 6Go). The percentage of BU-11 cells undergoing apoptosis was significantly reduced (p < 0.001), but not completely abrogated (p < 0.01), when these pro/pre-B cells were cultured with supernatants from DMBA-treated stromal cells in the presence of AhR-/- stromal cells. These data are reminiscent of those obtained when adding DMBA-3,4-dihydrodiol, an early DMBA metabolite, directly to BU-11/stromal cell cocultures in that both an AhR-dependent and an AhR-independent component are evident (Mann et al., 1999Go).



View larger version (11K):
[in this window]
[in a new window]
 
FIG. 6. Stromal cell-derived, apoptosis-inducing activity is partially AhR dependent. BU-11 cells grown on primary stromal cells isolated from the bone marrows of AhR+/+ or AhR-/- littermate mice were treated with conditioned supernatant from vehicle- or 10-5 M DMBA-treated BMS2 cells. Forty-eight h later, BU-11 cells were harvested and assayed for apoptosis as in Figure 1Go. Data were obtained from three experiments and are expressed as the mean ± SE. A cross (+) indicates a significant difference in the percentage of apoptosis observed in cocultures of BU-11 cells with AhR+/+ as compared with AhR-/- stromal cells (p < 0.001). An asterisk (*) indicates a significant level of apoptosis relative to the corresponding vehicle control (p < 0.01; Student’s t test).

 
Stromal Cell-Derived Apoptosis-Inducing Activity Is Associated with a CYP1A1-Inducing Activity
Although a role for protein was demonstrated by trypsin and heat inactivation of the apoptosis-inducing activity and is supported by its apparent size (>= 50 kDa), the requirement for AhR+/+ stromal cells in the apoptosis assay coculture simultaneously suggests a role for a relatively small AhR ligand which requires an AhR+/+ target stromal cell to mediate its activity. These apparently contradictory observations would be resolved if the apoptosis-inducing activity reflects a DMBA metabolite(s) associated with and stabilized by a >= 50 kDa protein(s). Supporting this possibility, some metabolites of DMBA, such as DMBA-3,4-dihydrodiol, both activate the AhR and induce apoptosis in BU-11/BMS2 cocultures (Mann et al., 1999Go). If such a metabolite contributes to the apoptosis-inducing activity in the supernatant, it would be expected that treatment of cells with supernatants or >= 50 kDa fractions of supernatants from DMBA-treated stromal cells would induce AhR-regulated, CYP1A1 gene transcription. Since little or no CYP1A1 mRNA is induced by AhR ligands in BMS2 cells (Mann et al., 1999Go), a well-characterized CYP1A1-inducible murine hepatic line, Hepa-1, was used for these studies.

As expected, treatment of Hepa-1 cells with 10-5–10-7 M DMBA for 18 h, as a positive control, significantly induced CYP1A1 mRNA (Fig. 7AGo, lanes 1–5, and Fig. 7BGo). Similarly, supernatants from DMBA- but not vehicle-treated BMS2 cells induced significant levels of CYP1A1 mRNA (Fig. 7AGo, lanes 6 and 7, and Fig. 7BGo). Furthermore, the apoptosis-inducing CYP1A1-inducing activity in supernatants from DMBA-treated stromal cells was retained following overnight dialysis or concentration with a > 50 kDa Amicon filtration unit (Fig. 7CGo). Collectively, these data support the working hypothesis that a DMBA-metabolite(s) produced by treatment of stromal cells with >= 10-6 M DMBA and associated with a >= 50 kDa protein(s) mediates pro/pre-B cell apoptosis in BU-11/BMS2 cocultures. However, the target of this putative complex is not the pro/pre-B cells themselves but rather the AhR+ stromal cells capable of delivering a death signal to the associated lymphocytes, presumably through cell–cell contact.



