PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 1/98 armor-mu TRANSCRIPTIONAL REGULATION OF IgM EXPRESSION BY 2,3,73- TETRACHLORODIBENZO-p—DIOXIN AND THE AhR/DRE SIGNALING PATHWAY By Courtney E. W. Sulentic A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1999 ABSTRACT TRANSCRIPTIONAL REGULATION OF IgM EXPRESSION BY 2,3,7,8- TETRACHLORODIBENZO—p-DIOXIN AND THE AhR/DRE SIGNALING PATHWAY By Courtney E. W. Sulentic Suppression of the humoral immune response by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is well established in a number of mouse models, including the B6C3F1 mouse, with several studies identifying the B-cell as a primary target of TCDD. The actual molecular mechanism responsible for the TCDD-mediated effects on B-cell function is unclear; however, many of the biological effects produced by TCDD are thought to be mediated by the aryl hydrocarbon receptor (AhR). The AhR signaling cascade involves TCDD binding to the AhR followed by nuclear translocation, dimerization with the AhR nuclear translocator (ARNT) and binding of the AhR nuclear complex to dioxin responsive elements (DRE) within the promoter regions of genes sensitive to TCDD. Although well characterized with the induction of metabolic enzymes, such as CYP1A1, this mechanism had not been directly linked to the effects of TCDD on B-cell function. The objectives of this investigation were four-fold. The first was to determine if the AhR and ARNT are functionally expressed in B6C3F1 mouse splenocytes. Northern and Western blot analysis of mRN A and whole cell lysate, respectively, isolated from mouse Splenocytes identified message and protein for both the AhR and ARNT. Additionally, TCDD induces binding of these proteins to the DRE as detected by an electrophoretic mobility shift assay (EMSA). The second Objective was to characterize the CH12.LX and BCL-l murine B-cell lines as potential models in determining the relationship between TCDD-induced alteration of B-cell function and events mediated by an AhR/ARNT-DRE mechanism. Initial screening by Western analysis demonstrated a marked expression Of the AhR protein in the CH12.LX cell line but not in the BCL—l cell line which was confirmed at the transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR). ARNT mRNA and protein are highly expressed in both cell lines. In addition, the AhR and ARNT protein are functional in CH12.LX cells as demonstrated by TCDD-induced Cypl a1 induction. TCDD did not induce CypIaI induction in the BCL—l cells. Furthermore, TCDD treatment resulted in a concentration- dependent suppression of LPS-induced IgM secretion in CH12.LX cells which was not Observed in the AhR-deficient BCL-l cells. The third objective was to further investigate the role of the AhR in TCDD-induced inhibition of IgM secretion through a characterization of the structure activity relationship (SAR) between inhibition of both immunoglobulin (Ig) secretion and Ig heavy chain transcription and AhR-mediated enzyme induction in CH12.LX cells. The effects Of several polychlorinated dibenzo-p- dioxin (PCDD) congeners on each endpoint followed a SAR. Among the three endpoints, there was also a general concordance between the EC50 and IC50'S for a particular congener. In addition, the PCDD congeners had no effect on IgM secretion, II expression or Cyp] a1 expression in the AhR-deficient BCL-l cells. The final objective was to determine if the AhR nuclear complex can bind to two DRE-like sites identified within the Ig heavy chain 3'Ot enhancer. EMSA-western analysis with the CH12.LX cells demonstrated TCDD-induced binding Of the AhR nuclear complex tO both DRE-like sites as well as TCDD-induced binding of several NF—KB/Rel proteins to a KB Site which overlaps one of the DRE-like Sites. Interestingly, KB binding in the BCL-l cells was induced by TCDD demonstrating an AhR-independent effect on KB binding. However, taken together, the results of this investigation are consistent with an AhR-mediated inhibition of IgM expression which may result from an altered regulation Of the Ig heavy chain gene perhaps due to TCDD-induced binding of the AhR-nuclear complex to DRE- like Sites within the 3'0: enhancer. DEDICATION To my husband, Jon, whose unwavering support, friendship and love have formed the foundation for this work. To my unborn daughter, who has already brought a great deal Of joy and perspective to my life. To my parents, William and Joan, for their many years of support and guidance and for teaching me the value Of education and hard work. To my Sister, Heather, for always being my best friend. iv ACKNOWLEDGMENTS There are many people I wish to acknowledge. First and foremost is my advisor, Dr. Norbert Kaminski, for his support and guidance and for providing a challenging and comfortable learning environment. Dr. Michael Holsapple, who first sparked my interest in immunotoxicology and initiated the funding for this project, has been a valuable contributor as both a coauthor and committee member. The rest of my committee members, Dr. Kathryn Brooks, Dr. Margarita Contreras and Dr. Lawrence Fischer, contributed very lively discussions and many helpful suggestions during, as well as outside of, scheduled committee meetings. The whole Kaminski lab, past and present, have been invaluable for their constructive criticism in lab meetings, entertaining lab discussions and comic relief. I should especially thank Robert Crawford and Robin Condie for their vast scientific knowledge and for their patience with a beginner's endless questions. I also wish to acknowledge Dr. Richard Pollenz, Dr. Gregory Fink, Dr. Ronald Johnson and Dr. Kurunthacha Kannan who have offered valuable advice, help and/or reagents critical to my project. TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................ x LIST OF FIGURES ...................................................................................................... xi LIST OF ABBREVIATIONS ....................................................................................... xiii INTRODUCTION ........................................................................................................ 1 LITERATURE REVIEW ............................................................................................. 3 I. B-cell activation and differentiation ..................................................... 3 A. B-cells and the immune system ................................................ 3 B B-cell activation ........................................................................ 4 C. Regulation of immunoglobulin expression ............................... 7 D Regulators of the immunoglobulin heavy chain 3'Ot enhancer .................................................................... ll 1. The B-cell-specific activator protein ............................ 11 2. The NF-KB/Rel protein family ..................................... 12 H. Toxic effects of TCDD ......................................................................... 14 A. General toxicity of TCDD ........................................................ 14 B. Immunotoxicity Of TCDD ........................................................ 16 III. Role of the AhR in TCDD-mediated immune suppression ........................................................................................... 18 A. The AhR Signaling pathway ..................................................... 18 B. The AhR and TCDD-mediated immunotoxicity ...................... 21 C. The AhR, BSAP and inhibition of CD19 expression ............... 23 MATERIALS AND METHODS ................................................................................. 25 1. Chemicals ............................................................................................. 25 H. Animals ................................................................................................. 25 vi III. Cell Lines .............................................................................................. 25 IV. Northern Blot Analysis ......................................................................... 26 A. RNA Isolation and Analysis ..................................................... 26 B. cDNA Probes ............................................................................ 27 V. Reverse Transcription-Polymerase Chain Reaction ............................. 27 A. RNA Isolation ........................................................................... 27 B. Qualitative RT-PCR Analysis ................................................... 28 C. Quantitative RT-PCR Analysis ................................................. 29 VI. Enzyme-Linked Immunosorbent Assay for IgM .................................. 30 VII. Whole Cell Lysate Protein Preparation ................................................ 30 VIII. Western Blot Analysis .......................................................................... 31 IX. Slot Blot Analysis ................................................................................. 32 X. Nuclear Protein Preparation .................................................................. 32 XI. Electrophoretic Mobility Shift Assay ................................................... 33 A. Analysis of Protein-DNA Complexes ...................................... 33 B. Synthetic DRE Oligonucleotides .............................................. 34 XII. EMSA-Westem Analysis ...................................................................... 34 XIII. Statistical Analysis of Data ................................................................... 35 EXPERIMENTAL RESULTS ...................................................................................... 36 1. Identification of AhR and ARNT in B6C3F1 mouse Splenocytes ............................................................................................ 36 A. Northern blot analysis of mouse splenocytes ........................... 36 B. Western blot analysis of mouse splenocytes ............................ 36 C. Slot blot and quantitative RT-PCR analysis of mouse splenocytes .................................................................... 40 D. EMSA analysis of mouse splenocytes ...................................... 40 II. Characterization Of the AhR and ARNT in the CH12.LX and BCL-l B-cell lines ................................................................................ 43 A. AhR and ARNT expression in two B-cell lines ........................ 43 vii B. AhR and ARNT regulate gene transcription in the CH12.LX B-cell line ................................................................. 48 C. TCDD alters immune function in CH12.LX B-cells but not in AhR-deficient BCL—l B-cells ......................................... 48 D. Differential expression of AhR in LPS-differentiated CH12.LX B-cells ...................................................................... 51 E. TCDD does not alter Ahr expression in naive or LPS- stimulated CH12.LX cells ........................................................ 51 F. Ahr upregulation does not enhance the sensitivity of activated CH12.LX cells to TCDD ........................................... 60 III. The SAR of several PCDD-mediated endpoints and AhR binding affinity ..................................................................................... 64 A. PCDD-mediated inhibition of LPS—induced IgM secretion in CH12.LX B-cells follows an SAR which is concordant with AhR ligand binding affinity and CypIaI induction ................................................................................... 64 B. Specific PCDD congeners have no affect on Cypl a1 expression or LPS-induced IgM secretion from the AhR-deficient BCL-l B-cells ........... 4 ...................................... 68 C. LPS-induced u expression in CH12.LX cells is inhibited by PCDD congeners and follows an SAR for AhR binding .............................................................................. 68 D. Inhibition of IgM protein secretion and II expression is AhR-dependent ..................................................................... 