Department of Environmental Medicine, University of Rochester, Rochester, New York 14642
Received October 7, 1999; accepted November 15, 1999
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ABSTRACT |
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Key Words: tetrachlorodibenzo-p-dioxin; TCDD; hemopoiesis; Sca-1; c-Kit; hemopoietic stem cells; flow cytometry.
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INTRODUCTION |
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Tetrachlorodibenzo-p-dioxin (TCDD), which binds to the aromatic hydrocarbon receptor (AhR), a ligand-activated bHLH protein (Rowlands and Gustafsson, 1997), affects cell differentiation processes (Ma and Whitlock, 1996
) and cell cycle status (Dohr and Abel, 1997
; Ge and Elferink, 1998
Weiss et al., 1996
). Such alterations may be related to the cross-species effects of TCDD on numerous immunologic parameters (Grassman et al., 1998
; Kerkvliet, 1995
). Perhaps at the forefront of immunomodulatory significance, TCDD is known to produce decreased host resistance at animal body burden levels similar to those present in humans (DeVito et al., 1995
; Kerkvliet et al., 1996
). Due to past focus upon mature/peripheral immune effector cells, the established research base has provided relatively limited insight into the effects of TCDD upon the immune system reconstitution process, i.e., hemopoiesis. The potential toxic ramifications of this and related xenobiotics upon immune system regeneration, therefore, are not well understood. While both an endogenous ligand and the normal function of the AhR are unknown, evidence is suggestive that it may play a significant role in immune system development and responsiveness (Fernandez-Salguero et al., 1995
; Gonzalez et al., 1995
; Brenner, 1996
).
In bone marrow, AhR-dependent immunological toxicities include myelopoietic alterations (Luster et al., 1985), reduced expression levels of the bone marrow lymphocyte developmentally-associated genes, RAG-1 and TdT (Fine et al., 1990; Frazier et al., 1994
; Silverstone et al., 1994
) and reduced prothymocyte reconstitution capacity (Fine et al., 1990). Our group has recently observed that AhR presence within bone marrow hemopoietic cells is required for TCDD-induced alterations to hemopoietic progenitors (Staples et al., 1998
). Furthermore, within bone marrow-derived cells, the presence of the AhR is also essential for TCDD-induced thymic atrophy and shifts in intrathymic cellular phenotypic profiles (Staples, et al., 1998
). Through cross-talk mechanisms, this transcription factor may further influence these and other regulatory processes that affect lympho-hemopoiesis. Hemopoietic alterations elicited by TCDD may result from a number of AhR-mediated responses such as reductions to estrogen receptor expression (Tian et al., 1998
), repression of NFkB-regulated cytokine responses (Tian et al., 1999
) and/or changes induced in proteins serving as important regulators of the cell cycle (Kolluri et al., 1999
).
However, the actual modulated gene targets responsible for TCDD-induced immune alterations are largely unknown. This is due, in part, to a lack of understanding of the specific cellular targets. Given the progenitor status of TCDD-affected marrow cells expressing RAG-1 and TdT, and the capability of hemopoietic progenitor cells for intrathymic colonization (Scollay et al., 1988), we have proposed that alterations to very early lympho-hemopoietic differentiation events, i.e., at the level of near-pluripotent HSC, are occurring in the marrow as a result of TCDD treatment (see Fig. 1
). Alterations to early events in differentiation have recently been reported in terms of altered enumerations of c-Kit- and Sca-1-defined marrow progenitor stages (Staples et al., 1998
). Evaluation of these hemopoietic cell-surface markers also has precedent in works that have characterized stem-cell responses to cytotoxic agents such as 5-fluorouracil (Nishio et al., 1996
; Randall and Weissman, 1997
).
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The studies herein describe a one-month time course of TCDD effects on hemopoietic stem cells. In addition, we have assessed the TCDD sensitivity of these cells with dose-response studies. These data suggest that the acute exposure of bone marrow to relatively low concentrations of TCDD results in altered patterns of lympho-hemopoietic differentiation.
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MATERIALS AND METHODS |
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Experimental animals.
Male C57BL/6J mice were obtained from The Jackson Laboratories (Bar Harbor, ME) at 56 weeks of age. All mice were housed and cared for in accordance with The Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), and were provided 5001 Rodent Diet (Purina Mills, Brentwood, MO) and water ad libitum.
Treatment protocol.
