2,3,7,8-Tetrachlorodibenzo-p-dioxin Does Not Directly Alter the Phenotype of Maturing B Cells in a Murine Coculture System

doi:10.1006/taap.2002.9396

Toxicology and Applied Pharmacology

Volume 180, Issue 3, 1 May 2002, Pages 164-177

https://doi.org/10.1006/taap.2002.9396 Get rights and content

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), acting through the aromatic hydrocarbon receptor (AhR), elicits numerous toxicological effects, including immunosuppression. Previous work from our laboratory has suggested that TCDD exposure in mice is associated with altered lymphopoietic development, in particular altered B-cell phenotype in the bone marrow. It remains to be determined which specific hematopoietic populations or subpopulations within the marrow cavity are directly targeted by TCDD. To examine the effects of TCDD on developing B cells in vitro, a stromal coculture model was used. Primary bone marrow cells from male, 6- to 7-week-old C57Bl/6 mice were cocultured separately on two AhR-containing stromal cell lines (M2–10B4 and S17) that support B-cell development in the presence of IL-7. The cocultures were treated with 0 to 10 nM TCDD. Shifts in phenotype were quantified by cell surface marker staining and flow cytometry. Four populations in the maturing B cell (very early pre-pro-B, pre-pro-B, pro-B, and pre-B) were defined for quantification. The results show that the only statistically significant effect of TCDD was within the pre-pro-B population in cultures with the S17 stromal cell line. The increase in number of cells with this phenotype was seen in cultures with both wild-type and AhR−/− primary bone marrow cells. These results suggest that the maturing B220+ B cell is not the direct target for TCDD-induced bone marrow B-cell alterations.