72 IV. Alteration of protein binding at the 3'OL Ig heavy chain enhancer by TCDD .............................................................................................. 74 A. TCDD induces binding to a DRE-like Site located within the 3'OtE(hsl,2) and 3'OL-hs4 enhancers ..................................... 74 B. TCDD induces binding to a KB site located within the 3'Ot-hs4 enhancer ....................................................................... 81 DISCUSSION ............................................................................................................... 88 1. Functional AhR and ARNT in murine splenocytes .............................. 88 II. AhR-dependent suppression by TCDD of IgM secretion in activated B-cells .................................................................................... 92 III. Transcriptional regulation Of IgM expression by TCDD and the AhR/DRE signaling pathway .......................................................... 96 viii LITERATURE CITED ................................................................................................. 102 ix LIST OF TABLES Table Page 1. Quantitative analysis of Ahr and Amt mRN A as determined by RT-PCR ....................................................................................................... 42 2. Congener Specific IC50 for inhibition of LPS-induced [.1 expression and protein secretion or EC50 for induction of Cypl a1 ................................... 66 LIST OF FIGURES Figure Page 1. The 3'Ot enhancer of the mouse Ig heavy chain gene ........................................ 9 2. The AhR signaling pathway ............................................................................. 19 3. Northern blot analysis of Ahr and Amt mRN A expression in splenocytes and liver. ....................................................................................... 37 4. Western blot analysis Of the AhR and ARNT in splenocytes and liver ............................................................................................................ 39 5. Slot blot analysis Of the AhR and ARNT in splenocytes and liver .................. 41 6. Binding of Hepa 1c1c7 or splenocyte nuclear proteins to a DRE .................... 44 7. Western blot analysis for the AhR and ARNT protein in the CH12.LX and BCL-l cell lines ........................................................................ 46 8. Basal expression of Ahr and Amt transcripts in the CH12.LX and BCL-l cell lines ......................................................................................... 47 9. Dose response and time course of TCDD-induced CypIaI induction in CH12.LX cells .............................................................................. 49 10. Effect of TCDD on CypI a1 induction in BCL—l cells ..................................... 50 11. Effect of TCDD on LPS-induced IgM secretion in CH12.LX cells ................. 52 12. Effect of TCDD on LPS-induced IgM secretion in BCL-l cells ...................... 53 13. Effect of LPS-induced differentiation on Ahr expression in the CH12.LX cells .................................................................................................. 54 14. Ahr expression in LPS-stimulated CH12.LX and BCL—l cells ........................ 55 15. AhR protein expression in LPS-stimulated CH12.LX cells ............................. 56 16. Amt expression in LPS-stimulated CH12.LX cells .......................................... 57 17. Effect of TCDD on Ahr expression in the CH12.LX cells ............................... 58 18. Effect of TCDD on LPS-induced Ahr expression in the CH12.LX cells .................................................................................................. 59 19. Time course of CypI a1 induction in CH12.LX cells cotreated with LPS and TCDD ......................................................................................... 61 xi 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Effect of a 24 hr LPS cotreatment on TCDD-induced CypIaI induction in CH12.LX cells .............................................................................. 62 TCDD-induced Cypl a1 induction in LPS pretreated CH12.LX cells .............. 63 Concentration-dependent effect Of selected chlorinated dibenzo-p- . dioxin congeners on CypI a1 expression in CH12.LX cells ............................. 65 Concentration-dependent effect Of selected chlorinated dibenzo-p- dioxin congeners on LPS-induced IgM secretion from CH12.LX cells ........... 67 Effect of selected chlorinated dibenzo-p-dioxin congeners on CypIaI expression in BCL-l cells ................................................................... 69 Effect Of selected chlorinated dibenzo—p-dioxin congeners on LPS-induced u expression and IgM secretion in BCL-l cells ......................... 7O Concentration-dependent effect of a 24 hr incubation with selected chlorinated dibenzo-p-dioxin congeners on [.1 expression in CH12.LX cells ............................................................................ 71 Concentration-dependent effect of a 48 hr incubation with selected chlorinated dibenzo-p-dioxin congeners on [.1 expression in CH12.LX cells ............................................................................ 73 Oligomers derived from the 3'0: enhancer used in the EMSA analysis ............................................................................................................. 75 TCDD-induced binding to a DRE-like Site within the 3'OtE(hsl,2) enhancer ........................................................................................ 76 TCDD-induced binding to a DRE-like site within the 3'Ot-hs4 enhancer ............................................................................................... 78 TCDD-induced binding of NF-KB/Rel proteins from CH12.LX cells to a KB binding site within the 3'0t-hs4 enhancer ..................................... 82 TCDD-induced binding of NF-KB/Rel proteins from BCL-l cells to a KB binding site within the 3'Ot-hs4 enhancer ..................................... 85 xii 2,4-D 2,4,5-T 3'OtE(hs 1 ,2) 3'a-hs4 AFC AhR Ahrbb Ahrdd BCA B-cell bHLH BSA BSAP BCR COTE CAT CD cDNA CMI CYP1A1 DCDD LIST OF ABBREVIATIONS 2,4-dichlorophenoxyacetic acid 2,4,5-trichlorophenoxyacetic acid 3'Ot enhancer, hypersensitive site 1 and 2 3'0: enhancer, hypersensitive site 4 antibody forming cell aryl hydrocarbon receptor high-responsive Ahr allele low-responsive Ahr allele AhR-interacting protein AhR nuclear translocator bicinchoninic acid bursal or bone marrow derived cell basic helix-loop-helix bovine serum albumin B-cell -Specific activator protein B-cell receptor 3'Ot enhancer, 5' of hs(1,2), 3 and 4 chloramphenicol acetyl transferase cluster of differentiation complimentary DNA heavy chain constant region cell-mediated immunity cytochrome P-4501A1 Ig diversity region 2,7-dichlorodibenzo-p-dioxin xiii DMSO DNA DNP—Ficoll DRE Eu ELISA EMSA ER EROD GC/MS HAH HPLC hs3 hsp HxCDD LBP LPS dimethyl sulfoxide deoxyribonucleic acid dinitrophenyl haptenated ficoll dioxin responsive element u heavy chain enhancer enzyme-linked immunosorbent assay electrophoretic mobility shift assay estrogen receptor 7-ethoxyresorufin O—deethylase gas chromatography/mass spectrophotometry halogenated aromatic hydrocarbons humoral immunity high performance liquid chromatography 3'a enhancer, hypersensitive Site 3 heat shock protein 1 ,2,3,4,7 ,8-hexachlorodibenzo—p—dioxin immunoglobulin Ig heavy chain inhibitory KB IKB kinase Interleukin interferon-7 internal standard Ig joining region LPS-binding protein lipopolysaccharide heavy chain gene for IgM xiv MHC MCDD mlcls mRN A NF-KB OD PAGE PBS PCDD PEST PLC RT-PCR SAR SDS sRBC TCDD T—cell TNP-LPS TriCDD major histocompatibility complex 1-monochlorodibenzo-p-dioxin molecules messenger RNA nuclear factor-K light chain of B—cells Optical density polyacrylamide gel electrophoresis phosphate-buffered saline polychlorinated dibenzo-p—dioxins proline-glutamine-serine-threonine phospholipase C ribonucleic acid reverse transcription-polymerase chain reaction structure activity relationship sodium dodecyl sulfate sheep red blood cell tris-buffered saline 2,3,7,8-tetrachlorodibenzo-p«dioxin thymic derived cell T-dependent T-independent trinitrophenyl haptenated LPS 2,3,7-trichlorodibenzo-p-dioxin transfer RNA Ig variable region heavy chain variable region vehicle XV INTRODUCTION Alterations in immune function are among the earliest and most sensitive responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (T CDD) exposure in most animal models. Previous cell separation-reconstitution experiments have identified the B-cell as a primary target of TCDD. In addition to immune suppression, TCDD has been Shown to mediate a variety of toxic effects in a broad range of tissues, the best characterized of which is the liver. In the liver, TCDD acts as a tumor promoter as well as an inducer of the drug metabolizing isozyme, cytochrome P-4501A1 (CYP1A1). Induction of CYP1A1 by TCDD is mediated through the binding of TCDD to the cytosolic aryl hydrocarbon receptor (AhR). Once bound by TCDD, the ligand-receptor complex translocates to the nucleus where it forms a heterodimer with the AhR nuclear translocator (ARNT). The TCDD/AhR-ARNT protein complex is capable Of functioning as a transcriptional regulator through direct binding to DNA at the dioxin-responsive enhancer (DRE). There is evidence for an AhR-dependency of TCDD-mediated immune suppression based on earlier studies which utilized: a) Ah high-responsive (Ahbb) and Ah low-responsive (Ahdd) mouse strains (1); b) congenic mice at the Ah locus (2); and c) structure activity relationships between various AhR ligands and inhibition Of the antibody forming cell (AFC) response (3, 4). However, these findings were tempered by the fact that: (a) although present in lymphoid tissues, the AhR and ARNT had not been demonstrated in immunocompetent cells; (b) using rat and guinea pig spleen extracts, TCDD did not induce binding of the AhR/ARNT heterodimer to the DRE (5); (c) the low affinity AhR ligand, 2,7-dichlorodibenzo-p-dioxin (DCDD) and TCDD produce comparable inhibition of the anti-sheep red blood cell (sRBC) IgM AFC response following subchronic treatment of mice in vivo and following direct addition to naive splenocytes in vitro (6); and (d) subchronic TCDD treatment produces a marked immunosuppression in both DBA/2 (Ah low-responsive) and B6C3Fl (Ah high- responsive) mice (7). From these results, it is unclear whether the AhR is a mediator of the immunotoxic effects of TCDD. The purpose of this investigation was to test the following hypothesis: TCDD acts directly on B-cells to suppress immunoglobulin secretion; this suppression is mediated by the AhR which adversely regulates immunologically relevant genes possessing DRES in their 5' regulatory regions. Initial experiments focused on identifying functional components of the AhR signaling pathway in B6C3F1 mouse splenocytes which has been the historical model of our laboratory for studying the effects of TCDD on immune competence. To more efficiently test our hypothesis, subsequent experiments focused on developing and characterizing a B-cell line model which has provided considerable insight into the mechanism of TCDD-induced alteration of B-cell function. LITERATURE REVIEW 1. B-cell activation and differentiation A. B-cells and the immune system A functional immune system plays a central role in maintenance of health. Its ability to maintain protection from infectious diseases is dependent on distinguishing foreign or "nonself" antigens from "self" antigens and then to neutralize and/or eliminate the foreign antigen. The immune system is not confined to a Single site in the body unlike most organ systems. It is composed of numerous lymphoid organs and cells. Immune cells circulate through the blood and lymph and are capable of migrating to virtually any location in the body. Primary lymphoid organs provide