Mice were weight-normalized across TCDD and olive oil (control) treatment groups and were administered a single dose of 30 µg/kg TCDD or olive oil via intraperitoneal injection (ip). Three or 4 animals were used in each treatment group. TCDD- and vehicle-treated animals were euthanized by CO2 overdose at 1, 2, 3, 6, 9, 12, 16, 20, or 31 days post injection. For experiments to determine HSC dose-responsiveness to TCDD, 4 to 6 animals per TCDD treatment group were administered either 0, 0.3, 3, 6, 15, or 30 µg/kg of TCDD and were euthanized at day 2 following treatment.
Bone marrow cell isolation.
Femurs and tibiae were removed from mice and the marrow cavities flushed with 10 ml of Hanks' Minimum Essential Medium (Life Technologies, Grand Island, NY) (HMEM), containing 5% fetal bovine serum and Penicillin-Streptomycin (P/S; 100U/ml penicillin and 0.1 mg/ml streptomycin; Life Technologies). The marrow cells were placed in single cell suspension and debris was eliminated by filtering the suspension through 80-gauge nylon mesh (TETKO Inc., Briarcliff Manor, NY). The filtered cells were then pelleted by centrifugation at 250 x g for 6 min. The pellet was resuspended in 1 ml of erythrocyte lysis buffer (0.17 M NH4Cl, 10 mM KHCO3, 1 mM EDTA) for 4 min on ice. Intact cells were then washed once, repelleted, and resuspended in HMEM to a volume of 5 ml for cell counting. The cell yield was enumerated with a Neubauer hemocytometer. Cell viability was evaluated with trypan blue dye (0.08%). The viability by dye exclusion was determined to be greater than 90% in all experiments. Bone marrow from each animal was prepared and analyzed separately.
Antibodies.
The following monoclonal antibodies (mAbs) were used at predetermined saturating levels for labeling of hemopoietic progenitor cells: biotin-conjugated anti-TER119 (clone TER119); biotin-conjugated anti-B220 (clone RA3-6B2); biotin-conjugated anti-Gr-1 (clone RB6-8C5); biotin-conjugated anti-Mac-1 (clone M1/70); biotin-conjugated anti-CD3
(clone 500A2); biotin-conjugated anti-CD8
(clone 536.7); FITC-conjugated anti-c-Kit (clone 2b8); and PE-conjugated anti-Sca-1 (clone E13-161.7). All antibodies were obtained from Pharmingen (San Diego, CA).
Cell staining and flow cytometry analysis.
Freshly isolated bone marrow cells were pelleted by centrifugation and washed in Hanks' Balanced Salts Solution with Ca2+ and Mg2+ (Life Technologies) containing 0.2% bovine serum albumin (BSA) (HBSS-0.2% BSA). Aliquots of 8 x 106 bone marrow cells were sequentially incubated with anti-Fc III/IIR (clone 2.4G2; Pharmingen) and then in a mixture of biotin-conjugated mAbs (anti-Mac-1, anti-Gr-1, anti-Ter-119, anti-CD3
, anti-CD8
, and anti-B220) diluted with HBSS-0.2% BSA, for 30 min. Additionally, the mixture contained PE-conjugated Sca-1 mAb, and FITC-conjugated c-Kit mAb. The cells were washed once with HBSS + 0.2% BSA and were then incubated for 20 min in Dulbecco's PBS containing 1% BSA and streptavidin-conjugated Red670 Pharmingen (San Diego, CA). After one rinse in HBSS-0.2% BSA, the cells were fixed in 1% paraformaldehyde in PBS. Data on the fixed bone marrow cells were acquired on a Becton Dickinson FACScan flow cytometer using the Becton Dickinson Lysis II program and analyzed with Becton Dickinson Attractors 3.0.0 software. A mean of the log fluorescence-3 value was initially determined for each sample from a data file of 25 x 103 fixed bone marrow cells. A fluorescence-3 intensity equal to 1/10 of the mean fluorescence-3 value was then used to define a low fluorescence-3 gate (see Fig. 2
). A second data file of 50 x 103 fixed bone marrow cells that satisfied the pre-determined low fluorescence-3 gate for each sample was acquired. The (c-Kit and Sca-1-defined) subpopulations of lineage-negative (lin) cells contained within the low fluorescence-3 gate were enumerated per the total number of bone marrow cells required to obtain the lin sample. Discrete bivariate regions were established with position parameters held constant among samples with Attractors algorithms, such that alterations within distinct c-Kit/Sca-1 lin subpopulations would be assessed, i.e., lin events defined by intermediate c-Kit/Sca-1 staining were excluded (Fig. 2
, lower panels).
