* Department of Immunology, Graduate School of Biomedical Science, Hiroshima University, Minami-ku, Hiroshima 734-8551, Japan;
Japanese Society for the Promotion of Science Research Fellowship for Young Scientists; and
CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
Received September 30, 2002; accepted November 11, 2002
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ABSTRACT |
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Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; hematopoietic stem cells (HSCs); CD34-KSL (CD34-, c-kit+, Sca-1+, lineage negative) cells; long-term reconstitution activity; aryl hydrocarbon receptor; AhR.
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INTRODUCTION |
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TCDD is believed to exert its effects primarily through a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). AhR belongs to the basic helixloophelix (bHLH) superfamily of DNA binding proteins and heterodimerizes with the AhR nuclear translocator (ARNT) to form an AhR/ARNT transcription factor complex (Hoffman et al., 1991; Reyes et al., 1992
). This complex binds to specific DNA sites in the regulatory domains of numerous target genes and mediates the biological effects of exogenous ligands (Okey et al., 1994
). Indirubin and indigo were reported recently as potent natural ligands of AhR (Adachi et al., 2001
). Studies using TCDD and its related congeners that activate AhR also suggest that the receptor has immunological functions. The recent generation of AhR knockout mice has provided evidence of a role for this protein in hepatic fibrosis development and in the immune system (Fernandez-Salguero et al., 1995
; Mimura et al., 1997
; Schmidt et al., 1996
). Although it is also known that AhR is present in most cell and tissue types of the body, its functions in hematopoietic stem cells (HSCs) have not been examined in detail.
HSCs possess the ability to self-renew or differentiate into multiple distinct cell lineages. Substantial HSC self-renewal has been demonstrated in transplantation models (Iscove and Nawa, 1997; Pawliuk et al., 1996
), in stroma-containing long-term marrow cultures supplemented with thrombopoietin (Yagi et al., 1999
) and upon the ectopic expression of HOXB4 (Antonchuk et al., 2002
; Sauvageau et al., 1995
). Although there are some reports that in vitro HSC self-renewal has been achieved using various culture conditions (Audet et al., 2001
; Ema et al., 2000
; Holyoake et al., 1996
; Miller and Eaves, 1997
), the processes that govern HSC self-renewal remain poorly understood. Although the functional diversity of HSCs has been demonstrated unequivocally, it is less clear how such diversity is generated. The prevailing view is that HSC heterogeneity is regulated by both extrinsic and intrinsic events (Enver et al., 1998
; Metcalf, 1998
). Extrinsic (environmental) signals are derived predominantly from stromal cells and their products. Stromal cells are organized into niches that differ in their ability to maintain HSCs (Blazsek et al., 1995
; Wineman et al., 1996
). Thus, homing of HSCs to different types of stromal cell niches should contribute to HSC heterogeneity. In addition to extrinsic signals, intrinsic mechanisms control HSC decisions. Intrinsic mechanisms could induce HSC heterogeneity if HSCs make random decisions at the time of each HSC division. For example, each HSC has a choice of many fates, including self-renewal, differentiation, apoptosis, and migration. Evidence for stochastic processes comes from the analysis of the differentiation of myeloid and lymphoid precursors (Busslinger et al., 2000
; Ogawa, 1999
). For example, multipotent myeloid precursors generate more restricted precursors in an apparently random fashion (Ogawa, 1999
). Recent studies of murine HSCs clearly demonstrated that expression of the surface antigens of stem cells is under the influence of the developmental stage and the kinetic state of the stem cells (Ogawa, 2002
). It was demonstrated using the monocloncal anti-CD34 antibody RAM34 that only the CD34- fraction of lineage negative (Lin-), Sca-1+, c-kit+ bone marrow (BM) cells, known as CD34-KSL cells, of normal adult mice are capable of long-term hematopoietic reconstitution in lethally irradiated mice (Osawa et al., 1996
).