View larger version (38K):
[in this window]
[in a new window]
 
FIG. 7. Stromal cell-derived, apoptosis-inducing activity is associated with a CYP1A1-inducing activity. Hepa-1 cell monolayers were treated with vehicle, 10-5-10-8 M DMBA, or conditioned supernatants from vehicle or 10-5 M DMBA-treated BMS2 cells. Eighteen h later, total RNA was isolated, 5 µg was reverse transcribed, and the cDNA was subjected to PCR amplification with CYP1A1- and ß-actin-specific primers. (A) Data from a representative experiment (from a total of three experiments) are shown. (B) CYP1A1 band densities were normalized to ß-actin band densities. Data from three experiments are presented as the mean ± SE. An asterisk (*) indicates a significant increase in band densities relative to the corresponding vehicle controls (p < 0.05; Dunnett’s test). (C) Hepa-1 cells were treated with untreated conditioned supernatant from vehicle or DMBA-treated BMS2 cells, conditioned supernatants dialyzed overnight against fresh media using 1-kDa cutoff dialysis tubing, or concentrated with a > 50-kDa Amicon filter and reconstituted to their original volumes. Representative data from a total of three experiments are presented.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a series of studies, we have evaluated the effects of common and prototypic PAHs on the developing immune system (Hardin et al., 1992Go; Mann et al., 1999Go, 2001Go; Near et al., 1999Go; Quadri et al., 2000Go; Ryu et al., 2003Go; Yamaguchi et al., 1997aGo,bGo). Although these studies focused on developing B cells as the ultimate targets of PAH toxicity, the results indicate that the immediate targets of PAHs are AhR+ bone marrow stromal cells. Since all eight lineages of bone marrow hematopoietic cells interact with stromal cells in the bone marrow microenvironment, the results suggest that the in vivo effects of PAH exposure may not be limited to the developing B cell compartment. Indeed, injection of either B[a]P or DMBA into mice results in the rapid destruction of the entire bone marrow hematopoietic compartment (Yamaguchi et al., 1997aGo).

The in vitro model of this bone marrow toxicity indicates that apoptosis is responsible for depletion of at least the B cell compartment. Several components of the intracellular apoptosis pathway activated in either nontransformed, stromal cell-dependent pro/pre-B cells (Mann et al., 1999Go, 2001Go; Ryu et al., 2003Go) or in transformed 70Z3 pre-B cells (Heidel et al., 1999Go; Page et al., 2002Go, 2003Go) following culture with stromal cells and PAH have been elucidated. For example, we have shown that down-regulation of NF-{kappa}B and NF-{kappa}B-regulated c-myc is required for maximal pro/pre-B cell apoptosis (Mann et al., 2001Go; Ryu et al., 2003Go). Unlike B cell receptor-mediated clonal deletion signals (Wu et al., 1996aGo,bGo, 1999Go), pro/pre-B cell apoptosis induced by PAHs does not involve upregulation of p21WAF or p27Kip1, indicating a potentially unique death signaling pathway activated by a stromal cell-derived death stimulus (Ryu et al., 2003Go). Other investigators have shown the involvement of caspase-3, -8, and -9 in DMBA-induced apoptosis in a transformed pre-B cell line (Page et al., 2002Go). Similarly, some of the effects of PAH on stromal cells required for induction of the death signal are known. For example, bone marrow stromal cells must activate their AhR in order to generate the death signal (Mann et al., 1999Go; Near et al., 1999Go; Quadri et al., 2000Go; Yamaguchi et al., 1997bGo). In addition, PAH metabolism likely plays a role in generating the stromal cell-derived death signal (Heidel et al., 1999Go; Mann et al., 1999Go).

Despite this information, little is known of the signal delivered by the PAH-treated stromal cell to the target B cell. Three general possibilities were considered in the design of the experiments presented here: (1) elaboration of a soluble protein (e.g., a cytokine), (2) production of a PAH metabolite that is directly toxic to early B cells, and (3) delivery of a death signal via cell–cell contact.