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Statistics.
The 2-tailed Student's t-test for unpaired variables was used to evaluate differences between treatment groups and their respective vehicle-treated controls. Differences in the results were considered statistically significant at p < 0.05.
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RESULTS |
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TCDD exposure increased the absolute number of bone marrow lin Sca-1+ c-Kit+ cells relative to control over 24 h through 31 days following treatment (Fig. 3A). Maximal effects to this phenotype were apparent at the 6 day time point, and values through 31 days did not differ substantially from the 6 day lin Sca-1+ c-Kit+ increase (Fig. 3A
). In contrast to the persistent increases in marrow lin Sca-1+ c-Kit+ cells, we observed a very rapid-onset elevation in the number of lin Sca-1+ c-Kit marrow cells, which peaked at the day 3 measurement and then exhibited rapid decline to near-control values (Fig. 3A
) through day 12. The number of lin Sca-1+ c-Kit cells subsequently rose above control values through day 31 (Fig. 3A
). Finally, with a singular exception of an increased number of lin Sca-1 c-Kit+ cells at the 20 day time point, no significant shifts in Sca-1-defined cell numbers were observed (Fig. 3B
).
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TCDD produced dose-dependent increases in lin Sca-1+ c-Kit+ hemopoietic stem cells in marrow assays conducted 2 days following TCDD treatment (Fig. 5). The effective and non-effective TCDD doses to lin Sca-1+ c-Kit+ hemopoietic stem cells were 6.0, and 3.0 µg/kg, respectively (Fig. 5A
). Effects on lin Sca-1+ c-Kit hemopoietic cells were observed at 0.3, and not observed at 6.0 µg/kg of TCDD, but were significant at the higher concentrations (Fig. 5A
). At the 2 day timepoint, significant dose-responsive shifts in Sca-1-defined cell numbers were observed only at 3 and 15 µg/kg (Fig. 5B
). In a more limited study, we investigated dose-related effects at 6 days following TCDD exposure. Increases in Sca-1+ c-Kit+ cells were apparent following 30 µg/kg and 3 µg/kg doses, while Sca-1+ c-Kit cell numbers remained unaltered at the latter dose (data not shown). Following 0.3 µg/kg TCDD treatment, no effects to Sca-1+ cells were observed, however, the number of Sca-1 c-Kit cells was significantly decreased to a level 25 percent below that of the control (data not shown). Total bone-marrow cellularity was not significantly affected by any TCDD dose level assayed at the 2- or 6-day time points (data not shown), nor were any effects on marrow cellularity observed following the (maximal) 30 µg/kg dose assayed over 31 days. Thymic weights were not significantly altered at the 2-day time point following 30 µg/kg TCDD. While one instance of diminished thymic weight occurred 2 days following 15 µg/kg TCDD, no significant alterations occurred at lower doses assayed at the 2-day time point. At the 6-day time point, TCDD caused significant loss of thymic weight at doses ranging from 30 to 3 µg/kg, but not at 0.3 µg/kg (data not shown).
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DISCUSSION |
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Recent evidence suggests that bone marrow common lymphoid progenitor cells (CLP) develop from short-term and/or multipotent HSC and differentiate from these stages with the onset of IL-7 receptor (IL-7R) expression (Kondo et al., 1997). Separate efforts have isolated a marrow T progenitor with multipotent (lymphoid and myeloid) reconstituting activity, based upon expression of the Sca-2 antigen (Antica et al., 1994
), and present on CD4lo T precursors within the thymus (Wu et al., 1991a
,b
) but not expressed on long-term HSC subsets (Spangrude and Scollay, 1990
). Phenotypically, long- and short-term repopulating lin HSC (McKinstry et al., 1997
; Morrison et al., 1997; Osawa et al., 1996; Spangrude et al., 1988
; Smith et al., 1991
), Sca-2 multipotent progenitors (Antica et al., 1994
) and CD4lo intrathymic precursors (Antica et al., 1993
, 1994
; Matsuzaki et al., 1993
) demonstrate high-level, cell-surface Sca-1 and c-Kit expression, while low expression levels of these proteins occur on the CLP (Kondo et al., 1997
). While the exact nature of HSC differentiation to pro-T lymphoid cells remains unclear, reconstitution analyses that have compared all marrow cells with T progenitor potential reveal that only long-term HSC possess extensive self-renewal capacity (Antica et al., 1994
; Kondo et al., 1997
; Matsuzaki et al., 1993
; Wu et al., 1991). These and other thymic reconstitution studies (Rodewald et al., 1994
) suggest that there may be numerically-limited differentiation and cell surface phenotypic transitions which separate HSC from a CD4lo intrathymic precursor. Thus, it follows that a reasonable and initial assessment of TCDD toxicity to marrow lympho-hemopoietic cells can be made based upon an analysis of lin, Sca-1 and c-Kit -defined cells.