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It is worth noting that there is no obvious dose-effect relationship in the production of part cytokines (Fig. 3). However, this is not rare that cytokines secretion does not always follow the increasing concentration or time of stimulation (Wyman et al., 2002; Podechard et al., 2008; Jensen et al., 2003; Ishimaru et al., 2009). Although the cytokines/chemokines have long been one of the most concerned research hot spots, it remains not entirely clear how these cytokines produced and interact.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known immunotoxic environmental pollutant. However, most immunotoxicology studies of TCDD were based on the animal models and the inner mechanisms have just focused on a few genes/proteins. In this study, the immune functions of THP-1-derived macrophages was measured with in-vitro bioassays after 24-h exposure of TCDD including environmentally relevant concentrations. RNA-seq and Weighted Gene Co-expression Network Analysis were used to characterize the immunotoxicity molecular mechanisms. Our study is the first report on the TCDD-induced effects of cell adhesion, morphology, and multiple cytokines/chemokines production on THP-1 macrophages. After TCDD treatment, we observed an inhibited cell adherence, probably attributed to the suppressed mRNA levels of adhesion molecules ICAM-1, VCAM-1 and CD11b, and a decrease in cell pseudopodia and expression of F-actin. The inflammatory cytokines TNF-α, IL-10 and other 8 cytokines/chemokines regulating granulocytes/T cells and angiogenesis were disrupted by TCDD. Alternative splicing event was found to be a sensitive target for TCDD. Using WGCNA, we identified 10 hub genes (TNF, SRC, FGF2, PTGS2, CDH2, GNG11, BDNF, WNT5A, CXCR5 and RUNX2) highly relevant to these observed phenotypes, suggesting AhR less important in the effects TCDD have on THP-1 macrophages than in other cells. Our findings broaden the understanding of TCDD immunotoxicity on macrophages and provide new potential targets for clarifying the molecular mechanisms.
The Aryl Hydrocarbon Receptor and Immunity
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The aryl hydrocarbon receptor (AhR), originally discovered as a xenobiotic receptor involved in upregulating metabolic enzymes, has become increasingly linked to physiological processes such as cell cycle regulation and cellular differentiation as well as disease states associated with these processes. The immune system is composed of numerous effector cells and protein mediators with specialized functions, all of which are highly dependent on cell cycle regulation and cellular differentiation and therefore offer a plethora of potential targets for modulation by the AhR. Indeed, the vast majority of studies support a direct or indirect influence of the AhR in most aspects of immunity. The focus of this chapter is to discuss these studies. Although AhR activation by dioxin and nondioxin ligands is a primary method for evaluating AhR-mediated effects, there is the possibility for off-target effects by AhR ligands (i.e., not AhR mediated) or for effects that do not accurately reflect the physiological function of the AhR (i.e., exogenous vs. endogenous ligands). Therefore, more emphasis will be given to immune effects that have been demonstrated to be AhR dependent by either the use of a chemical AhR antagonist or the knockdown or knockout of the AhR.
A single mid-gestation exposure to TCDD yields a postnatal autoimmune signature, differing by sex, in early geriatric C57BL/6 mice
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Citation Excerpt :
The present mice showed multiple TCDD-related differences in B cells at 48 weeks-of-age including an increase in the earliest bone marrow B progenitor (B220loCD24−AA4.1+) and a decrease in total bone marrow-derived lymphoid cells expressing B220. The increase in the earliest B progenitor cells may agree with an increase in pre-pro-B cells detected in a S17 stromal line exposed to TCDD (Wyman et al., 2002). Functional or other changes in bone marrow B-lineage cells that may accompany these alterations in phenotypic distribution have not been studied but may be important.
We recently observed an autoimmune profile in 24-week-old C57BL/6 mice that received a 2.5 or 5.0 μg/kg mid-gestation dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Mustafa et al., 2008). The clinical signs were consistent with a lupus-like syndrome and included: increased autoantibody levels, renal IgG and C3 immune complex deposition with associated inflammation, and increased peripheral Vβ⁺ T cells. No studies currently exist following the progression of such disease into middle or advanced ages, when human autoimmune diseases may manifest. Therefore in the present study, littermates of mice from the previous 24 week prenatal TCDD study were allowed to age to 48 weeks, considered early geriatric in mice. Similarities and differences in the disease profile based on age and sex were observed. Peripheral autoreactive Vβ⁺ T cells were increased in both sexes at 48 weeks, in contrast to males only at 24 weeks. Activated T cells from 48-week-old prenatal TCDD females over-produced the pro-inflammatory cytokine IFN-γ while males over-produced IL-10, effects again not seen at 24 weeks. Splenic transitional-2 B cells (CD21^intCD24^hi) were increased in males while transitional-1 B cells (CD23^neg CD1^neg) were increased in females at 48 weeks. Autoantibodies to cardiolipin and CD138⁺ spleen plasma cells were significantly increased in the aged males but not females. Anti-IgG and anti-C3 immune complex renal deposition were also significantly increased in the prenatal TCDD males but not females. These selective changes in the aged male mice may be noteworthy, in that the prevalence of SLE in humans shifts dramatically toward males with aging. The collective findings in aged mice suggest that prenatal TCDD permanently biases the postnatal immune response in C57BL/6 mice toward autoimmunity, and support a significant B cell component to the induced renal autoimmune disease.
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That the effect was not specific for just the T-lymphoid precursor population was suggested by studies showing that numbers of cells in the immature B cell compartment also decreased following TCDD exposure [20]. However, the further differentiation of committed, but immature, B cells to more mature cells was not affected by the direct exposure of these cells to TCDD under culture conditions [21]. These studies suggested that either more immature progenitors or stem cells were directly affected by TCDD or that TCDD was targeting cell populations in the microenvironment in vivo that are, in part, responsible for directing B cell differentiation and which may not be active or present under culture conditions.
The aryl hydrocarbon receptor (AhR) is known mainly as the mediator for the toxicity of certain xenobiotics. However, there is also much information to indicate that this transcription factor has important biological functions. Here we review the evidence that the AhR has a significant role in the regulation of hematopoietic stem cells (HSCs). Data to support this come from studies with xenobiotic AhR ligands, phenotypic analyses of mice lacking AhR, examining the presence and regulation of the AhR within HSCs, knowledge of genes and signaling pathways regulated by the AhR, and investigations of hematopoietic disorders. Based on this information, we hypothesize that AhR expression is necessary for the proper maintenance of quiescence in HSCs, and that AhR down-regulation is essential for “escape” from quiescence and subsequent proliferation of these cells. This implicates the AhR as a negative regulator of hematopoiesis with a function of curbing excessive or unnecessary proliferation. This provides an important advantage by preventing the premature exhaustion of HSCs and sensitivity to genetic alterations, thus preserving HSC function and long-term multi-lineage generation over the lifespan of the organism. This also implicates a role of the AhR in aging processes. AhR dysregulation may result in the altered ability of HSCs to sense appropriate signals in the bone marrow microenvironment leading to hematopoietic disease. It is also reasonable to hypothesize that this protein has an important function in the regulation of other tissue stem cell populations. Suggestive evidence is consistent with a role in skin and neural stem cells.
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¹: Current address: Mitretek Systems, Inc., 7525 Colshire Dr., McLean, VA 22102.

²: To whom correspondence should be addressed at Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, 575 Elmwood Ave., Box EHSC, Rochester, NY 14642.

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Regular Article2,3,7,8-Tetrachlorodibenzo-p-dioxin Does Not Directly Alter the Phenotype of Maturing B Cells in a Murine Coculture System

Abstract

Toxicol. Appl. Pharmacol.

Blood

J. Immunol. Methods

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Adv. Cancer Res.

Arch. Biochem. Biophys.

Clin. Immunol. Immunopathol.

Blood

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Immunity

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Lymphoid development from stem cells and the common lymphocyte progenitors

Cold Spring Harbor Symp. Quant. Biol.

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

J. Immunol.

Leukocyte activation induces aryl hydrocarbon receptor up-regulation, DNA binding, and increased Cyp1a1 expression in the absence of exogenous ligand

Mol. Pharmacol.

The influence of S17 stromal cells and interleukin 7 on B cell development

Eur. J. Immunol.

Regulation of hematopoiesis by bone marrow stromal cells and their products

Annu. Rev. Immunol.

Regulation of B cell differentiation by bone marrow stromal cells

Int. J. Cell Cloning

Isolation and characterization of a stromal cell line

Curr. Top. Microbiol. Immunol.

Regular Article
2,3,7,8-Tetrachlorodibenzo-p-dioxin Does Not Directly Alter the Phenotype of Maturing B Cells in a Murine Coculture System