If DMBA-treated stromal cells produce a soluble death-inducing protein, it would be predicted that supernatants from treated cells would induce pro/pre-B cell death. The ability to induce death in BU-11/BMS2 cultures with supernatant from DMBA-treated BMS2 cells initially seemed consistent with this hypothesis. Experiments with size exclusion filters indicated that the death-inducing activity was >= 50 kDa, ruling out the possibility of DMBA contamination or free DMBA metabolites as the mediators of the apoptosis signal. Furthermore, the activity contained in the >= 50 kDa fraction was ablated completely with trypsin or 60°C heat treatment, clearly indicating the involvement of one or more proteins.

Despite these results, several experiments indicated that elaboration of a conventional soluble protein such as a cytokine either was not involved or not sufficient for pro/pre-B cell apoptosis. Experiments with several recombinant cytokines associated with cell death (e.g., TNF-{alpha}, TNF-ß, lymphotoxin-ß, TGF-ß1, ß2, ß3) or their respective receptor knockout mice failed to identify a soluble cytokine capable of inducing pre-B or pro/pre-B cell death (data not shown). Furthermore, induction of pro/pre-B cell apoptosis was diminished significantly when BU-11 cells were cultured with supernatants from DMBA-treated stromal cells and AhR-/- bone marrow stromal cells. This partial AhR-dependency would not be expected of an apoptosis-inducing effector cytokine. Rather, it is reminiscent of results obtained with DMBA-3,4-dihydrodiol, in that BU-11 cell apoptosis induced by that AhR-binding metabolite is only partially AhR-dependent (Mann et al., 1999Go). Furthermore, the production of a CYP1A1-inducing activity by DMBA-treated stromal cells (Fig. 7Go) suggests the presence of either DMBA or an AhR-activating DMBA metabolite, e.g., DMBA-3,4-dihydrodiol or DMBA-9,10-diolepoxide (Mann et al., 1999Go).

The most likely explanation for these results is the association of a DMBA metabolite with a >= 50 kDa protein(s) such that the putative metabolite is stabilized and capable of acting at some distance in cell cultures. The ability of {alpha}-NF, an AhR and CYP1A1 inhibitor, to block production of the death-inducing activity strongly suggests that the stromal cell is required to produce the metabolite, which putatively associates with protein. It is not known if the metabolite is bound to proteins present in the culture supernatant or proteins produced by stromal cells themselves. However, the retention of death-inducing activity only in fractions >= 50 kDa suggests some limitation in what protein(s) is targeted. The identities of the protein(s) involved in stabilizing the putative DMBA metabolite and the metabolite mediating the death-inducing activity are currently under investigation.

Finally, the results suggest that products of PAH-exposed stromal cells, i.e., metabolite-protein complexes, may affect the function of distant cells, including stromal elements, within the bone marrow microenvironment. Such an interaction between stromal cells may represent a positive feedback loop in effect at higher PAH doses (e.g., >= 10-6 M DMBA). Moreover, the failure of concentrated supernatant from DMBA-treated stromal cells to kill pro/pre-B cells grown in the absence of stromal cells and the failure of pro/pre-B cells to die when separated from DMBA-treated stromal cells by a culture transwell (not shown) indicate that the production of a soluble death-inducing effector factor is not likely and that stromal cell-pro/pre-B cell contact is required, particularly at lower DMBA doses (< 10-6 M), for initiation of the B cell suicide program. The nature of this cell–cell signaling is under study.


    NOTES
 
This work was supported by NIEHS Grant RO1-ES06086 and Superfund Basic Research Program Grant #1P42-ES07381, Program Project #1P01ES11624, and P01HL68705.

1 To whom correspondence should be addressed at Department of Environmental Health, Boston University School of Public Health, 715 Albany St. (R-408), Boston, MA, 02118. E-mail: dsherr{at}bu.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Burchiel, S. W., Davis, D. A. P., Ray, S. D., and Barton, S. L. (1993). DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma. Fundam. Appl. Toxicol. 21, 120–124.[CrossRef][ISI][Medline]

Burchiel, S. W., Davis, D. A. P., Ray, S. D., Archuleta, M. M., Thilsted, J. P., and Corcoran, G. B. (1992). DMBA-induced cytotoxicity in lymphoid and nonlymphoid organs of B6C3F1 mice: Relation of cell death to intracellular calcium and DNA damage. Toxicol. Appl. Pharmacol. 113, 126–132.[ISI][Medline]