a microenvironment capable of supporting the production or maturation of immunocompetent cells. For example, all leukocytes and platelets are produced in the bone marrow. Maturation of these cells, except T-cells, occurs in the bone marrow as well; T-cells mature in the thymus. The site of antigen contact with virgin lymphocytes (T- and B-cells) occurs in secondary lymphoid organs such as the Spleen and lymph nodes which filter the blood and the fluid surrounding the tissues, respectively, for antigens. Antigen recognition results in lymphocyte activation, proliferation and differentiation into effector or memory cells. There are two functional divisions of mammalian immunity, innate and acquired immunity. Innate immunity encompasses the intrinsic, nonspecific host defenses and involves anatomic, physiologic, endocytic and phagocytic, and inflammatory defense mechanisms. Acquired immunity is mediated by lymphocytes following exposure to antigen and exhibits specificity, diversity, memory, and self/nonself recognition. Acquired immunity is further divided into cell-mediated immunity (CMI) and humoral immunity (HI). CMI defends the host against intracellular bacteria, viruses, and cancer. This immunity is mediated by antigen-specific T-cells which mature in the thymus and by various nonspecific cells of the immune system. HI defends the host against extracellular bacteria, parasites, and foreign macromolecules and is mediated by soluble antibodies which are secreted from differentiated B-cells (plasma cells or AFCS). Secreted antibodies coat a specific antigen and facilitate antigen clearance by recruiting complement components and nonspecific immune cells which destroy and remove the antigen. Antibodies or immunoglobulins (Ig) are composed of two identical heavy chains and two identical light chains. There are five distinct classes of Ig which possess a characteristic type of heavy chain. For example, the heavy chains for IgM, IgG, IgA, IgE and IgD are encoded, respectively, by it, y, or, e, and 8 heavy chain genes. Each class of Ig appears to have unique biological properties. For example, IgM is pentameric or hexameric resulting in high antigen valence, has relatively low affinity for antigen and is the major Ig involved in a primary antibody response. IgG is monomeric, has high affinity for antigen, can cross the placental barrier and is the hallmark of a secondary antibody response. IgE is also monomeric and is involved in allergic and anti-parasitic responses. IgA is monomeric or dimeric, is very efficient at bacterial lysis and is the main secretory antibody. IgD is monomeric, is a major surface component on many B-cells and has unknown biological properties. In vivo and in vitro antigen activation of B—cells leading to differentiated AFCS requires the coordinated interaction of several different immune cells, such as macrophages, T-cells, and B-cells for the T-dependent antigen, sRBC; macrophages and B-cells for the T-independent antigen, dinitrophenyl (DNP)-ficoll; and only B-cells for the polyclonal activator, lipopolysaccharide (LPS). B. B-cell activation AS alluded to above, B-cells can be activated in a T-dependent or -independent manner. Antibody responses to most protein antigens are dependent on T-helper cells since most of these responses are not elicited in athymic mice (8). In a T-dependent (TD) response, naive B-cells circulate through blood and lymph until they contact a specific antigen which usually occurs in the spleen or lymph nodes. Antigen recognized by the B- cell receptor (BCR) is internalized, processed and presented on the B-cell surface in association with major histocompatibility complex (MHC) class H molecules. The same antigen is phagocytosed by other antigen presenting cells (macrophages and/or dendritic cells), processed and presented to T-cells, again, in the context of MHC class H molecules. T-cells that recognize the processed antigen are activated to search for B-cells presenting the same processed antigen. T- and B-cells with the same antigen Specificity form a stable association through an interaction of surface ligands and receptors on either cell. This association allows the T-cell to provide the B-cell with activation and differentiation Signals. These signals initiate from membrane-bound molecules and soluble cytokines and interact with surface molecules on B-cells. T-cell signals induce B-cells to proliferate and terminally differentiate into plasma cells. It should be noted that a T-cell can provide differentiation signals to a B-cell in an antigen nonspecific manner. Though both cells are initially activated by different antigens, they are Still capable of associating through cell- surface molecules. This association results in B-cell differentiation and secretion of antibodies specific for the antigen that originally activated the B-cell, not the T-cell. Though this type of humoral response is generally very weak, it may be associated with background antibody titers as well as certain autoimmune reactions. An initial response or primary response to a TD antigen results in a burst of IgM production at the Site of B-cell activation; however, some B-cells, prior to differentiation, migrate into the follicular region Of secondary lymphoid organs and initiate germinal centers. Activated B-cells forming germinal centers undergo rapid proliferation along with rapid somatic mutation of their Ig genes. Following somatic mutation, germinal center B- cells experience positive selection for high affinity membrane-bound lg. lg class switching which is independent from affinity maturation (somatic mutation and selection) also occurs in germinal center B-cells (9). Positively selected B-cells will either terminally differentiate to plasma cells or to quiescent memory cells. Most plasma cells live for only a few days before programmed cell death; however, these cells may migrate to the gut or bone marrow, where they secrete antibody and may live for more than 20 days (8). The second and subsequent responses to a particular antigen are more rapid and Of a longer duration than a primary antibody response. In addition, the antibody titer is greater in a secondary response and consists primarily Of IgG as Opposed to IgM from the primary response. This enhanced secondary antibody response is due to immunologic memory. T-independent (TI) antigens have been classified as TI-l or TI-2 based on mechanistic differences in antibody responses to these antigens. TI-l antigens such as LPS, are generally polyclonal B-cell activators; whereas, TI-2 antigens such as viral particles, are usually repeating polymers that are not polyclonal activators of B-cells (8). LPS is the best studied Of the bacterial components; however more progress has been made on how macrophages recognize LPS as opposed to B-cells. Macrophages respond to lower concentrations of LPS (1 ng/ml) as compared to those concentrations (10 ug/ml) required to initiate a B—cell polyclonal response. This greater sensitivity of macrophages to LPS is due to expression of the cluster of differentiation 14 (CD14). CD14, a glycosyl- phosphatidylinositol-linked protein, has no transmembrane or cytoskeletal domains and therefore, has no signaling capacity. CD14 has high affinity for LPS which is generally transferred to CD14 from the LPS-binding protein (LBP) (10). Macrophages lacking CD14 still respond to LPS but like B-cells, require higher concentrations. This has led to the theory that macrophages and B-cells express Similar low affinity LPS receptors and that at high concentrations LPS can directly bind tO this receptor and stimulate both macrophages and B-cells (10). Most B-cells are stimulated by LPS to proliferate and differentiate resulting in the polyclonal B-cell activator phenomenon. LPS at low concentrations only stimulates macrophages and B-cells if another receptor (i.e., CD14 for macrophages and BCR with LPS specificity for B-cells) is present. This second receptor has high affinity for LPS allowing the adjacent low affinity LPS signaling receptor to bind LPS and induce biological responses in macrophages (production of cytokines and other mediators) and B-cells (proliferation and differentiation). This is only a model of LPS activation and little is known about the existence of an LPS signaling receptor or about the molecular details of LPS activation (8, 10). The strength of an antibody response varies among antigens; some antigens are more efficient than others at inducing BCR signaling. For example, a soluble antigen triggers a moderate antibody response but requires additional signals from accessory cells (macrophages and T-cells). A cell-bound antigen produces a strong antibody response due to the polyvalent nature Of the antigen and to cell-cell adhesion. A polysaccharide antigen elicits a very Strong antibody response through crosslinking of many BCRs. This response has the least requirement for additional signals from T-helper cells and macrophages. Bacterial cell walls contain both repetitious polysaccharides and polyclonal B-cell activators. These antigens are thus potent stimulators of B-cell activation due to a combination of strong Signaling via the BCR of antigen Specific B-cells (TI-2 response) with the nonspecific induction of B-cell terminal differentiation by polyclonal activators (T I-l response) (8). C. Regulation of immunoglobulin expression The transcriptional ,1 heavy chain enhancer, Eu, lies between the variable (V1.1) and constant (CH) regions of an assembled u heavy chain and was identified by its effects on immunoglobulin heavy chain (IgH) gene transcription and has been shown to enhance the variable (V)-diversity (D)-joining (J) recombination process (11, 12). Although Eu, in conjuction with a IgH promoter, can result in tissue-specific expression of an Ig gene, they are not sufficient for high level IgH gene expression in viva (13-15). This Observation and the identification of several Ig-secreting cells that function normally in the absence of Eu, suggested the existence of additional IgH locus enhancers (16-19). A DNA segment with transcriptional enhancer activity was identified at the 3'-end of the murine IgH locus (20, 21). This enhancer, 3'OIE(hsl,2) [3'Ot enhancer, hypersensitive sites (hs) 1 and 2], lies 13 kb downstream of the on gene and has been implicated in the induction of germline transcripts and class switching to most downstream CH genes (22). It was soon discovered that the 3'OtE(hsl,2) enhancer was one of four enhancers contained within an approximately 50 kb DNA segment (23-25). These enhancers [Ca3'E, 3'0tE(hs1,2), hs3 and 3'Ot-hs4], collectively referred to as the 3'Ot enhancer, are thought to form a locus control region and have been divided into two separate structural and functional units (21, 26) (see Figure 1). Unit I is approximately 25 kb and includes the Ca3'E, 3'aE(hsl,2), and hs3 enhancers. This unit has extensive dyad symmetry with 1067 bp of Ca3'E and hs3 being virtually identical (26, 27). All three enhancers of Unit I are DNase I hypersensitive late in B-cell differentiation (activated B-cells or plasma cells) (20, 26, 28). The 3'aE(hsl,2) enhancer has transcriptional activity only in B-cells and plasma cells as determined by transient transfection assays (20, 29-33) and studies with transgenic mice (13). In contrast, Ca3'E and hs3, individually, have no activity in pre-B-cells (immature B-cells), B-cells or plasma cells (23, 25, 26, 33); however each enhancer in duplicate or paired together has substantial activity in plasma cells (25, 26). Interestingly, a chloramphenicol acetyl transferase (CAT) construct containing Ca3'E and Eu had greater activity than Eu alone in a plasma cell line. Synergy between these two regulatory domains did not occur in a pre-B- or B-cell line (33). Synergy also occurs with the enhancers of Unit I since addition of all three enhancers results in greater activity in a B-cell and plasma cell line than with 3'OtE(hsl,2) alone (33). Addition Of Eu to a construct containing Unit I dramatically