When the effects of TCDD treatment on the percentage of marrow hemopoietic cell number were assayed over 31 days, the most persistent and significant alteration throughout was an increase in the lin cell subset of pluripotent phenotype which expressed high-level cell surface expression of Sca-1 and c-Kit antigens (Fig. 3A). Also evident, were consistent early (24 h through 9 day) TCDD-induced increases in the percentage of lin Sca-1+ c-Kit cells (Fig. 3A
). One interpretation of these data is that the proliferation of hemopoietic cells is enhanced following in vivo exposure to TCDD. Alternately, TCDD may directly or indirectly arrest development of the lin Sca-1+ c-Kit+ population resulting in a decreased differentiation potential of these cells and an increase in their relative number. The latter interpretation is consistent with the reported marrow decreases in RAG-1 and TdT, which are highly expressed in the developmentally more mature prothymocyte, pro-B and pre-B subsets (Li et al., 1993
). Taken together with our previous studies (Staples et al., 1998
), these data suggest that TCDD, via the AhR, acts at a developmental step at or prior to the CLP stage (see Fig. 1
).
Previous studies from our labs have shown a TCDD-induced increase in the percentage of lin Sca-1+ c-Kit+ CD44hi CD25- intrathymic precursor cells. Increased percentages of such cells, present within the CD3, CD4 and CD8 triple-negative (TN) compartment of the thymus, have been observed following TCDD treatments either in vivo, or of fetal thymic organ culture (Lai et al., 1998; Staples et al., 1998
). TCDD-induced shifts in TN profile have been shown to exhibit dependence upon the presence of the AhR within hemopoietic cells and were hypothesized to result from an arrest in early thymocyte differentiation (Lai et al., 1998
; Staples et al., 1998
). In bone marrow obtained from the same chimeric animals, shifts in HSC phenotypic profile, defined in terms of Sca-1/c-Kit, were similarly stem cell AhR genotype-dependent and perhaps also reflect the proposed arrest which occurs in thymic pre-T lymphocyte differentiation. With TCDD treatment, expression of RAG-1, RAG-2 and TdT in CD4- CD8-
TCR cells is reduced (Lai et al., 1998
), i.e., present in lower numbers of these TcR-rearranging pre-T cells. This event seems analogous to the reduced RAG-1 and TdT expression in pro-lymphoid descendants of HSC within the bone marrow (Fine et al., 1990; Silverstone et al., 1994
), yet it remains to be determined whether the same primary gene and/or cell cycle alterations are involved.
The in vivo TCDD responsiveness of hemopoietic progenitors was assessed as a function of dose to further characterize alterations to the hemopoietic compartment by TCDD. The dose-response results were analogous to those of the TCDD time course, in that more persistent as well as comparatively lower-dose associated lin Sca-1+ c-Kit+ hemopoietic stem cell elevations occurred compared to those exhibited by the more committed/less pluripotent lin Sca-1+ c-Kit cells (Fig. 5A). However, it is important to note that the less pluripotent lin Sca-1+ c-Kit cell population maximal response occurred earlier (Fig. 3A
).
