The immune system has been identified as a sensitive target for the toxic effects produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Furthermore, the B cell has been identified as a sensitive cellular target of TCDD by previous cell-type fractionation studies from this laboratory. The mechanism responsible for the immunotoxic effects produced by TCDD is unclear; however, many of the biological effects of TCDD are thought to be mediated by the aryl hydrocarbon receptor (AhR). Here, we describe two B cell lines that differ considerably in their expression of the AhR and in their sensitivity to TCDD. Our results demonstrated a marked expression of the AhR protein in the CH12.LX B cell line but not in the BCL-1 B cell line. Transcripts for the AhR were not detected by reverse transcriptase-polymerase chain reaction in the BCL-1 cells. The AhR nuclear translocator (ARNT) protein was highly expressed in both cell lines. In addition, the AhR and ARNT are functional in CH12.LX cells as demonstrated by TCDD-induced CYP1A1 induction. TCDD did not induce CYP1A1 in BCL-1 cells. Furthermore, TCDD treatment resulted in suppression of lipopolysaccharide (LPS)-induced IgM secretion in CH12.LX cells. Conversely, TCDD-induced inhibition of IgM secretion was not demonstrated in LPS-stimulated BCL-1 cells, implicating a role for the AhR in the inhibition of B cell effector function. LPS-induced differentiation of the CH12.LX cells also resulted in a marked induction of Ahr expression which was not induced in LPS-stimulated BCL-1 cells. These studies have implicated the AhR as a critical factor in TCDD-induced inhibition of IgM secretion and have demonstrated an induction of AhR gene and protein expression after B cell activation.

Suppression of humoral immune responses by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was first reported in the mid-1970s. Since this initial observation, much effort has been devoted by many laboratories toward elucidation of the cellular and molecular mechanisms responsible for the profound impairment of humoral immune responses by TCDD, which is characterized by decreased B cell to plasma cell differentiation and suppression of immunoglobulin production. These efforts have led to a significant body of research demonstrating a direct effect of TCDD on B-cell maturation and function as well as a requisite but as yet undefined role of the aryl hydrocarbon receptor (AhR) in these effects. Likewise, a number of molecular targets putatively involved in mediating B-cell dysfunction by TCDD, and other AhR ligands, have been identified. However, our current understanding has primarily relied on findings from mouse models, and the translation of this knowledge to effects on human B cells and humoral immunity in humans is less clear. Therefore, a current challenge is to determine how TCDD and the AhR affect human B cells. Efforts have been made in this direction but continued progress in developing adequate human models is needed. An in-depth discussion of these advances and limitations in elucidating the cellular and molecular mechanisms putatively involved in the suppression of B-cell function by TCDD as well as the implications on human diseases associated in epidemiological studies with exposure to TCDD and dioxin-like compounds is the primary focus of this review.Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; B cell; humoral immunity; immune suppression; AhR.Halogenated aromatic hydrocarbons such as the polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls are persistent environmental toxicants. The most potent of this class of chemicals is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is produced as a by-product during a variety of industrial and municipal processes, many of which involve the combustion of organic materials in the presence of chlorine. TCDD has generated much public concern due to several highly publicized incidents of human and wildlife exposure (reviewed by White and Birnbaum, 2009). Additionally, the sensitivity of rodent models to TCDD-induced toxicity has fueled concern regarding the potential health risks to humans. TCDD produces a broad range of biological effects in animal and cellular models including death, a generalized wasting syndrome, lymphoid involution (especially of the thymus), hepatotoxicity, cardiotoxicity, teratogenicity, developmental toxicity, carcinogenesis, neurotoxicity, immunotoxicity, and various biochemical effects (reviewed by Birnbaum and Tuomisto, 2000).Alterations in immune function, primarily immune suppression, have been observed in virtually every species studied and occurs at doses that do not produce obvious signs of toxicity in vivo and at noncytotoxic concentrations in vitro (Holsapple et al., 1991b;Kerkvliet, 2002). These alterations include ...