increases enhancer activity in a plasma cell line; again this synergy did not occur in a pre-B- or B-cell line (33). £8558 v3.5.4” 98 AmefimBh 05 563, 3:82 mm 8% Batman < .5825 359% 05 35:8 fl E5 582858 mm: c5 ANAmSm—dh dado 05 83:8 H as .38: E5583 93 mo 389:8 mm 628:5 5h 05. .oeow Ego ban: 3 8:08 05 me 32:23 6h 25. A 9—53 a. 55 g 0 § _||_ _Al Al |v_ __ 2:3 _ 2:3 Unit H of the 3'Ot enhancer contains a Single enhancer, hs4, which is DNase I hypersensitive at both the pre-B and plasma cell stage (24, 28). This enhancer is transcriptionally active in a pre-B- and B-cell line but lacks activity in a plasma cell line (33). Interestingly, addition of Eu does not enhance the activity of hs4 and in a pre-B-cell line the combined activity is less than Eu alone (33). The intact 3'0t enhancer (Unit I and H) has greater activity in a pre-B—cell, B-cell or plasma cell line than any of the individual enhancers alone or in combination (33). This activity was also greater than Eu alone in the B-cell and plasma cell line but equal to By. in the pre-B-cell line (33). Addition of Eu greatly enhanced activity of the 3'OI enhancer in a pre-B- and B-cell line but not in a plasma cell line (33). The interplay between the individual enhancers appears to be complex, stage specific and not fully understood. In general, synergy of the entire 3'0: enhancer with Eu appears to be more important in pre-B- and B-cell lines. The hs4 enhancer appears to be more active in pre-B- and B-cell lines as compared to plasma cells whereas Ca3'E, 3'OLE(hS1,2) and hs3 seem to be more active in B-cell and plasma cell lines. It has been proposed that hs4 may have distinct functions at the pre-B cell stage, perhaps to facilitate early stages of development (c. g. V(D)J recombination), and that at the B-cell and plasma cell stage the 30: enhancers may function cooperatively to control events such as Ig transcription and class switching (34, 35). In fact, a variant of a plasmacytoma which expresses very low levels of IgA has a complete. deletion of the 3'Ot enhancer (24, 36). Replacement of the 3'OLE(hs1,2) with a neo gene in a plasma cell line results in decreased IgH gene expression (37). A similar experiment in mice resulted in deficient class switching to certain isotypes; secretion of IgM, IgG1 and IgA was normal (22). However, an approximately 2-fOld increase or decrease in u expression has been observed following activation or repression, respectively, of the 3'OtE(hS1,2) enhancer (13, 20, 38). Addition of the other enhancer domains might result in a more profound effect on [.1 expression as was seen with OI expression following a complete deletion of the 3'Ot enhancer (24, 36). 10 The Specific regulation of the 3'Ot enhancer has not been elucidated; however, several DNA binding proteins, including BSAP, NF-KB, Oct proteins and a G-rich DNA binding protein have been Shown to affect 3'0tE(hsl,2) and hs4 enhancer activity at various B-cell stages (31, 32, 34, 39). Interestingly, LPS activates the 3'(X.E(hsl,2) enhancer (13) and LPS will induce a class switch to IgG3 and IgGZb isotypes (40). Conversely, the aforementioned isotypes are Significantly decreased in 3'OtE(hsl,2) knockout mice (22). D. Regulators of the immunoglobulin heavy chain 3'01 enhancer 1. The B-cell-specific activator protein The B-cell-Specific activator protein (BSAP) is encoded by the Pax5 gene and is a member Of the highly conserved Pax-gene family of transcription factors (41). Within the hematopoietic system, BSAP expression and DNA binding activity are restricted to the B- cell lineage and are initiated in pro-B cells continuing throughout the B—cell maturation pathway until the cells terminally differentiate into plasma cells in which BSAP is no longer expressed (42). Various binding sites for BSAP have been identified in the promoter regions of B-cell Specific genes such as genes encoding CD19 (43), Blk (44), lambda 5, and VpreBl (45). These genes play important roles in early B-cell development (lambda 5 and Vpch1) and surface Ig Signaling (CD19 and Elk). In addition BSAP appears to positively regulate promoters of these genes implying an important role Of BSAP in immature B-cell development. This implication was confirmed by the demonstration that Pax5-knockout mice failed to produce small pre-B-, B-, and plasma cells (46). BSAP binding sites have also been identified 5' to and within the switch regions of Ig heavy chain genes (47-49). Wakatsuki and coworkers (50) demonstrated an inhibition of isotype switching induced by various stimuli, most notably, LPS plus interleukin-4 (H.- 4), after downregulation of BSAP by antisense Oligonucleotides. However, the authors 11 caution that this effect may be a more general effect on cell proliferation since proliferation has been shown to be a prerequisite of isotype switching. In addition to these positive effects of BSAP on gene regulation, BSAP has been shown to negatively regulate the murine Ig heavy chain 3'OIE(hsl,2) enhancer in B-cells (31, 32, 34). Suppression of this enhancer by BSAP may lead to downregulation of Ig gene transcription and would explain the lack of BSAP expression in plasma cells whose main function is high-rate Ig gene transcription (50). Studies utilizing the CH12.LX B-cell lymphoma demonstrated that BSAP negatively regulates the 3'OIE (hs1,2) by suppressing the downstream binding of a 40-kDa protein (NF-OIP) that positively affects enhancer activity and Ig gene transcription (31). BSAP also regulates another 3'Ot enhancer, 3'Ot- hs4. In contrast to 3'OtE(hsl,2), BSAP has been shown to positively regulate 3'Ot-hs4 in the A—20 B-cell line, implying distinct functions of these 3'OL enhancers which are dependent on the maturation state of the B-cell (34). Opposite effects of BSAP at these two enhancers is probably due to a difference in protein-protein interaction rather than differences in BSAP binding. 2. The N F-KB/Rel protein family The NF-KB/Rel protein family has been divided into two groups based on structure, function and mode of synthesis (51, 52). The p50 (NF-KB 1) and p52 (NF-K32) proteins which are synthesized as precursor proteins of 105 and 100 kDa, respectively, comprise the first group, while the second group consists of p65 (RelA), c-Rel (Rel) and RelB which are not synthesized as precursors. Both groups possess the Rel homology domain which is responsible for protein dimerization, nuclear localization and DNA binding. In addition to the Re] homology domain, p65, c-Rel and RelB, unlike p50 and p52, also have one or more transcriptional activation domains. Inactive NF-KB/Rel proteins are anchored in the cytoplasm as homo- or heterodimers bound to a member of the 12 IKB protein family (IKB-0L, IKB-[3, IKB-y, IKB-e) or as heterodimers of a processed NF- KB/Rel protein and an unprocessed NF-KB/Rel precursor (53, 54). The IKB protein family contain an ankyrin repeat motif important for an interaction between NF-KB/Rel proteins and a C-terminal PEST sequence which may be involved in protein degradation (55). NF- KB/Rel inducers such as, phorbol esters, calcium ionophores, inflammatory cytokines, bacterial LPS, antigen receptor cross-linking of T- and B-cells, oxidative and physical stress and viral infection, appear to activate NF-KB/Rel proteins via several different signal transduction pathways that converge at phosphorylation of IKB and of NF-KB/Rel precursors (51, 54-57). IKB is phosphorylated by an IKB kinase (IKK) which is a large molecular weight complex (500-900 kDa) composed of several polypeptides, two of which hold the kinase activity (IKKOI and IKKB) (54, 58). Phosphorylation Of IKB and of NF- KB/Rel precursors appears to result in ubiquitination and subsequent proteosome processing and release of NF-KB/Rel which translocates to the nucleus and activates target genes (59, 60). A wide variety of genes are targeted by NF-KB/Rel proteins including genes encoding viruses, immunoreceptors, cell adhesion molecules, cytokines, hematopoietic growth factors, acute phase proteins and transcription factors (51). In B-cells, NF-KB/Rel proteins are involved in cellular activation and Ig gene expression. In fact, NF-KB was first identified by its binding to a specific DNA site within the Ig K light chain intronic enhancer (61). Interestingly, in pre-B cell lines NF-KB must be activated by PMA or LPS; whereas in B-cell and plasma cell lines, NF-KB is a constitutively active nuclear protein complex (51, 59). The constitutive form of NF-KB is primarily composed of c-Rel-pSO heterodimers; p65-p50 heterodimers are inactive and are at normal levels in the cytosol (62, 63). Constitutive nuclear levels Of c-Rel-p50 may be due to an increased instability of IKBOI and by increased transcription of the c-Rel gene (63). In addition, proliferative responses as well as Ig production to certain stimuli was impaired in B-cells from both p50 and c-Rel knockout mice (64-66). These specific effects were not observed with p65 and RelB knockout mice (67-69). In agreement with these 13 results, Michaelson and coworkers (34) have demonstrated KB binding sites within the 3'OLE(hS1,2) and 3'OI-hs4 domains of the Ig heavy chain 3'Ot enhancer. NF-KB/Rel proteins bind to both Of these KB sites (34). In a B-cell line, c—Rel and p50 bind to the 3'0tE(hsl,2) KB site and RelB, p50 and p52 bind to the 3'a-hs4 KB site. As homodimers or heterodimers with each other, p50 and p52 are negative regulators of gene transcription. The protein composition for the 3'OtE(hsl,2) KB site is slightly altered in a plasma cell line in that RelB, in addition to c-Rel and p50 was identified. Protein binding to the 3'Ot-hs4 KB site in plasma cells was not assessed. Interestingly, NF-KB/Rel proteins negatively regulate, in conjunction with BSAP, 3'OtE(hsl,2) activity in two B—cell lines but positively regulate this enhancer in a plasma cell line as determined by transient transfection assays (34). In contrast, NF-KB/Rel proteins positively regulate the 3'Ot-hs4 activity in the two B- cell lines as well as in the plasma cell line (34). In addition, octamer binding to sites within the 3'OtE(hsl,2) and 3'OL-hs4 enhancers results in the same regulation profile as NF- KB/Rel proteins except that octamer binding had no effect on 3'Ot-hs4 activity in a plasma cell line (31, 34, 39). II. Toxic effects of TCDD A. General toxicity of TCDD Halogenated aromatic hydrocarbons (HAH) such as the polychlorinated dibenzo-p— dioxins (PCDD), dibenzofurans and biphenyls are persistent environmental toxins. TCDD has been considered the prototype of HAHS because of its biological potency in experimental animals. TCDD is a true contaminant that is produced during the combustion of organic materials in the presence Of chlorine and for example, is formed during the production of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5- trichlorophenoxyacetic acid (2,4,5-T) which are the main components of Agent Orange (a 14 1:1 mixture of 2,4-D and 2,4,5-T). TCDD contamination of Agent Orange has led to the exposure of soldiers and civilians to TCDD during and after the Vietnam war (70). In addition, residents of Alsea, Oregon (70); Times Beach, Missouri (71, 72); and Seveso, Italy (73, 74) have been exposed to TCDD during defoliation of forests with 2,4,5-T; spraying of dirt roads for dust control with TCDD-contaminated chemical wastes; and accidental release of TCDD contaminated chemicals from an industrial plant, respectively. These highly publicized incidents Of human exposure have generated considerable public concern toward this contaminant. TCDD is also formed during combustion Of municipal and industrial wastes, wood pulp and paper bleaching, wood and coal burning, and metal recycling. The toxic effects Of TCDD on humans remains unclear and presently only sufficient evidence exists for a causal relationship between TCDD exposure and the development of soft tissue sarcoma, chloracne and porphyria cutanea (70). A causal relationship between TCDD exposure and the development of various cancers, teratogenicity, neurotoxicity and