Surprisingly, there have been only a few reports concerning the relationship between HSC and TCDD that describe an increase in the number of hematopoietic progenitor cells upon TCDD treatment (Frazier et al., 1994; Murante and Gasiewicz, 2000
; Staples et al., 1998
; Thurmond and Gasiewicz, 2000
). Therefore, we decided to investigate more precisely whether TCDD influences the long-term reconstruction activity of CD34-KSL cells in BM.
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MATERIALS AND METHODS |
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Animals.
C57BL/6J (B6, CD45.2) mice were obtained from CREA JAPAN, INC. (Meguro, Tokyo, Japan) and B6 SJL Ptprca Pep3bBoyJ (B6 Ptprc, CD45.1) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and kept in accordance with the Laboratory Animal Science guidelines of Hiroshima University. AhR-/- mice were kindly provided by Dr. Y. Fujii-Kuriyama (Tohoku University, Sendai, Japan). All mice were acclimated in-house for one week prior to treatment. Mice older than 10 weeks of age were used in the experiments.
TCDD treatment.
The experiments shown here were performed more than three times with at least five mice per group. Animals were administered either vehicle or TCDD at day zero and were euthanized at h 0, 3, and 12, and on days 1, 3, 7, 14, 28, 56, and 112. TCDD-treated animals were administered by gavage 40 µg/kg body weight (bw) TCDD and vehicle-treated animals were administered by gavage an equivalent dose of corn oil. The following TCDD doses were used: 0 (vehicle), 0.16, 0.63, 2.5, 10, 40, and 80 µg/kg bw. Animals were administered either vehicle or TCDD at day zero and were euthanized on day 7.
Antibodies.
The antibodies used in immunofluorescence staining included RAM34 (anti-CD34), D7 (anti-Sca-1), 2B8 (anti-c-kit), 104 (anti-CD45.2), and A20 (anti-CD45.1). Antibodies using for lineage markers included RA3-6B2 (anti-CD45R/B220), 145-2C (anti-CD3e), RM4-5 (anti-CD4), 53-6.7 (anti-CD8a), M1/70 (anti-Mac-1), RB6-8C5 (anti-Gr-1), and TER119. All the antibodies were purchased from Pharmingen (San Diego, CA).
Purification and analysis of CD34-KSL cells.
BM cells were flushed from femurs and tibiae of mice. Cell suspensions were then filtered through a sterile 100 µm Cell Strainer (No. 2360; Falcon, Lincoln Park, NJ) and stained with biotinylated antilineage makers. For analysis, cells were then stained with fluorescein isothiocyanate (FITC) anti-CD34, phycoerythrin (PE) anti-Sca-1, allophycocyanin (APC) anti-c-kit antibodies and streptavidin-PerCP (Pharmingen). Four-color analysis was performed on a FACS Calibur (Becton Dickinson, San Jose, CA) using CELLQuest software (Becton Dickinson). For cell sorting, bone marrow was depleted of lineage positive cells using MACS (Milteny Biotech GmbH, Bergisch Gladbach). Cells were stained with biotinylated antilineage markers and then were allowed to bind streptavidin-magnetic beads (Milteny Biotech). Ten µl of beads were used per 107 cells. Cells were then applied to a C-type MACS column and nonadherent (lineage negative; Lin-) cells were stained with the subsequent antibodies as described above (Randall and Weissman, 1998). CD34-KSL cells were sorted by FACS Vantage SE (Becton Dickinson). Dead cells were excluded by propodium iodide (PI) from analysis and sorting.
Competitive repopulation assay.
