* Department of Environmental Health,
Department of Microbiology, Boston University Schools of Medicine and Public Health, Boston, Massachusetts;
Lady Davis Institute for Medical Research, Montreal, Canada; and
The Burdock Group, Vero Beach, Florida
Received July 24, 2003; accepted September 5, 2003
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ABSTRACT |
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Key Words: polycyclic aromatic hydrocarbon; B cell apoptosis; stromal cells.
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INTRODUCTION |
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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., 1987; Uno et al., 2001
). There is a considerable body of evidence supporting the hypothesis that the AhR influences PAH-mediated immunosuppression (Dertinger et al., 2001
; Kerkvliet, 1995
; Laiosa et al., 2002
; Lawrence et al., 1996
; Mann et al., 1999
; Near et al., 1999
; Staples et al., 1998
; Thurmond et al., 2000
; Vorderstrasse and Kerkvliet, 2001
; Yamaguchi et al., 1997a
,b
), as well as PAH-induced malignant transformation (Poland et al., 1974
; Safe and Krishan, 1995
). 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., 1996
; Kawabata and White, 1987
; Ladics et al., 1991
; Mann et al., 1999
; Thurmond et al., 1988
).
Of particular concern for environmental exposures is the extreme sensitivity of the developing immune system to PAHs and HAHs (Holladay and Smith, 1995; Lai et al., 2000
; Urso and Johnson, 1988
). 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., 1992
; Mann et al., 1999
, 2001
; Near et al., 1999
; Quadri et al., 2000
; Yamaguchi et al., 1997a
,b
). 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., 10-610-8 M) is blocked by AhR inhibitors (Quadri et al., 2000; Yamaguchi et al., 1997b
) and requires that the stromal cells express a functional AhR (Mann et al., 1999
; Near et al., 1999
). 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., 1999
, 2000
). 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-, 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., 1999
). 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.
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MATERIAL AND METHODS |
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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., 1984). 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 24 x106/ml. Nonadherent cells were removed 7 days later by vigorous washing. Adherent cells were treated with 0.05% trypsin0.53 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), 10-510-8 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% trypsin0.53 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 10-510-8 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., 2003). 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 68 h before treating individual wells with vehicle (0.01% DMSO), 10-510-8 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., 1999
, 2001
).
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 Students t-test and one-factor ANOVAs were used to analyze the data. For the ANOVAs, the Dunnetts or Tukey/Kramer multiple comparisons tests were used to determine significant differences.
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RESULTS |
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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 10-510-7 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., 1999, 2001
; Near et al., 1999
; Quadri et al., 2000
). 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- 510-7 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. 1A, 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. 1A
, right histograms). Similar results were obtained when BU-11 cells were cultured on primary bone marrow stromal cells (below).
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-Naphthoflavone (
-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 -naphthoflavone (
-NF), an AhR and cytochrome P-4501A1 inhibitor (Mann et al., 1999
; Quadri et al., 2000
; Yamaguchi et al., 1997b
). To determine if
-NF similarly blocks release of the death-inducing activity, BMS2 cells were pretreated with vehicle or 10-6 M
-NF. Vehicle or 10-510-6 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 1
. As seen when added directly to DMBA-treated cocultures (Mann et al., 1999
; Quadri et al., 2000
; Yamaguchi et al., 1997b
),
-NF completely blocked the induction of the soluble death-inducing activity from DMBA-treated BMS2 cells (Fig. 2
). These results suggest a role for either the AhR or PAH-metabolizing enzymes in production of an apoptosis-inducing activity.
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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. 4, 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. 4
, 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. 4
, right histograms), a result consistent with a role for a heat-labile protein.
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As expected, treatment of Hepa-1 cells with 10-510-7 M DMBA for 18 h, as a positive control, significantly induced CYP1A1 mRNA (Fig. 7A, lanes 15, and Fig. 7B
). Similarly, supernatants from DMBA- but not vehicle-treated BMS2 cells induced significant levels of CYP1A1 mRNA (Fig. 7A
, lanes 6 and 7, and Fig. 7B
). 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. 7C
). 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 cellcell contact.
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DISCUSSION |
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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., 1999, 2001
; Ryu et al., 2003
) or in transformed 70Z3 pre-B cells (Heidel et al., 1999
; Page et al., 2002
, 2003
) following culture with stromal cells and PAH have been elucidated. For example, we have shown that down-regulation of NF-
B and NF-
B-regulated c-myc is required for maximal pro/pre-B cell apoptosis (Mann et al., 2001
; Ryu et al., 2003
). Unlike B cell receptor-mediated clonal deletion signals (Wu et al., 1996a
,b
, 1999
), 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., 2003
). 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., 2002
). 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., 1999
; Near et al., 1999
; Quadri et al., 2000
; Yamaguchi et al., 1997b
). In addition, PAH metabolism likely plays a role in generating the stromal cell-derived death signal (Heidel et al., 1999
; Mann et al., 1999
).
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 cellcell 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-, 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., 1999
). Furthermore, the production of a CYP1A1-inducing activity by DMBA-treated stromal cells (Fig. 7
) 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., 1999
).
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
-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 cellcell signaling is under study.
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NOTES |
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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.
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