Courtney E. W. Sulentic

The Long Winding Road toward Understanding the Molecular Mechanisms for B-Cell Suppression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

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.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Induces Transcriptional Activity of the Human Polymorphic hs1,2 Enhancer of the 3′Igh Regulatory Region

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant known to inhibit Ab secretion and Ig expression. Inhibition of Ig expression may be partially mediated through repression of the 3′Igh regulatory region (3′IghRR). TCDD inhibits mouse 3′IghRR activation and induces aryl hydrocarbon receptor binding to dioxin response elements within the 3′IghRR enhancers hs1,2 and hs4. The human hs1,2 enhancer (hu-hs1,2) is polymorphic as the result of the presence of one to four invariant sequences (ISs), which have been correlated with several autoimmune diseases. The IS also contains a dioxin response element core motif. Therefore, the objective was to determine whether hu-hs1,2 activity is sensitive to TCDD. Using a mouse B cell line (CH12.LX), we compared the effects of TCDD on mouse hs1,2 versus hu-hs1,2 activity. TCDD inhibited mouse hs1,2 similarly to the mouse 3′IghRR. In contrast, hu-hs1,2 was activated by TCDD, and antagonist studies supported an aryl hydrocarbon receptor-dependent activation, which was replicated in a human B cell line (IM-9). Absence of Pax5 binding sites is a major difference between the human and mouse hs1,2 sequence. Insertion of the high-affinity Pax5 site in hu-hs1,2 markedly blunted reporter activity but did not alter TCDD’s effect (i.e., no shift from activation to inhibition). Additionally, deletional analysis demonstrated a significant IS contribution to hu-hs1,2 basal activity, but TCDD-induced activity was not strictly IS number dependent. Taken together, our results suggest that hu-hs1,2 is a significant target of TCDD and support species differences in hs1,2 regulation. Therefore, sensitivity of hu-hs1,2 to chemical-induced modulation may influence the occurrence and/or severity of human diseases associated with hu-hs1,2.

Hydrogen Peroxide Modulates Immunoglobulin Expression by Targeting the 3’Igh Regulatory Region through an NFκB-Dependent Mechanism

Abstract Reactive oxygen species such as hydrogen peroxide (H2O2) appear to play a role in signal transduction in immune cells and have been shown to be synthesized upon antigen-mediated activation and to facilitate cellular activation in B- and T-cells. However, an effect of H2O2 on B-cell function (i.e. immunoglobulin (Ig) expression) has been less well-characterized. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in Ig heavy chain (Igh) gene expression. Igh transcription in B lymphocytes is an essential step in antibody production and is governed through a complex interaction of several regulatory elements, including the 3′Igh regulatory region (3′IghRR). Utilizing an in vitro mouse B-cell line model, this study demonstrates that exposure to low μM concentrations of H2O2 can enhance 3′IghRR-regulated transcriptional activity and Igh gene expression, while either higher concentrations of H2O2 or the expression of a degradation resistant inhibitory κB (IκBα super-repressor) can abrogate this effect. Furthermore, suppressive H2O2 concentrations increased protein levels of the p50 NFκB sub-unit, IκBα, and an IκBα immunoreactive band which was previously characterized as an IκBα cleavage product exhibiting stronger inhibitory function than native IκBα. Taken together, these observations suggest that exposure of B lymphocytes to H2O2 can alter Igh transcriptional activity and Ig expression in a complex biphasic manner which appears to be mediated by NFκB and altered 3′IghRR activity. These results may have significant implications to disease states previously associated with the 3′IghRR.

The effect of shear flow on nanoparticle agglomeration and deposition in in vitro dynamic flow models

Abstract Traditional in vitro toxicity experiments typically involve exposure of a mono- or co-culture of cells to nanoparticles (NPs) in static conditions with the assumption of 100% deposition (i.e. dose) of well-dispersed particles. However, cellular dose can be affected by agglomeration and the unique transport kinetics of NPs in biological media. We hypothesize that shear flow can address these issues and achieve more predictable dosage. Here, we compare the behavior of gold NPs with diameters of 5, 10 and 30 nm in static and dynamic in vitro models. We also utilize transport modeling to approximate the shear rate experienced by the cells in dynamic conditions to evaluate physiological relevance. The transport kinetics show that NP behavior is governed by both gravity and diffusion forces in static conditions and only diffusion in dynamic conditions. Our results reveal that dynamic systems are capable of producing a more predictable dose compared to static systems, which has strong implications for improving repeatability in nanotoxicity assessments.