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a known disruptor of B-cell differentiation and a ligand for the aryl hydrocarbon receptor (AhR), induces binding of the AhR to dioxin responsive elements (DRE) in sensitive genes. The Ig heavy chain (IgH) gene is a sensitive target of TCDD and may be transcriptionally inhibited by TCDD through inhibition of the 3′IgH transcriptional regulatory region (3′IgHRR). While the 3′IgHRR contains binding sites for several transcription factors, two DRE motifs were also identified which may be responsible for TCDD-induced inhibition of 3′IgHRR activation and may implicate the AhR as an important regulator of IgH expression. The objectives of the present study were to determine if 3′IgHRR modulation is limited to TCDD or if structurally diverse chemicals (AhR ligands and non-AhR ligands) from environmental, industrial, dietary or pharmaceutical origin are also capable of modulating the 3′IgHRR and to verify a correlation between effects on a stable 3′IgHRR reporter and the endogenous IgH protein. Utilizing a CH12.LX mouse B-cell line that stably expresses a 3′IgHRR-regulated transgene, we identified an inhibition of both 3′IgHRR activation and IgH protein expression by the non-dioxin AhR activators indolo(3,2-b)carbazole, primaquine, carbaryl, and omeprazole which followed a rank order potency for AhR activation supporting a role of the AhR in the transcriptional regulation of the 3′IgHRR and IgH expression. However, modulation of the 3′IgHRR and IgH expression was not limited to AhR activators or to suppressive effects. Hydrogen peroxide and terbutaline had an activating effect and benzyl isothiocyanate was inhibitory. These chemicals are not known to influence the AhR signaling pathway but have been previously shown to modulate humoral immunity and/or transcription factors that regulate the 3′IgHRR. Taken together these results implicate the 3′IgHRR as a sensitive immunological target and are the first to identify altered 3′IgHRR activation by a diverse range of chemicals.

Gold nanoparticles (Au-NPs) have been designated as superior tools for biological applications owing to their characteristic surface plasmon absorption/scattering and amperometric (electron transfer) properties, in conjunction with low or no immediate toxicity towards biological systems. Many studies have shown the ease of designing application-based tools using Au-NPs but the interaction of this nanosized material with biomolecules in a physiological environment is an area requiring deeper investigation. Immune cells such as lymphocytes circulate through the blood and lymph and therefore are likely cellular components to come in contact with Au-NPs. The main aim of this study was to mechanistically determine the functional impact of Au-NPs on B-lymphocytes. Using a murine B-lymphocyte cell line (CH12.LX), treatment with citrate-stabilized 10 nm Au-NPs induced activation of an NF-κB-regulated luciferase reporter, which correlated with altered B lymphocyte function (i.e. increased antibody expression). TEM imaging demonstrated that Au-NPs can pass through the cellular membrane and therefore could interact with intracellular components of the NF-κB signaling pathway. Based on the inherent property of Au-NPs to bind to -thiol groups and the presence of cysteine residues on the NF-κB signal transduction proteins IκB kinases (IKK), proteins specifically bound to Au-NPs were extracted from CH12.LX cellular lysate exposed to 10 nm Au-NPs. Electrophoresis identified several bands, of which IKKα and IKKβ were immunoreactive. Further evaluation revealed activation of the canonical NF-κB signaling pathway as evidenced by IκBα phosphorylation at serine residues 32 and 36 followed by IκBα degradation and increased nuclear RelA. Additionally, expression of an IκBα super-repressor (resistant to proteasomal degradation) reversed Au-NP-induced NF-κB activation. Altered NF-κB signaling and cellular function in B-lymphocytes suggests a potential for off-target effects with in vivo applications of gold nanomaterials and underscores the need for more studies evaluating the interactions of nanomaterials with biomolecules and cellular components.

Transcriptional regulation of the Ig heavy chain gene involves several regulatory elements, including the 3Ј␣ enhancer, which is composed of four distinct regulatory domains. DNA binding sites for several transcription factors, including B cell-specific activator protein, nuclear factor for immunoglobulin chain in B cells, and octamer have been identified within the 3Ј␣ enhancer domains and are believed to be important in regulating 3Ј␣ enhancer activity. We have identified an additional DNA binding motif, the dioxin-responsive element (DRE), which can contribute to 3Ј␣ enhancer regulation. 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD), a known disrupter of B cell differentiation (i.e., decreased plasma cell formation, inhibition of heavy chain expression, and suppression of IgM secretion), induces binding of the aryl hydrocarbon receptor (AhR) nuclear complex to DREs. TCDD also induces AhR binding to the hypersensitive (hs)4 domain of the 3Ј␣ enhancer. Interestingly, TCDD enhances LPS-induced activation of the hs4 domain but profoundly inhibits LPS-induced activation of the complete 3Ј␣ enhancer. Furthermore, site-directed mutational analysis demonstrated that a DRE and B element in the hs4 domain is modulated by TCDD in lipopolysaccharide-activated B cells. We propose that the AhR is a novel transcriptional regulator of the 3Ј␣ enhancer, which can mediate, at least in part, the effects of TCDD on the 3Ј␣ enhancer and its domains, putatively contributing to a marked suppression of IgM production.Regulation of the murine immunoglobulin heavy chain (IgH) locus is governed through a complex interaction of several regulatory elements whose activity is B cell-specific and dependent on the state of B cell maturation. The most 5Ј regulatory element is the V H promoter, which lies immediately upstream of each variable region and contributes to B cell-specific activity of the Ig heavy chain locus. Located between the rearranged VDJ segments and the C constant region is the E, which contributes to B cell-specific activity and is involved early in B cell development where it regulates