Christou, M., Moore, C., Gould, M., and Jefcoate, C. (1987). Induction of mammary cytochromes P-450: An essential first step in the metabolism of 7,12-dimethylbenz[a]anthracene by rat mammary epithelial cells. Carcinogenesis 8, 73–80.[Abstract]

Davilla, D. R., Romero, D. L., and Burchiel, S. W. (1996). Human T cells are highly sensitive to suppression of mitogenesis by polycyclic aromatic hydrocarbons and this effect is differentially reversed by {alpha}-naphthoflavone. Toxicol. Appl. Pharmacol. 139, 333–341.[CrossRef][ISI][Medline]

Dertinger, S. D., Nazarenko, D. A., Silverstone, A. E., and Gasiewicz, T. A. (2001). Aryl hydrocarbon receptor signaling plays a significant role in mediating benzo[a]pyrene- and cigarette smoke condensate-induced cytogenetic damage in vivo. Carcinogenesis 22, 171–177.[Abstract/Free Full Text]

Hardin, J. A., Hinoshita, F., and Sherr, D. H. (1992). Mechanisms by which benzo[a]pyrene, an environmental carcinogen, suppresses B cell lymphopoiesis. Toxicol. Appl. Pharmacol. 117, 155–164.[ISI][Medline]

Heidel, S. M., Holston, K., Buters, J. T., Gonzalez, F. J., Jefcoate, C. R., and Czupyrynski, C. J. (1999). Bone marrow stromal cell cytochrome P4501B1 is required for pre-B cell apoptosis induced by 7,12-dimethylbenz[a]anthracene. Mol. Pharmacol. 56, 1317–1323.[Abstract/Free Full Text]

Heidel, S. M., MacWilliams, P. S., Baird, W. M., Dashwood, W. M., Buters, J. T., Gonzalez, F. J., Larsen, M. C., Czuprynski, C. J., and Jefcoate, C. R. (2000). Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene. Cancer Res. 60, 3454–3460.[Abstract/Free Full Text]

Holladay, S. D., and Smith, B. J. (1995). Alterations in murine fetal thymus and liver hematopoietic cell populations following developmental exposure to 7,12-dimethylbenz[a]anthracene. Environ. Res. 68, 106–113.[CrossRef][ISI][Medline]

Kawabata, T. T., and White, K. L. (1987). Suppression of the in vitro humoral immune response of mouse splenocytes by benzo(a)pyrene metabolites and inhibition of benzo(a)pyrene-induced immunosuppression by {alpha}-naphthaflavone. Cancer Res. 47, 2317–2322.[Abstract]

Kerkvliet, N. I. (1995). Immunological effects of chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 103, 47–53.[ISI][Medline]

Ladics, G. S., Kawabata, T. T., and White, K. L. (1991). Suppression of the in vitro humoral immune response of mouse splenocytes by 7,12-dimethylbenz[a]anthracene metabolites and inhibition of immunosuppression by {alpha}-naphthoflavone. Toxicol. Appl. Pharmacol. 110, 31–44.[ISI][Medline]

Lai, Z. W., Fiore, N. C., Hahn, P. J., Gasiewicz, T. A., and Silverstone, A. E. (2000). Differential effects of diethylstilbestrol and 2,3,7,8- tetrachlorodibenzo-p-dioxin on thymocyte differentiation, proliferation, and apoptosis in bcl-2 transgenic mouse fetal thymus organ culture. Toxicol. Appl. Pharmacol. 168, 15–24.[CrossRef][ISI][Medline]

Laiosa, M. D., Lai, Z. W., Thurmond, T. S., Fiore, N. C., DeRossi, C., Holdener, B. C., Gasiewicz, T. A., and Silverstone, A. E. (2002). 2,3,7,8-tetrachlorodibenzo-p-dioxin causes alterations in lymphocyte development and thymic atrophy in hemopoietic chimeras generated from mice deficient in ARNT2. Toxicol. Sci. 69, 117–124.[Abstract/Free Full Text]