We applied and performed a competitive repopulation assay to which the CD45 (Ly5) system was adapted (Ema and Nakauchi, 2000); 100 sorted CD34-KSL cells from TCDD- or vehicle-treated B6 (CD45.2), AhR-/- (CD45.2), and AhR WT (CD45.2) mice were mixed with 1 x 105 BM competitor cells (B6 Ptprc, CD45.1) and were transplanted into B6 Ptprc mice irradiated at a dose of 10 Gy. After transplantation, peripheral blood cells of the recipients were stained with FITC anti-CD45.2 and PE anti-CD45.1. The cells were simultaneously stained with biotinylated anti-B220 or biotinylated anti-TER119, a mixture of biotinylated anti-Mac-1 and Gr-1, or a mixture of APC-conjugated anti-CD4 and -CD8. Biotinylated antibodies were developed with streptavidin-APC (Molecular Probes, Eugene, OR). The cells were analyzed on a FACS, and percentage chimerism was taken as the quadrant ratio of donor cells (CD45.2+ cells). When percent chimerism was more than 1% with all lineage reconstitution, recipient mice were considered to be multilineage reconstituted (positive mice). Dead cells were excluded by PI staining.
Two step single cell-methylcellulose colony assay.
Sorted CD34-KSL cells were deposited as single cells into 96-well microtiter plates by FACS Vantage SE and Clone Cyt (Becton Dickinson). The single cells were incubated in 100 µl of culture medium (MethocultTM GF, StemCell Technologies Inc., Vancouver, British Columbia, Canada) at 37°C in a humidified atmosphere 5% CO2 for seven days. Cultures were scored for colony-forming units using an inverted microscope. We evaluated and designated those containing more than 50 cells/colony as first colonies. Next, the first colonies were collected and sorted to deposit 100 cells/well and incubated for another seven days. Cultures were scored for colony-forming units in the same manner, and these colonies were designated second colonies.
Statistical analysis.
Means ± SD were compared with a Students t-test. Significance levels were set at p = 0.010.05 (*), p = 0.010.001 (**), and p < 0.001 (***).
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RESULTS |
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To understand the mechanisms of this functional deterioration, we thought it could be a possibility that long-term reconstitution activity of HSC might shift from CD34-KSL fraction to CD34+KSL fraction, usually supporting short-term reconstitution activity, by stimulation of TCDD. One thousand CD34+KSL cells from both TCDD- and vehicle-treated adult mice were transplanted by the same method as described above. No long-term reconstitution activity was observed in either group (data not shown). Therefore, it is reasonable to conclude that the effect of TCDD on long-term reconstitution activity is targeted on the CD34-KSL cell itself rather than population shift.
Next we performed an in vitro two-step single-cell methylcellulose colony assay on CD34-KSL cells to analyze the effects of TCDD on self-renewal activity at the single cell level. Single CD34-KSL cells were sorted into individual wells filled with culture medium formulated to optimize the detection of murine hematopoieic progenitor cells, BFU-E, CFU-GM, CFU-G, CFU-M, and CFU-GEMM. Although we could not find a difference of cellularity of the colony between vehicle plates and TCDD plates (the plates where single CD34-KSL cells from vehicle- or TCDD-treated mice were sorted into each well), the first colony forming ability of CD34-KSL cells from the TCDD-treated mice was only one-third that of cells from the vehicle-treated mice (Fig. 3D p, = 0.0024, n = 6). Next we examined second colony forming activity. This process is an indicator of the self-renewal or progenitor-forming ability of HSCs. The second colony forming ability of CD34-KSL cells from the TCDD-treated mice was one-tenth of that of vehicle-treated mice, and the size of colonies generated from CD34-KSL cells from the TCDD-treated mice was 50% of that of vehicle-treated mice.
From these results, it is also conceivable that the long-term reconstitution activity of HSCs in CD34-KSL populations is damaged by TCDD, despite the increase in cell number.
Absence of Aryl Hydrocarbon Receptor Prevents the Elimination of Stem Cell Activity
As AhR is known to be a major dioxin receptor, we also examined the role of AhR in the TCDD-mediated elimination of stem cell activity. We performed the same experiments as described above using AhR-knockout (AhR-/-) mice. As shown in Figure 4A, we could not detect a significant TCDD-induced increase in the number of CD34-KSL cells in AhR-/- mice, although a three- to four-fold increase in cell number was observed in wild type (WT) mice. It is noteworthy that the total number of BM cells was not changed between AhR-/- and WT mice.