Variation in progenitor c-Kit expression level has been correlated with differential cycling status of marrow progenitor cells (Morrison and Weissman, 1994). Following TCDD treatment, it may be presumed that more actively-cycling, less pluripotent cells are normally, as in oil-treated animals, replenished by more quiescent HSC, while commitment processes contribute to diminished less pluripotent, i.e. lin Sca-1+ c-Kit or lin Sca-1 c-Kit+, cell number. It is unknown whether the TCDD-induced effect(s) to such more committed/less pluripotent cells took place within contexts of their relatively higher cycling activity and more limited self-renewal ability (Bradford et al., 1997
), such that the response displayed by these cells was faster and more transient than the more pluripotent, i.e. lin Sca-1+ c-Kit+ cells, and/or whether this response occurred as a result of a differential sensitivity to marrow TCDD concentration. Nevertheless, the collective data of the time course and dose-response studies show that marrow lin Sca-1 cells reveal segregate-patterned responses to TCDD exposure, per cellular expression of c-Kit.
Although the molecular mechanisms leading to cellular alterations remain unclear, in vitro studies in AhR-deficient cells implicate involvement of the Ah receptor and/or a putative endogenous ligand for the AhR in the regulation of cell cycle progression through G1 (Kolluri et al., 1999; Ma and Whitlock, 1996
; Weiss et al., 1996
). Moreover, physical interactions of the AhR with Retinoblastoma and NF-
B proteins suggest AhR involvement in cell cycle control and differentiation (Ge and Elferink, 1998
; Tian et al., 1999
). More recently, it has been suggested that TCDD may affect the transcription of the p27Kip1 cyclin/cdk inhibitor in thymocytes (Kolluri et al., 1999
). In addition, HSC effects on TCDD could possibly be related to the fact that early hemopoietic proliferation/differentiation processes are subject to pleiotropic regulation via cytokine signals transduced through tumor necrosis factor (TNF) receptor isoforms (Jacobsen et al., 1994
, 1996
; Zhang et al., 1995
). Notably, lin Sca-1+ c-Kit+ HSC are elevated in TNF
R null mice, while TNF
R null HSC lack suppressed proliferative responses in vitro when co-incubated with TNF
(Zhang et al., 1995
). Hence, our elevated lin Sca-1+ c-Kit+ HSC profiles that followed from TCDD treatment coincide with a predicted effect to HSC drawn from the recently described mechanism in which activated AhR led to repressed NF-
B transcription factor induction and decreased TNF
activity (Tian et al., 1999
).
We have previously determined a 49-fg TCDD/mg marrow tissue concentration 6 days following a 30 µg/kg single i.p. TCDD dose in young adult female Balb/c mice (Fine et al., 1990). This concentration was associated with physiologic alterations to this marrow: significantly-reduced marrow TdT biosynthesis, prothymocyte capacity and 12 day CFU-S (Fine et al., 1990). Other studies have shown B6C3F1 adult female mouse thymus and spleen TCDD levels to be similar in terms of the percent of administered (by gavage) TCDD dose (10 µg/kg) per gram tissue, over variations in both exposure time and dose (Diliberto et al., 1995), while the bone marrow levels, measured over 35 days, approximated 50% of the levels in thymus and spleen. Young adult C57BL/6J male mice administered 10 µg/kg TCDD ip, rather than by gavage, exhibited near-equivalency in thymic and splenic tissue TCDD levels with that observed in the B6C3F1 mice (Gasiewicz et al., 1983
). Given these findings, and those which showed marrow TCDD concentrations ranging from 252 to 40 fg/mg, measured between the 7 to 35 days post-dose period in the female B6C3F1 mice, (Diliberto et al., 1995
), we extrapolate that a similar, or slightly lower, prolonged-effect course of marrow TCDD exposure in these studies was established. It is significant that the marrow alteration-inducing TCDD concentrations established in these studies compare to an approximate average total body burden of 10 fg/mg for total TCDD equivalents in humans (DeVito, et al., 1995
).
In summary, this study has shown that the levels of hemopoietic stem cells are elevated shortly following TCDD treatment, with persistence of this effect extending through 31 days. HSC further demonstrate a marked low-dose sensitivity to TCDD in terms of phenotypic profile shifts. It remains unclear whether observed shifts in less pluripotent lin cells resulted secondarily from pluripotent HSC alterations or if the more developmentally mature progenitors serve directly as TCDD targets. Although no definitive mechanisms can be put forth at this time, differentiation processes of hemopoietic stem cells are likely to be affected by TCDD. Experiments are planned to focus on the mechanisms of dysregulated differentiation processes of lympho-hemopoiesis by TCDD through phenotypic, gene, and functional analyses of isolated stem cell subpopulations.
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ACKNOWLEDGMENTS |
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NOTES |
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