Lawrence, B. P., Leid, M., and Kerkvliet, N. I. (1996). Distribution and behavior of the Ah receptor in murine T lymphocytes. Toxicol. Appl. Pharmacol. 138, 275–284.[CrossRef][ISI][Medline]

Mann, K. K., Doerre, S., Sherr, D. H., and Quadri, S. (2001). The Role of NF-{kappa}B as a survival factor in environmental chemical-induced pre-B cell apoptosis. Mol. Pharmacol. 59, 302–309.[Abstract/Free Full Text]

Mann, K. K., Matulka, R. A., Lawrence, B. P., Kerkvliet, N. I., and Sherr, D. H. (1999). The role of cytochrome P-450 enzymes in 7,12-dimethylbenz[a]anthracene-induced apoptosis. Toxicol. Appl. Pharmacol. 161, 10–22.[CrossRef][ISI][Medline]

Near, R. I., Mann, K. K., Matulka, R. A., Shneider, A. M., Gogate, S. U., Trombino, A. F., and Sherr, D. H. (1999). Regulation of pre-B cell apoptosis by aryl hydrocarbon receptor/transcription factor-expressing stromal/adherent cells. Proc. Soc. Exp. Biol. Med. 221, 242–252.[Abstract]

Page, T. J., O’Brien, S., Holston, K., MacWilliams, P. S., Jefcoate, C. R., and Czuprynski, C. J. (2003). 7,12-Dimethylbenz[a]anthracene induced bone marrow toxicity is p53 dependent. Toxicol. Sci. 2, 85–92.[CrossRef]

Page, T. J., O’Brien, S., Jefcoate, C. R., and Czuprynski, C. J. (2002). 7,12-Dimethylbenz[a]anthracene induces apoptosis in murine pre-B cells through a caspase-8-dependent pathway. Mol. Pharmacol. 62, 313–319.[Abstract/Free Full Text]

Pietrangeli, C. E., Hayashi, S., and Kincade, P. W. (1988). Stromal cell lines which support lymphocyte growth: characterization, sensitivity to radiation and responsiveness to growth factors. Eur. J. Immunol. 18, 863–872.[ISI][Medline]

Poland, A., Glover, E., Robinson, J., and Nebert, D. (1974). Genetic expression of aryl hydrocarbon hydroxylase activity: induction of monooxygenase activities and cytochrome P–450 formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice genetically "nonresponsive" to other aromatic hydrocarbons. J. Biol. Chem. 249, 5599–5606.[Abstract/Free Full Text]

Quadri, S., Qadri, A., Mann, K. L., and Sherr, D. H. (2000). The bioflavonoid galangin blocks aryl hydrocarbon receptor (AhR) activation and polycyclic aromatic hydrocarbon-induced pre-B cell apoptosis. Mol. Pharmacol. 58, 515–525.[Abstract/Free Full Text]

Rigdon, R. H., and Neal, J. (1969). Relationship of leukemia to lung and stomach tumors in mice fed benzo[a]pyrene. Proc. Soc. Exp. Biol. Med. 130, 146–148.

Ryu, H.-Y., Mann, K. K., Schlezinger, J. J., Jensen, B., and Sherr, D. H. (2003). Environmental chemical-induced pro/pre-B cell apoptosis: Analysis of c-Myc, p27Kip1, and p21WAF1 reveals a death pathway distinct from clonal deletion. J. Immunol. 170, 4897–4904.[Abstract/Free Full Text]

Safe, S., and Krishan, V. (1995). Cellular and molecular biology of aryl hydrocarbon (Ah) receptor-mediated gene expression. Arch. Toxicol. 17, 99–115.