immunotoxicity remains uncertain (70). However, a wide range of toxic responses have been observed in most animal models after exposure tO TCDD and these include death, generalized wasting syndrome, lymphoid involution (especially of the thymus), hepatotoxicity, teratogenicity, developmental toxicity, carcinogenesis, neurotoxicity and immunotoxicity (reviewed in references 75 and 76). In addition to these toxic effects, TCDD causes oxidative stress, hormonal alterations, changes in phosphorylation patterns, and alteration of gene expression such as induction of metabolic enzymes, most notably specific isozymes of cytochrome P450 such as CYP1A1 (75, 77-81). There has been considerable effort directed towards characterizing the mechanism by which TCDD causes induction of metabolic enzymes; however, no causal relationship has been demonstrated between this induction and the toxic effects of TCDD. Notable variation in the effects of TCDD within and between species has been observed. In terms of LD50, the guinea pig is the most sensitive species to TCDD whereas 15 the hamster is the least sensitive (75). Within species variation has been best characterized with different strains and F1 crosses of mice (82-84). For example, C57Bl/6 and B6C3F1 mice are on the average 10- to 20-fold more sensitive to TCDD than DBA/2 and AKR mice (1, 85). Although there is considerable debate over the actual sensitivity Of humans to the toxic effects of TCDD, the persistence of this contaminant in the environment in addition to its potent toxicity in laboratory animals has resulted in continued public concern regarding the potential health effects of human exposure to TCDD. B. Immunotoxicity of TCDD Alteration Of immune function is among the earliest and most sensitive responses to TCDD exposure in most animal models and occurs at doses which do not produce overt organ toxicity. TCDD has been shown to alter both innate and acquired immunity (reviewed in 86). Because of the marked thymic involution observed in most species after exposure to TCDD, early immunotoxicological studies focused on TCDD-induced alteration of CMI. Initial studies demonstrated an almost complete loss Of cortical derived thymocytes in rats and mice after peri- and post-natal exposure to TCDD (87, 88). However, Greenlee and coworkers (89) later demonstrated that TCDD treatment of the thymus directly induces terminal differentiation of the thymic epithelial cells which plays a major role in thymocyte maturation. These authors conclude that terminal differentiation of the thymic epithelial cells may result in a loss of their ability to support thymocyte maturation. In contrast, Staples and coworkers (90) through irradiation and reconstitution experiments, have identified hemopoietic components which give rise to macrOphages and dendritic cells as opposed to stromal components as the primary mediator of thymic atrophy. In addition several laboratories have Observed a significant suppression of B-cell function (TD antibody response) to acute TCDD exposure at concentrations minimally affecting both thymic weight (1, 91-93) and T-cell function (proliferative response, graft 16 versus host reaction and cytotoxic T lymphocyte response) (94, 95) suggesting a greater sensitivity of H1 to TCDD-induced immune alteration. Interestingly, time of addition studies demonstrate that TCDD must be added to splenocyte cultures within 24 hr of antigen stimulation to produce a significant and potent inhibition Of the plaque forming cell response to sRBC (93). Since this TD antibody response requires both macrophages and T-cells as accessory cells for the production of antigen-specific antibody, a series of studies aimed at characterizing the specific cellular target(s) Of TCDD were initiated. Separation/reconstitution studies and characterization Of in vitro responses to various defined antigens requiring differential cellular cooperativity identified the B-cell as a cellular target for TCDD-mediated humoral suppression (96-98). In these studies the polyclonal response to LPS, the TI responses to trinitrophenyl (TNP)-LPS (96), and DNP-Ficoll and the TD response to sRBC were all comparably suppressed by TCDD (97); notably, only the B-cell is required for all three types of these responses. Results from separation/reconstitution studies in which splenocytes from mice exposed to vehicle or TCDD were separated into various cell populations (macrophages, T-cells and B-cells) and reconstituted in various combinations in cell culture, demonstrated that a significant suppression of the sRBC immune response required TCDD exposed B-cells (97). These findings further supported the conclusion that the B-cell is a cellular target of TCDD. In addition, an increase in protein phosphorylation (99, 100) and Ca2+ influx (101, 102) occur following TCDD-treatment of primary B-cells; these cellular changes have been implicated in TCDD-induced suppression of the antibody response. Because of the above results, our laboratory has focused primarily on elucidating the mechanism responsible for TCDD- mediated B-cell dysfunction. However, it should be recognized that TCDD does have effects on other leukocyte populations and that these effects may also contribute to an alteration in B—cell function. For instance, Shepherd and Kerkvliet (103) have recently identified decreased levels of T-cell derived cytokines as well as H.-12 in TCDD-treated mice. H.-12 drives T-cell differentiation and is produced by antigen presenting cells. From 17 this and previous results, the authors have hypothesized that the effect of TCDD on T-cell function is a consequence Of dysfunctional antigen presenting cells resulting in decreased T helper cell differentiation (103, 104). However, cell separation/reconstitution experiments suggest that TCDD has no effect on the ability of macrophages to support the plaque forming cell response to sRBC and has only a modest effect on T-cell accessory function (97). III. Role of the AhR in TCDD-mediated immune suppression A. The AhR signaling pathway The putative mechanism Of action for TCDD and structurally related compounds is believed to involve the cytosolic AhR and its binding partner, ARNT (75, 76, 105-107) (Figure 2). This mechanism has been primarily elucidated by studying HAH-induced upregulation of drug metabolizing enzymes, such as CYP1A1, in liver and liver-derived cell-lines. The AhR is a 95-110 kDa basic helix-loop-helix (bHLH) type of ligand- dependent transcription factor (105, 106). In the absence of ligand, the AhR is primarily located in the cytoplasm and is complexed with heat shock protein (hsp) 90, c-src and the AhR-interacting protein (AIP) (108-112). Binding of TCDD to the AhR results in disassociation of the cytoplasmic complex and translocation of the liganded AhR into the nucleus where it forms a heterodimer with a structurally related 87 kDa bIH.H protein called ARNT (111, 113-119). Recent results suggest that hsp90 translocates with the AhR into the nucleus where it is displaced by ARNT (Pollenz, personal communication 1999). The AIP and c-src proteins are localized in the cytosol and presumably disassociate from the AhR following ligand binding (109, 110). It has been suggested that c-src is activated following release from the AhR which is consistent with the increase in c-src activity and in protein phosphorylation identified by several laboratories following TCDD treatment (81, 18 99, 100, 120, 121). The other AhR cytosolic partner, AIP, has homology with the FK506-binding protein family and is thought to have a positive influence on AhR-mediated signaling (110). In any case, the ligand-AhR/ARNT complex can act as a transcription factor by binding to DNA at the DRE in the promoter region of sensitive genes such as CYP1A1, glutathione S-transferase, and menadione oxidoreductase (75, 76, 113, 122- 126). This mechanism has only been characterized with the induction of metabolic enzymes and as previously mentioned, this upregulation has not been directly correlated with the toxicity of TCDD. In fact, the most TCDD-susceptible species based on the LDso concentration is the guinea pig which does not exhibit a notable induction of CYP1A1 activity with TCDD treatment (127). In contrast, the least TCDD-susceptible species, the hamster, shows a marked induction of CYP1A1 activity with TCDD treatment (81). Indeed, several other genes such as those encoding plasrninogen activator inhibitor-2, interleukin-1B, transforming growth factor-or and -B, epidermal growth factor receptor, estrogen receptor, c-fos, c-jun, and recombination activating gene have been shown to be upregulated or downregulated following TCDD treatment (128-133). The modulation of non-metabolic genes such as those above, may account, in varying degrees, for the various toxicities observed in animals treated with TCDD. In addition, DRE-like sites have been found in the promoter regions of several of these genes supporting the possibility for transcriptional regulation through the AhR which would be mechanistically analogous to CYP1A1 induction (134). Furthermore, several laboratories have demonstrated novel protein-protein associations involving the AhR and several other proteins some of which are NF-KB, Spl, transcription factor HB, retinoblastoma protein and as mentioned above, the Src family kinase, c-src (109, 135-138). This suggests a possible interaction of the AhR with different signaling pathways and thus, a potential for DRE-independent mediation of some of TCDD'S effects. Four different allelic forms of the murine AhR have been identified and may account for strain differences in sensitivity to TCDD (139). These alleles are separated into 20 two groups based on ligand binding affinity. There are three high affinity alleles, Ahb" (95 kDa), AM” (104 kDa) and Ahb'3 (105 kDa) as well as one low affinity allele, Ahd (104 kDa). Marked species variability in biochemical responses to TCDD may also be a result of differences in the AhR across Species. The most extensively studied group of ligands for the AhR have been the HAHS which are xenobiotics. The actual endogenous ligand for the AhR is unknown; however, the endogenous metabolite bilirubin has been Shown to directly regulate CYP1A1 gene expression in an AhR-dependent manner (140). Dietary and therapeutic AhR ligands such as indolocarbazoles, tryptophan metabolites and omeprazole have also been identified ( 141-143). It has been suggested that indolocarbazoles may be physiological AhR ligands due to their prevalence in the diet and to a virtually equal potency in activating the AhR as TCDD (142). The biological consequences of indolocarbazole and AhR interactions is unknown. B. The AhR and TCDD-mediated immunotoxicity Despite a lack of direct evidence, there is a wide spread belief among researchers in this area that most if not all of the effects produced by TCDD are mediated through binding of the ligand-bound AhR/ARNT complex to DRE motifs present in the 5' regulatory regions of target genes. This belief has been supported by the generally observed parallel relationship between CYP1A1 induction and toxic responses induced by HAH exposure (75, 144, 145). In terms of TCDD-induced immunotoxicity there is evidence for and against the involvement of an AhR/ARNT-DRE-mediated mechanism. Structure-activity relationship (SAR) studies have demonstrated that with few exceptions, high affinity AhR ligands are more immunosuppressive than low affinity ligands (3). Immune suppression following acute exposure to TCDD was shown to segregate with the AhR using mouse strains susceptible (Ah high-responsive) or resistant (Ah low-responsive) to enzyme induction. Ah high-responsive C57BL/6 and C3H/HeN strains were highly sensitive to the 21 immunosuppressive effects of TCDD; whereas, Ah low-responsive DBA/2 and AKR strains were less sensitive to immunosuppression (1, 2, 146). Additional studies using congenic mice at the Ah locus demonstrated that B6 mice expressing the wild-type phenotype Ahbb allele are more sensitive to TCDD-induced