Transgenic Expression of Spi-C Impairs B Cell Development and Function by Affecting Genes Associated With BCR Signaling

Spi‐C is an Ets family transcription factor closely related to PU.1 and Spi‐B. Expression of Spi‐C is developmentally regulated in the B‐cell lineage, but its function remains unknown. To determine the function of Spi‐C in B‐cell development, we generated mice expressing a B‐cell‐specific Spi‐C transgene under the control of the IgH intronic enhancer. Spi‐C transgenic mice had 50% fewer B cells than wild‐type littermates. Flow cytometric analyses showed that splenic transitional B cells and bone marrow pre‐B or immature B cells from transgenic mice were dramatically reduced compared with those of wild type. Both nonspecific and Ag‐specific serum IgM levels were significantly increased in transgenic mice, while serum IgG levels were significantly decreased compared with wild type. Spi‐C transgenic B cells proliferated poorly after stimulation by anti‐IgM or anti‐CD40 in vitro, although they responded normally to LPS stimulation. Using real‐time RT‐PCR, we found that several BCR signaling‐related mediators were downregulated at pre‐B‐cell and mature B‐cell stages in transgenic mice, while an inhibitor of BCR signaling was upregulated. Taken together, these data indicate that ectopic expression of Spi‐C can impair B‐cell development and function by affecting genes associated with BCR signaling.

The Aryl Hydrocarbon Receptor and Immunity

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 dioxin response element in the multiple cloning site of the pGL3 luciferase reporter influences transcriptional activity.

Luciferase reporter plasmids (pGL3 backbone, Promega) have been utilized to characterize the transcriptional effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands. Following ligand activation, the AhR and its dimerization partner AhR nuclear translocator (ARNT) regulate transcription by binding dioxin response elements (DREs) in regulatory regions of dioxin-sensitive genes. Upon sequencing of our luciferase reporters, we unexpectedly identified a DRE core motif within the multiple cloning site (mcsDRE) of the pGL3 luciferase plasmid backbone in a subset of our reporters. Therefore, the objective of this study was to determine if the mcsDRE inadvertently influences reporter activity. Utilizing deletional analysis we determined that the mcsDRE did significantly alter the transcriptional effect induced by TCDD. Since many chemicals have been shown to interact with the AhR and influence transcription through the DRE, the presence of the mcsDRE in the pGL3 luciferase plasmid may inappropriately influence promoter and enhancer analysis. As such, insertion of regulatory elements into pGL3 reporters should be designed to avoid retaining the mcsDRE core motif (GCGTG) and currently utilized pGL3 reporters should be evaluated for the presence of the mcsDRE.

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

Analysis of Modulation of Immunoglobulin Gene Expression

Immunoglobulins (Ig) are critical in maintaining host immunity to a variety of pathogens. Regulation of Ig expression is a complex process involving transcriptional regulation of different Ig gene loci by many transcription factors and transcriptional regulatory regions. This complexity suggests many possible molecular targets for immunotoxicants. Therefore, thorough evaluation of chemical‐induced modulation of Ig expression may necessitate multiple experimental approaches evaluating: (1) number of B cells secreting antibodies by antibody‐forming cell response or plaque assay; (2) concentration of total secreted antibodies by enzyme‐linked immunosorbent assay (ELISA); (3) cellular proliferation and viability by cell count measurements, [3H] thymidine incorporation, and trypan blue exclusion; (4) Ig mRNA expression by quantitative real‐time reverse transcriptase–polymerase chain reaction (RT‐PCR); (5) transcriptional activity of specific Ig regulatory regions by reporter gene analysis; and (6) transcription factor binding to specific Ig regulatory regions by electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP). These experimental approaches are discussed in the unit, with detailed description of EMSA, EMSA‐western analysis, and isolation of nuclear protein. Curr. Protoc. Toxicol. 36:18.14.1‐18.14.21.