Staples, J. E., Murante, F. G., Fiore, N. C., Gasiewicz, T. A., and Silverstone, A. E. (1998). Thymic alterations induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin are strictly dependent on aryl hydrocarbon receptor activation in hemopoietic cells. J. Immunol. 160, 3844–3854.[Abstract/Free Full Text]

Thurmond, L. M., House, R. V., Lauer, L. D., and Dean, J. H. (1988). Suppression of splenic lymphocyte function by 7,12-dimethylbenz[a]anthracene in murine lymphoid cells. Toxicol. Appl. Pharmacol. 93, 369–377.[ISI][Medline]

Thurmond, T. S., Staples, J. E., Silverstone, A. E., and Gasiewicz, T. A. (2000). The aryl hydrocarbon receptor has a role in the in vivo maturation of murine bone marrow B lymphocytes and their response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 165, 227–236.[CrossRef][ISI][Medline]

Uno, S., Dalton, T. P., Shertzer, H. G., Genter, M. B., Warshawsky, D., Talaska, G., and Nebert, D. W. (2001). Benzo[a]pyrene-induced toxicity: Paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels. Biochem. Biophys. Res. Commun. 289, 1049–1056.[CrossRef][ISI][Medline]

Urso, P., and Johnson, R. A. (1988). Quantitative and functional change in T cells of primiparous mice following injection of benzo(a)pyrene at the second trimester of pregnancy. Immunopharmacol. Immunotoxicol. 10, 195–217.[ISI][Medline]

Vorderstrasse, B. A., and Kerkvliet, N. I. (2001). 2,3,7,8-Tetrachlorodibenzo-p-dioxin affects the number and function of murine splenic dendritic cells and their expression of accessory molecules. Toxicol. Appl. Pharmacol. 171, 117–125.[CrossRef][ISI][Medline]

White, K. L., Jr., Lysy, H. H., and Holsapple, M. P. (1985). Immunosuppression by polycyclic aromatic hydrocarbons: A structure-activity relationship in B6C3F1 and DBA/2 mice. Immunopharmacology 9, 155–164.[CrossRef][ISI][Medline]

Whitlock, C. A., Robertson, D., and Witte, O. N. (1984). Murine B cell lymphopoiesis in long term culture. J. Immunol. Methods 67, 353–369.[CrossRef][ISI][Medline]

Wojdani, A., Attarzadeh, M., Wode-Tsadik, G., and Alfred, L. J. (1984). Immunocytotoxicity effects of polycyclic aromatic hydrocarbons on mouse lymphocytes. Toxicology 31, 181–189.[CrossRef][ISI][Medline]

Wu, M., Arsura, M., Lee, H., Schauer, S., Sherr, D. H., and Sonenshein, G. E. (1996a). Inhibition of NF-{kappa}B/Rel induces apoptosis of murine B cells. EMBO. J. 15, 101–107.

Wu, M., Bellas, R. E., Shen, J., Yang, W., and Sonenshein, G. E. (1999). Increased p27Kip1 cyclin-dependent kinase inhibitor gene expression following anti-IgM treatment promotes apoptosis of WEHI 231 B cells. J. Immunol. 163, 6530–6535.[Abstract/Free Full Text]

Wu, M., Lee, H., Bellas, R. E., Schauer, S. L., Arsura, M., Katz, D., FitzGerald, M. J., Rothstein, T. L., Sherr, D. H., and Sonenshein, G. E. (1996b). Inhibition of c-myc expression induces apoptosis of WEHI 231 murine B cells. Mol. Cell. Biol. 16, 5015–5021.[Abstract]

Yamaguchi, K., Matulka, R. A., Shneider, A., Toselli, P., Trombino, A. F., Yang, S., Hafer, L. J., Mann, K. K., Tao, X.-J., Tilly, J. L., et al. (1997a). Induction of pre-B cell apoptosis by 7,12 dimethylbenz[a]anthracene in long term bone marrow cultures. Toxicol. Appl. Pharmacol. 147, 190–203.[CrossRef][ISI][Medline]

Yamaguchi, K., Near, R. I., Matulka, R. A., Shneider, A., Toselli, P., Trombino, A. F., and Sherr, D. H. (1997b). Activation of the aryl hydrocarbon receptor/transcription factor and stromal cell-dependent pre-B cell apoptosis. J. Immunol. 158, 2165–2173.[Abstract]