suppression of the antibody response than congenic B6 Ahdd mice (2). The Ahbb allele encodes for an AhR with high affinity for ligand, while the Ahdd allele which is the wild-type phenotype for the DBA/2 mouse strain encodes for an AhR with low affinity for ligand (83). In addition Lorean and coworkers (147) identified nuclear [3H]-TCDD by sucrose density gradient centrifugation in human tonsilar cells, thus suggesting the translocation of the AhR-TCDD complex to the nucleus. However, these findings were tempered by the fact that although identified in lymphoid tissues, the AhR and ARNT had not been directly demonstrated in immunocompetent cells. This was an important distinction because of previous results by Greenlee and coworkers (89) demonstrating that the thymic epithelial cells express significantly more binding of [3H]-TCDD as compared to thymocytes and were directly targeted by TCDD. In addition, a study by Neumann and coworkers (148) demonstrated comparable binding of [3H]-TCDD between the splenic capsule and isolated splenocytes. In contrast to SARS observed between AhR binding and immunotoxicity, the low affinity AhR ligand, DCDD, and TCDD produced comparable inhibition of the TD antibody forming cell response following subchronic treatment of mice in viva and following direct addition to naive splenocytes in vitro (6, 149). The condition of TCDD exposure appears to be a factor in segregation of the immunotoxicity with the AhR in that, subchronic TCDD treatment produced a marked irnmunosuppression in DBA/2, B6C3F], and congenic Ahdd mice (7, 149) indicating a loss of the resistance seen in DBA/2 mice after acute TCDD exposure (1, 2, 146). In addition, binding of the AhR/ARNT heterodimer to the DRE following TCDD-treatment has not been detected in spleen using an electrophoretic mobility shift assay (EMSA) which measures the ability of a DNA regulatory protein to bind a specific DNA motif (5); again, questioning if the AhR and ARNT are present and 22 functional in immunocompetent cells. Furthermore, increased protein phosphorylation that did not segregate with the AhR (99) and an increase in Ca2+ influx which may be AhR- independent (101, 102, 132) occur in primary B-cells following TCDD treatment. In light of the above observations, it was unclear what role the AhR plays in mediating the immunotoxicity of TCDD. C. The AhR, BSAP and inhibition of CDI9 expression Masten and Shiverick (150) have identified a DRE core motif within a BSAP binding site that is located in the CD1 9 promoter region. CDI9 is a signal transducing protein that is expressed through the early stages of B-cell development but is lost upon B- cell differentiation to a plasma cell (151). Masten and Shiverick (150) have also demonstrated a TCDD-induced suppression of CD19 mRN A expression in a human B-cell line. They have proposed that this inhibition is a result of the AhR nuclear complex interfering with the binding of BSAP to a common DNA binding site in the CD19 promoter region. This speculation was supported by EMSA analysis and competition experiments in which unlabeled oligonucleotide corresponding to the BSAP binding site in the CD19 promoter region partially inhibits the formation of an AhR/ARNT-DRE complex and is effective at high concentrations in competing with the DRE for binding of the AhR nuclear complex. These results suggest that the AhR nuclear complex is capable of binding the BSAP binding motif with weak affinity in comparison to DRE binding. However, this interaction between BSAP and the AhR is limited to the human CDI9 promoter and to a moderate affinity BSAP site which may not play a prominent role in CD1 9 expression (43). Therefore, the inhibition of CD19 expression seen by Masten and Shiverick (150) is probably not due to an interaction between BSAP and the AhR. In addition, this particular DRE-containing BSAP site is not present in the mouse CDI9 promoter (43); however, we have identified a DRE-like site within the transcription initiation region of both the mouse 23 and human CD19 genes. It is unclear if these DRE sites mediate an affect on CD19 gene expression and will be a focus of future studies in our laboratory. 24 MATERIALS AND METHODS 1. Chemicals TCDD, 1,2,3,4,,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7-trichloro- dibenzo-p-dioxin (TriCDD) and 1-monochlorodibenzo-p-dioxin (MCDD), in 100% DMSO, were purchased from AccuStandard Inc. (New Haven, CT). The certificate of product analysis stated the purity of TCDD, HxCDD, TriCDD and MCDD to be 99.1, 100, 99.6, 100% respectively, as determined by AccuStandard, using gas chromatography/mass spectrophotometry (GC/MS). DMSO and LPS were purchased from Sigma Chemicals (St. Louis, MO). 11. Animals Virus-free female B6C3F 1 (C57BL/6 x C3H) mice, 5-6 weeks of age were purchased from the Frederick Cancer Research Center (Frederick, MD). On arrival, mice were randomized, transferred to plastic cages containing a saw dust bedding (5 mice per cage) and quarantined for 1 week. Mice were provided with food (Purina Certified Laboratory Chow) and water ad libitum. Animal holding rooms were kept at 21-24°C and 40-60% relative humidity with a 12 hour light/dark cycle. 111. Cell Lines The CH12.LX B-cell line derived from the murine CH12 B-cell lymphoma, which arose in B10.H-2aH-4bp/W ts mice (B 10.A x B 10.129), has been previously characterized (152) and was a generous gift of Dr. Geoffrey Haughton (University of North Carolina). The BCL-l B-cell line was derived from a murine B-cell lymphoma that spontaneously 25 arose in a BALB/c mouse (153). This cell line has been previously characterized (154) and was generously provided by Dr. Kathryn H. Brooks (Michigan State University). CH12.LX and BCL-l cell lines were grown in RPMI-1640 (Gibco BRL, Grand Island, NY) supplemented with heat-inactivated bovine calf serum (10% for CH12.LX cells and 5% for BCL-l cells) (Hyclone, Logan, UT), 13.5 mM HEPES, 23.8 mM sodium bicarbonate, 100 U/ml penicillin, 100 ug/ml streptomycin, 2 mM L-glutarnine, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 50 M B-mercaptoethanol. The mouse hepatoma cell line, Hepa 1c1c7, was generously provided by Michael S. Denison (University of California, Davis). Hepa lclc7 cells were cultured in aMEM media supplemented with 13.5 mM HEPES, 23.8 mM sodium bicarbonate, 100 units/ml of penicillin, 100 ug/ml streptomycin, 2 mM L-glutarnine, and 5% bovine calf serum. All cells were maintained at 37° C in atmosphere of 5% C02. IV. Northern Blot Analysis A. RNA Isolation and Analysis Total RNA was isolated using a modified method of Chomczynski and Sacci (155). Briefly, mouse spleen and liver was homogenized in denaturing solution (4 M guanidium isothiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 100 mM 2-mercaptoethanol) and extracted twice with phenolzchloroformzisoamyl alcohol (1:1:24). Nucleic acids were precipitated and resuspended in water. Poly(A) RNA was purified by PolyATtract (Promega, Madison, WI), precipitated, resuspended in water, and quantitated spectrophotometrically. Poly(A) RNA was fractionated in a 1.2% agarose-formaldehyde gel, transferred to nylon membrane (Amersham, Arlington Heights, H.), and cross-linked to the membrane using a UV Stratalinker 1800 (Stratagene, LaJolla, CA). Blots were prehybridized for 2-6 hr and hybridized overnight at 42°C in hybridization solution [50% 26 formamide, 5x SSPE (0.9 M NaClz, 0.05 M NaH2P04 and 0.005 M EDTA), 10x Denhardt's solution, 2% SDS (sodium dodecyl sulfate), 7% dextran sulfate, yeast tRNA at 130 ug/ml, and labeled probe], then washed twice for 5 min in 2x SSPE with 0.5% SDS, twice for 15 min in 1x SSPE with 1% SDS, and twice for 15 min in 0.1x SSPE with 0.1% SDS if needed. Blots were then exposed to Reflection film (Dupont NEN, Boston, MA) at -80°C in the presence of intensifying screens. Mouse spleen without capsule was prepared by carefully separating the splenic pulp from the splenic capsule. The spleen cells were homogenized in denaturing solution and mRN A was isolated as described above. B. cDNA Probes The plasmid pSportAhR (ATCC 63215) developed by Bradfield (106) contains a 3.12 kb insert of the cDNA for the mouse AhR gene which was cloned from the Hepa 1c1c7 cell line. The AhR cDNA probe was a 1.87 kb fragment cut with restriction enzymes Hind HI and BamH I from pSportAhR and corresponds to the 3' end of the cloned AhR gene. The 75 base ARNT oligomer probe was synthesized using an Applied Biosystem DNA synthesizer and purified by HPLC (Macromolecular Structure Facility, MSU). This oligomer represents nucleotides 360 to 435 of the human ARNT cDNA clone sequenced by Hoffman et al., ( 122). V. Reverse Transcription-Polymerase Chain Reaction A. RNA Isolation Total RNA for the reverse transcription-polymerase chain reaction (RT-PCR) was isolated using Tri Reagent (Molecular Research Center, Cincinnati, OH) as described by 27 Chomczynski (156, 157) or with the High Pure RNA Isolation system (Boehringer Mannheim Biochemicals, Indianapolis, IN). RNA samples were first analyzed for DNA contamination by PCR analysis without reverse transcriptase. RNA samples containing DNA were incubated with RN ase-free DNase for 15 min at 37°C in 10mM MgClz, 1mM DTT, 25 units RNasin, 10mM Tris, 1mM EDTA, then phenolzchloroform extracted, and precipitated in isopropanol. B. Qualitative RT-PCR Analysis Primer sequences for RT-PCR analysis were chosen using GeneWorks, IntelliGenetics, Inc. (Real Mountain View, CA). No Significant homology was detected when each sequence was searched in the Genebank database and the PCR products were observed as a single band of the expected size (Ahr 385 bp; Amt 340 bp; CypI al 228 bp; p. 404 bp) on an ethidium bromide-stained agarose gel. The forward and reverse primers were synthesized using an Applied Biosystems DNA synthesizer and purified by HPLC (Macromolecular Structure Facility, MSU) and were as follows: Ahr, TCATGGAGAGGT GCTI‘CAGG and GTCI'I‘AATCATGCGGATGTGG; Amt, TI‘CCGA'ITCCGATCTAAG ACC and TGTTCTGATCCTGCACTTGC; u, TGAGCAACTGAACCTGAGG and TGCATACACAGAGCAACTG. Primers for the CYP1A1 gene were a generous gift of Dr. Dale Morris (J. D. Searle). For the cDNA reaction, known amounts of total RNA was reverse transcribed by RT into cDNA using oligo(dT)15 primers. For the PCR amplification reaction, a PCR master mix consisting of PCR buffer, MgClz (4 mM for the Ahr and CypI a1 reactions and 2 mM for the Amt and p. reactions), 6 pmol each of the appropriate forward and reverse primer, and 2.5 units Taq DNA polymerase were added to the cDNA samples. For the Ahr and Amt reactions, samples were then heated to 94°C for 4 min and cycled 32 times at 94°C for 15 sec (disassociation), 59°C for 30 sec (annealing), and 72°C for 30 sec (elongation) after which an additional extension step at 72°C for 5 min 28 was included. The PCR reactions for CypI a1 and u were performed as described above except that the annealing temperature was 56°C for Cypl a1 and 60°C for H. In addition, the [.1 reaction was cycled 30 times. PCR products were electrophoresed in 1.5% agarose gels and visualized by ethidium bromide staining and assessed qualitatively. All RT-PCR reagents were purchased from Promega (Madison, WI) except the Taq DNA polymerase which was purchased from Perkin Elmer (Foster City, CA). C. Quantitative RT-PCR Analysis Quantitative RT-PCR was performed as outlined in Gilliland et al. (158, 159), except that the recombinant RNA (rcRN A) was used as an internal standard (18) instead of genomic DNA. Each IS was generated as previously described (160) and contain specific PCR primer sequences for Ahr, Amt, Cypl (11, or [.1 (PCR product sizes: Ahr 385 bp; Ahr- IS 256 bp; Amt 340 bp; Amt-IS 255 bp; CypIaI 228 bp; CypIaI-IS 336 bp; u 404 bp; It- IS 309 bp). Briefly, known amounts of total RNA sample and IS rcRNA were reverse transcribed Simultaneously, in the same reaction tube, into cDNA using oligo(dT)15 as primers. PCR amplification of the IS and sample cDNA was performed as described above for qualitative RT-PCR. PCR products were electrophoresed in 3% NuSieve 3:1 gels (FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide staining. Quantitation was performed by assessing the optical density for both of the DNA bands (i.e., IS versus target gene) using a Gel Doc 1000 video imaging system (Bio Rad, Hercules, CA). The number of transcripts was calculated from a standard curve generated by using the density ratio between the gene of interest and the different internal standard concentrations used (159). The point at which the ratio of IS to mRNA is equal to one signifies the "cross-over" point which represents the amount of Ahr, Amt, CypI a1, or [.1 molecules present in the initial RNA sample. At least two separate RNA isolations per experiment were analyzed for each of the tissues or cell lines. 