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These observations strongly suggest that both TCDD-induced increase in CD34-KSL cell number and suppression of CD34-KSL long-term reconstitution activity are AhR-dependent. Therefore, it is likely that TCDD affects long-term reconstitution activity through the AhR/ARNT pathway.
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DISCUSSION |
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The capacity for extensive self-renewal is commonly regarded as a property that is reserved for stem cells. Recently, a clonal BM-HSC transplantation system was used to study the function of HSCs. Here, we also used the BM-HSC transplantation system (competitive repopulation assay) to examine the effects of TCDD on long-term reconstitution activity. Our data demonstrate an unexpected elimination of long-term reconstitution activity in the population of CD34-KSL cells in BM. The abrogation seems to encompass all HSC behaviors, including self-renewal, cell death, homing, and lineage contribution (development). It strongly suggests that TCDD-induced alteration of all hematopoetic processes appeared from the beginning of hematopoiesis.
Therefore it is important to hypothesize that some regulation systems that are affected by TCDD might modulate hematopoiesis from the beginning. Recent studies have demonstrated the ability of intrinsic factors such as HOXB4, cyclin-dependent kinase (CDK) inhibitors, and Wnt signals to control HSC self-renewal and/or reconstitution activities. We hypothesized that misexpression of these intrinsic factors (genes) might abrogate HSC reconstitution activity after TCDD administration.
In the case of HOXB4, the quality of the HSCs induced by HOXB4 overexpression is not impaired, as demonstrated by their ability to fully repopulate all lineages (Antonchuk et al., 2002). Enhanced HSC regenerative ability in HOXB4-transduced bone marrow cells has also been demonstrated (Antonchuk et al., 2001
; Sauvageau et al., 1995
; Thorsteinsdottir et al., 1999
). Studies in RAT-1 cells showed that HOXB4 overexpression activates the expression of AP-1 complex genes Fra-1 and Jun-B, with subsequent upregulation of cyclin D1 (Krosl and Sauvageau, 2000
). It was also demonstrated that HOXB4 overexpression enhances ex vivo growth of total bone marrow cultures and that this effect is due to increased proliferation rather than reduced apoptosis (Antonchuk et al., 2001
). Furthermore, the HOXB4-mediated growth advantage was found to be largely restricted to the most primitive fraction of hematopoietic cells (Antonchuk et al., 2002
). The primitive cell-specific growth advantage suggests that HOXB4 overexpression either enhances HSC proliferation or self-renewal, or some combination of both. Indeed, we noticed that the upstream region of the HOXB4 gene possesses two xenobiotic response elements (TNGCGTG), which might interact with AhR/ARNT upon TCDD administration (M. Kanno, unpublished observation). Therefore, we speculate that AhR/ARNT might negatively control HOXB4 expression in HSCs.
Recently, the function of CDK inhibitor in HSCs has been studied quite extensively. In the absence of p21 (p21cip1/waf1), the G1 checkpoint-regulating CDK inhibitor, HSC proliferation and number are increased under normal homeostatic conditions. Furthermore, self-renewal of primitive cells is impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure (Cheng et al., 2000a,b
; Enan et al., 1998
; Kolluri et al., 1999
).
In the case of the Wnt pathway, a central role of Wnt in the establishment and maintenance of cell fates has been demonstrated in vertebrates and invertebrates. To examine whether Wnt are involved in the regulation of hematopoietic stem/progenitor cell populations (HSCPs), several groups have investigated Wnt and frizzled gene expression in hematopoietic tissues and the response of HSCPs to WNTs (Austin et al., 1997; Van Den Berg et al., 1998
).
There have been several reports describing two states of HSCs (resting and active). Seventy-five percent of HSCs are usually in a resting state, but external stimuli can induce cell cycle progression (Cheshier et al., 1999; Wright et al., 2001
). HOXB4 and Wnt might be the candidates for this stimulus. It is conceivable that the signal pathways used by these stimuli are antagonized by the modulation AhR/ARNT system activity. This process of HSC activation has yet to be explored.
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ACKNOWLEDGMENTS |
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NOTES |
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