29 VI. Enzyme-Linked Immunosorbent Assay for IgM Supernatants were harvested from naive or LPS (3 or 30 ug/ml)-stimulated CH12.LX or BCL-l cells following a 72 hr incubation at 37°C in 5% C02 and were analyzed for IgM by sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 100 pl of supernatant or standard (mouse IgM, Sigma, St. Louis, M0) were added to wells of a 96-well microtiter plate coated with anti-mouse Ig capture antibody (Boehringer Mannheim, Indianapolis, IN), and then incubated at 37°C for 1.5 h. After the incubation period, the plate was washed with 0.05% Tween-20:phosphate-buffered saline (PBS) and H20, followed by addition of a horseradish peroxidase anti-mouse IgM detection antibody (Sigma, St. Louis, MO) and another incubation at 37°C for 1.5 h. Unbound detection antibody was washed from the plate following the incubation period with 0.05% Tween-20 PBS then H20. ABTS substrate (Boehringer Mannheim, Indianapolis, IN) was added and colorimetric detection was performed over a 1 hr period using an EL808 automated microplate reader with a 405 nm filter (BIO-TEK, Winooski, VT). The DeltaSoft 3 computer analysis program (BioMetallics, Princeton, NJ) calculated the concentration of IgM in each sample from a standard curve generated from the absorbance readings of known IgM concentrations. VII. Whole Cell Lysate Protein Preparation Livers were perfused with HEDGM (25 mM HEPES (pH 7.5), 1 mM EDTA, 2 mM DTT, 10% (v/v) glycerol, 20 mM sodium molybdate), through the portal vein, isolated, pooled, and minced in buffer. The last wash was passed through a cheesecloth and 1.5 (w/v) of HEDGM/LAP (HEDGM with 100 M leupeptin, 40 U/ml aprotinin, and 200 M PMSF) was added. The sample was homogenized and centrifuged at 21,000 x g; supernatant was collected and then centrifuged at 105,000 x g. Single spleen cell 30 suspensions without red blood cells were prepared as previously described (161). Briefly, splenocytes were carefully removed from the splenic capsule, washed once with complete media (10% BCS) and treated with Gey's solution (5 ml Gey's per 1x108 cells) for 3 minutes on ice to lyse the RBCS. Gey's solution was then removed by diluting with complete media (10% BCS) and centrifuging at 200 x g for 10 min at 4°C. Cells were then washed once more in complete media (10% BCS). After the last wash, one cell volume of HEDM/LAP was added and the cells were homogenized with a tight fitting pestle. An equal volume of HED2GM/LAP (HEDM/LAP with 20% glycerol) was added and centrifuged at 105,000 x g for 1 hr at 4°C. Whole cell lysates from CH12.LX, BCL-l and Hepa 1c1c7 cells were prepared in HEDGM/LAP, homogenized and centrifuged as above. Hepa lclc7 cells were first removed from tissue culture flasks with 0.5 M EDTA in PBS. The supernatant was aliquoted and stored at -80°C prior to use in the western and Slot blot analysis. Protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Sigma, St. Louis, MO). VIII. Western Blot Analysis Cell lysate proteins were resolved by denaturing SDS-polyacrylamide gel electrophoresis (PAGE) with 7.5% polyacrylarrride (National Diagnostics, Atlanta, GA). The electrophoresed proteins were transferred to nitrocellulose (Amersham, Arlington Heights, Us). Protein blots were blocked in BLOTI‘O buffer [5% low fat dry milk in 0.1% Tween-202tris-buffered saline (TBS)] for 1-2 hr at 22°C. Primary antibodies to the AhR (17-10B) and ARNT protein (20-9B), previously characterized by Pollenz et al., (111), were a generous gift of Dr. Richard S. Pollenz (Medical University of South Carolina). Immunochemical staining was carried out as previously described (111). The AhR antibody and the ARNT antibody were diluted to 1 rig/ml in antibody dilution buffer [0. 1% 31 ficoll, 0.1% polyvinylpyrrolidone, 0.05% gelatin, 0.1% Nonidet p-40, and 0.5% bovine serum albumin (BSA) in borate buffered saline]. Detection was performed using the ECL method (Amersham, Arlington Heights, IL). The relative intensity for the protein of interest was measured by densitometry using a model 700 imaging system (Bio Rad) and was derived from the adjusted volume [optical density (OD) times the area]. IX. Slot Blot Analysis Varying amounts of whole cell protein lysates were directly filtered onto nitrocellulose membranes using a Bio-Dot SF microfiltration apparatus (Bio-Rad, Hercules, CA). The same procedures used in the Western blot analysis were employed for the detection of AhR and ARNT proteins in the slot blots. The negative control consisted of cell lysates which were incubated with the secondary antibody but not with the primary antibody. Slot blots were analyzed by densitometry as described above. X. Nuclear Protein Preparation Single spleen cell suspensions without red blood cells were prepared as described above. Splenocytes, Hepa1c1c7 cells, CH12.LX cells or BCL-l cells were incubated with DMSO (0.01%) or 30 nM TCDD in DMSO for 1 hr at 37°C then harvested by centrifugation at 1,200 rpm for 10 min. Hepa1c1c7 cells were first removed from culture flasks with 0.5 M EDTA in PBS. Following centrifugation, the cells were washed once with 1x PBS, then incubated in 10 mM I-IEPES (pH 7.5) for 5 min on ice, and centrifuged at 1,200 rpm for 5 min. One ml of MDH/LAP (3 mM MgC12, 1 mM DTT, 25 mM HEPES, 100 11M leupeptin, 40 U/ml aprotinin, and 200 1.1M PMSF) was added to the cell pellet and homogenized with a tight fitting pestle. Nuclei were pelleted by centrifuging at 1000 X g for 5 min, washed twice with MDHK/LAP (3 mM MgC12, 1 mM DTT, 25 mM 32 HEPES, 100 mM KCL, 100 1.1M leupeptin, 40 Ulml aprotinin, and 200 M PMSF), then resuspended in 100 pl of HEDGK/LAP (25 mM HEPES, 1 mM EDTA, 1 mM DTT, 10% glycerol, 400 mM KCl, 100 uM leupeptin, 40 U/ml aprotinin, and 200 11M PMSF), incubated on ice with agitation for 40 min and centrifuged at 14,000 X g for 15 min. The supernatant was aliquoted and stored at -80°C prior to use in the EMSA. Protein concentrations were determined using the BCA protein determination assay (Sigma, St. Louis, MO). XI. Electrophoretic Mobility Shift Assay A. Analysis of Protein-DN A Complexes Nuclear protein preparations were used in the EMSA as previously described (113, 119) with a few modifications. Briefly, nuclear extracts (6 ug of protein for the Hepa 1c1c7 and splenocyte experiments and 10 ug of protein for the CH12.LX and BCL-l experiments) were incubated with poly(dI-dC) (Boehringer Mannheim Biochemicals, Indianapolis, IN) at room temperature for 15 min. Radiolabeled DRE3, 3'OtE(hsl,2) or 3'0t-hs4 Oligomer was added (80,000 cpm for the Hepa 1c1c7 and splenocyte experiments and 40,000 cpm for the CH12.LX and BCL-l experiments) and incubated at room temperature for another 30 min. Final reaction concentrations for the Hepa lclc7 cells were as follows 25 mM HEPES (pH 7.5), 1 mM EDTA, 2 mM DTT, 10% glycerol, 110 mM KCl, and 1.8 ug poly(dI-dC). The splenocyte reaction mixture was identical to that used for the Hepa1c1c7 cells with the exception of the poly (dI-dC) concentration (0.4 pg). The CH12.LX and BCL-l reaction mixtures were also identical to that used for the Hepa1c1c7 cells with the exception of the KCL concentration (108 mM) and the poly (dI- dC) concentration (1.0 pg). Where indicated, 50- to 100- fold excess of unlabeled DRE3, 3'OLE(hsl,2) or 3'or-hs4 oligomer was added to the reaction. Binding of protein to the 33 DNA was resolved by a 4.0% nondenaturing PAGE gel, dried on 3MM filter paper (Whattman, Hillsboro, OR) and autoradiographed. B. Synthetic DRE Oligonucleotides Complementary pairs of synthetic DNA fragments corresponding to the AhR/ARNT binding site of mouse DRE3 (162), to two putative DRE sites in the mouse Ig 3'0: enhancer, 3'OLE(hsl,2) (TAGGGGTCTATTAACICACQACQCIAGGCCATC ATGGAGAG; positions 1096-1136) (GenBank accession No. X62778, 20) and 3'Ot-hs4 (AGCAGAGGGGGGGACWGAAAGCCCCA’ITCACCCAT; position 319- 360) (GenBank accession No. L39932, 24) and to the KB site from the 3'Ot-hs4 enhancer (hs4-KB; GATCTCTCTWTCTGA) were synthesized using an Applied Biosystems DNA synthesizer and purified by HPLC (Macromolecular Structure Facility, MSU). The DRE Oligonucleotides were annealed by heating equal concentrations of the sense and antisense strands at 88°C for 5 min and allowing the mixture to cool slowly at room temperature. The double-stranded oligomer was then end labeled using T4 polynucleotide kinase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and [y- 32P]ATP (Dupont NEN, Boston, MA). XII. EMSA-Western Analysis EMSA analysis was conducted as described above but included samples containing 10 pmol of cold DRE3, 3'OIE(hsl,2) or 3'Ot—hs4 instead of radiolabeled Oligomers. The nonradiolabeled portion of the EMSA gel was separated from the radiolabeled portion and transferred to nitrocellulose (Amersham, Arlington Heights, H.). Protein blots were blocked in BLO'I'I‘O buffer (1% low fat dry milk in 0.1% Tween-20 TBS) for 1-2 hr at 22°C. Primary antibody to the AhR (17-10B), previously characterized by Pollenz et al. 34 (111), was a generous gift of Dr. Richard S. Pollenz (Medical University of South Carolina). The ARNT antibody (NB 100-110) was purchased from Novus Biological (Littleton, CO). RelA (p65), p50 (NF-KBl), RelB, and c-rel antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunochemical staining was performed as described above for the Western analysis. Optical density for the protein of interest was measured by densitometry using a model 700 imaging system (Bio Rad). XIII. Statistical Analysis of Data The mean :t S.E.M. was determined for each treatment group of a given experiment. Where indicated, the statistical difference between treatment groups and the vehicle controls was determined by Dunnett's two-tailed t-test. For ECso and IC50 generation, a complete concentration-response curve for TCDD, HxCDD, and TriCDD was obtained for a given endpoint. Concentration-response curves were fit by a four parameter logistic concentration-response equation given as Y=[A1-A2)/[1+(X-XO)P]]+A2. The derived parameter, EC50 or IC50 (X0; concentration generating half-maximal response) was expressed as nM (mean i S.E.M). Statistical difference between EC50 and IC50 means was determined by a l-way ANOVA followed by a least significant difference test. P<0.05 was considered statistically Significant. 35 EXPERIMENTAL RESULTS 1. Identification of AhR and ARNT in B6C3F1 mouse splenocytes A. Northern blot analysis of mouse splenocytes Transcripts for the Ahr and Amt have been identified in rat spleen by ribonuclease protection analysis (163). The purpose of the following studies was to determine if Ahr and Amt were also expressed in splenic RNA isolated from B6C3F1 mice. Liver RNA from the same mouse strain was isolated as a comparative control Since the AhR and its putative mechanism of action have been primarily studied using hepatic tissue and hepatic cell lines. Northern analysis for the AhR using a 1.87 kb mouse cDNA probe identified a transcript of approximately 6.6 kb in mouse spleen and liver (Figure 3A). Splenocytes devoid of connective tissue were examined by Northern analysis for the AhR to address the possibility that the transcripts observed from the spleen may have originated primarily from the connective tissue encapsulating the spleen. Transcripts detected in the splenocytes matched the liver control (Figure 3B). Northern analysis using a radiolabeled 75 base oligomer synthesized from human cDNA for ARNT (122) revealed three transcripts of approximately 5.6 kb, 2.0 kb and 1.1 kb in both the spleen and liver (Figure 3C). B. Western blot analysis of mouse splenocytes Based on the identification of transcripts for both the Ahr and Amt in mouse splenocytes, Western blot analysis was conducted on whole cell lysates prepared from splenocytes devoid of connective tissue and red blood cells. Antibodies to the AhR revealed two major proteins of approximately 95 kDa and 104 kDa in splenocytes and liver (Figure 4A). These two sizes correspond, respectively, to the codorrrinant expression of 36 Figure 3. Northern blot analysis of Ahr and Amt mRN A expression in splenocytes and liver. Using a guanidium isothiocyanate method, total RNA was extracted from the whole Spleen, spleen without splenic capsule and liver. Poly(A) RNA was isolated from the total RNA using the PolyATtract mRNA isolation system. Poly(A) RNA from the spleen (6 ug, A and C), the Spleen without capsule (6 pg, B), and liver (2 11g, A, B and C) was loaded onto a 1.0% formaldehyde gel and electrophoresed at 100 volts for 4 hr. The gel was blotted onto a nylon membrane and hybridized with either a 32P-labeled 1.87 kb fragment from the cloned mouse Ahr (A and B) or a 32P-labeled 75 base oligomer synthesized from human cDNA for Amt (C). These figures are representative of at least two separate experiments. 37 Spleen L1ver Splenocytes Liver Ahr—9 new (— 75 id) —> <— 4.4 kb -—> <— 2.4 kb —> <— 13 kb —> A B 8 a o o — > J? :I' A <— 7.5 kb t m —> <— 4.4 kb Amt—> ('- 24 kb Amt —> <— 1.3 kb Figure 3. Northern blot analysis of Ahr and Amt mRNA expression in splenocytes and liver. 38 Splenocytes Splenocytes Liver Liver ezoomaa <— 68kDa —> A B Figure 4. Western blot analysis of the AhR and ARNT in splenocytes and liver. Protein was isolated from whole cell lysates of splenocytes (devoid of splenic capsule and red blood cells) or liver. One hundred micrograms of protein was loaded in each lane and resolved on 7.5% SDS-PAGE gels, transferred to nitrocellulose, and incubated with 2 rig/ml of anti-AhR, 17-10B (A) or anti-ARNT, 20-9B (B). Antibody binding was visualized by staining the blot with donkey anti-rabbit horseradish peroxidase-linked immunoglobulins. These figures are representative of more than three separate experiments. 39 the AM” (C57BL/6) and AM” (C3H) alleles in the B6C3F1 (C57BL/6 x C3H) mice (164). An approximately 70 kDa protein, previously identified as a proteolytic fragment of the 95 kDa AhR in C57BL/6 mice (165), was detected in lysates prepared from liver of the B6C3F1 mice. Antibodies to ARNT identified an approximately 87 kDa protein in both tissues (Figure 4B). C. Slot blot and quantitative RT-PCR analysis of mouse splenocytes Slot blot analysis and quantitation by densitometry of whole cell lysates prepared from the splenocytes and liver demonstrated approximately 2.0 i 0.5-fold more AhR protein present in liver than in splenocytes (Figure 5A); whereas, approximately 2.4 :1: 0.05-fold more ARNT protein was identified in splenocytes than that observed in liver (Figure 5B). Quantitative comparisons by RT-PCR of the Ahr and Amt transcripts in liver and Splenocytes demonstrated approximately 2.3-fold more Ahr transcripts in liver as compared to splenocytes and approximately 3.2-fold more Amt transcripts in Splenocytes as compared to liver (Table 1). In addition, a greater number of transcripts for Amt as compared to Ahr were identified in both the liver and splenocytes (Table 1). D. EMSA analysis of mouse splenocytes With the identification of both the AhR and ARNT proteins in splenocytes, the functionality of these proteins was assessed by EMSA. Hepa 1c1c7 cells served as a comparative control since this cell line has been extensively characterized with respect to the induction by TCDD of AhR nuclear translocation, dimerization with ARNT and DRE binding of the ligand-AhR/ARNT nuclear complex. Following a 2 hr treatment of Hepa lclc7 cells or Splenocytes with 30 nM TCDD, the AhR nuclear complex was confirmed to be functional in both nuclear preparations as demonstrated by its ability to bind to the DRE 40 988830 8838 93 a8 guacamame 8 gm 085. 52,8on8 8.5 c8 82% new god can mmad Am e5 #36 e8 wamd 2 83 808508 888%: 2a. .88 88 me $885 9522 2a 80:8 23 Q08 x ddv 0820.» 835:8 88m 83.8w 8:?» 85:8 038 2: 8808888 a 9:8 85:83 203 803 SE .éno%08§ Baéaaeaeom 82838 258.59. 3.38 e? as 2: 8:8... E 888.: as 885 38%... .3 mix fizz... 68 Ho 93 MST: EATER .3 ER: N HE? @8385 83 083808 05. 655808 0838858 a 98 e888 .3886 203 mammb :8 30:3 Sch 5208 no 8095888 wfifiaofi 80>: Ea 8S88Em 3 P792 28 m5. 8.. me $838 83 86 .m charm m WY: avg. 2:. wow Nvfi vw.m 0o." 02 OZ 8i..— OVHN ow.o_ 2.3 502 m9: om.m ofim 56 DZ oioecoim .2. ifi -r.:: (it... _ :83 SN 2: ex 8 2. am 2 m o 33 23.3 £205 4. Ed 8m 9% 48 a... go 93 oz oz as: :1” m: m: 85 8.0 Rd oz oz az 239.2% ED... .2387... . . ._ ...... ............ . . , .. .. ..... .. . I. . .I.\N ......... . . . ... .. . . . . .. .. . . 1 . .L 0;. . like! , a: . .. . . .. . . , a." kw.)— . . ....... o _ ......_ . , L 2.882% ... ....._,....r...,....... 1...». arm. u..u.n...,...... “SHAWL.” . u“... .t.. .. ”I... .. Una... .. ,x. _.,, . , ..h...... . ................,. .. .. . . . 3.5.1.. unzip?“ _.. .ufirwtuwawuvnrfiprrtuuvwwkvim-flip.ruhaxxbmrlfisthnih. 33 23.3 528m 41 .Amnav maouflofi <73 ouaaaom 925 88.“ <72 253 w: 2:272 28w Hows“ .«o 8:629: mo mm H macaw Rm 2: H o6 H a: x ed 1: x 1.. H me x S 8.582% cm as x mm H a: H 3 m: x 3 H u: x 3 .2: a 022 . E<..~E< 22¢. .32.. mama. denim B 8:52.... 3 <58 :5. Ba .5. .0 H325 3.3255 a 2%... 42 motif. This is shown in Figure 6, lane 3 for TCDD-treated Hepa 1c1c7 cells and in lane 5 for TCDD-treated splenocytes. Denison and Yao (162) have identified a constitutive protein-single-stranded DNA complex that migrates faster than the TCDD-inducible protein-DNA complex. We similarly observed this same constitutive complex in nuclear protein preparations from Hepa lclc7 cells and splenocytes (Figure 6, complex labeled C in lanes 2, 3, and 5). However, the gel retardation pattern observed with the TCDD-treated splenocytes was different from that observed with the Hepa lclc7 cells in that the TCDD- inducible protein-DNA complex resolved as a rather broad band in comparison to the complex formed in Hepa 1c1c7 cells (Figure 6, band labeled A and B in lane 5). Addition of excess unlabeled DRE abrogated the TCDD-induced mobility shift indicating binding specificity of the AhR nuclear complex for the DRE (Figure 6, lanes 6). II. Characterization of the AhR and ARNT in the CH12.LX and BCL-l B-cell lines A. AhR and ARNT expression in two B-cell lines Western analysis for the AhR and ARNT was performed using whole cell lysates from the CH12.LX and BCL-l B-cell lines. Interestingly, an approximately 95 kDa AhR was markedly expressed in CH12.LX cells but was not detected in BCL-l cells (Figure 7A). The 87 kDa ARNT protein was expressed in both B-cell lines (Figure 7B). To confirm a lack of AhR expression in BCL-l cells, total RNA isolated from BCL-l cells was analyzed by qualitative RT-PCR analysis, a more sensitive technique than Western analysis. In agreement with the above results, Ahr transcripts were not detected in BCL—l RNA (Figure 8A). Quantitative RT-PCR analysis of basal Ahr and Amt transcripts demonstrated a much greater expression of Amt in CH12.LX cells as compared to Ahr 43 Figure 6. Binding of Hepa lclc7 or splenocyte nuclear proteins to a DRE. Six micrograms of nuclear protein from Hepa 1c1c7 cells (Hepa), vehicle (lane 2) or TCDD- treated (lane 3), or from B6C3Fl splenocytes, vehicle (lane 4) or TCDD-treated (lanes 5 and 6), were incubated with a 32P-labeled, 26 bp DRE oligonucleotide. Protein-DNA complexes were resolved by EMSA. Unlabeled competitor DRE oligonucleotide was added at a 50-fold excess (lane 6) to show specific interaction of the proteins with the DRE oligonucleotide. Lane 1 is the labeled oligonucleotide without nuclear protein. Letters A and B identify TCDD-inducible protein-DNA complexes and letter C identifies constitutive protein-single-stranded DNA complexes. This figure is representative of more than three separate experiments. Hepa Splenocytes TCDD Cold DRE3 123456 Figure 6. Binding of Hepa 1c1c7 or splenocyte nuclear proteins to a DRE. 45 .aaofitumxo 3§m8 23 58 0.88 .«o o>u8u88m8 2a aimed .mconuqa Rafi...“ =5»: _ A8 28 £335 «fires in: 2 AS a? Base 23 am $3.5-QO $2 a 8 8208“ .82 58 a 822 a; a: at 522m as: :8 48:8 928.“ a a 3:8 €28 $25 “=8 52 ac: 2F £8 2828 3:5 30m 2a €38 3:5 5.36 3355 ace 322 as 28.: =8 225 as: :8 76m ca 5.36 as a 532m 925. 2a m5. ea 5 wins 83 E383 s 2:5 3H3 3H _I..I.d 1...... .29 .2 lb- L Iv- mo m L 3 A 10m BdaH ‘_ M. .fiaoauomxo Banana PE 56 Boa .«o u>uSaom8m2 8a £32 2F Anna 83208 <72 agaom 025 8m 85 BacaSm use 508 05 fizomemou an scam dz 38:3 203 weakest you 295 33.8me 533 E «.29an 472 we SCEQEV 838—08 3 mg-» 05 no 6858.598 08 muauomaab t8n8892 2a 338.— 2: .ANHE macaw—8m <2»— Sauna 23 .8.“ 3.5 335% H :38 Cum Jim w: ooifloqav 83.022: 3 use-» 05 so 388292 Ba flaw—Saab <58 BEAU 95% 8253: some Soc @8235 .5 an“: .«o muouahuoonoo 688.8 A6 .5 9E2“ 08: @8038 8m 5920 $8 Ed .36 £029 3 ODOR 2: 9m AS 5‘3 @335 0.83 2:523 mefl 33 V3.26 8:3 XANSfiV E cove—65 33.5 coosvumdauh. no 3.58 0:5 Es omaomme 88 .a 0.53% m < 80;: 3 as: a up a o 4 N 0d 0.0 mod mood I) <2 .od .0 O m M l m. 18:6 w .23.. t M o w -83 M 88.25- m 6 V . Exqm .w UN. an .. w v v [85.3 49 858:2:5 88:8 95 as: 0.58 :0 3:88:22 8a 838% due» Hogan: :80 80.: .8853 <2: no Baotou as? 336 8: £8328 and»: 9:8:85 .8 Va 8.: 594m: 886 4.5V 88:9 Ho 98:. E: Qm 5:3 @838 803 8:8 288:8 335 5.26 23 988:8 no:an TAU: 8:3 TAU: E 8:865 336 no 98:. :0 “cow: .3 9:53 98» :> «z 88 :> 3. 2:9.” 3.6.», p 5.0: x._.N EU 50 cells to secrete IgM and treatment of CH12.LX cells with TCDD resulted in a marked inhibition of LPS-induced IgM secretion at concentrations as low as 0.03 nM TCDD (Figure 11). In contrast, LPS-induced IgM secretion in AhR-deficient BCL-l cells was not inhibited at concentrations as high as 3.0 nM TCDD (Figure 12). D. Differential expression of AhR in LPS-differentiated CH12.LX B-cells The effect of LPS-induced differentiation of the CH12.LX and BCL-l cells on AhR gene expression was evaluated by RT-PCR. A marked upregulation of AhR gene expression occurred by 4 hr in LPS-activated CH12.LX cells (Figure 13). In contrast, LPS-induced activation of BCL-l cells did not result in expression of the AhR gene (Fig 14). Consistent with AhR gene expression, Western analysis for the AhR protein in LPS activated CH12.LX cells demonstrated an increase in protein expression by 8 hr (Figure 15). In addition, LPS treatment did not alter basal Amt mRN A expression (Figure 16). E. TCDD does not alter Ahr expression in naive or LPS-stimulated CH12.LX cells Since the 5' untranslated region of the AhR gene contains a consensus DRE (166), we explored the possibility that TCDD may regulate Ahr expression. Naive and LPS- stimulated CH12.LX cells were evaluated by RT-PCR for Ahr expression. A 3.0 nM TCDD treatment did not alter Ahr expression in naive cells over a 24 hr time course (Figure 17). Likewise, TCDD did not alter the induction of AhR expression by LPS (Figure 18). It is notable that at the 6 and 8 hr timepoints there is an increase in Ahr expression in NA and LPS-stimulated CH12.LX cells which is more pronounced with the LPS stimulation and is not affected by TCDD treatment (Figures 17 and 18). This effect is probably related 51 |gM(ng/1O5 cells) NA LPS VH 0.003 0.03 0.3 3.0 nM TCDD Figure 11. Effect of TCDD on LPS-induced IgM secretion in CH12.LX cell. CH12.LX cells (1x104 cells/ml) were treated with LPS (30 [lg/ml) and selected concentrations of TCDD or vehicle (VH, 0.01% DMSO). Supernatants were harvested at 72 hr and analyzed for IgM by sandwich ELISA. Results from triplicate determinations are represented as mean IgM (mg/105 cells) i standard error (n=3). These results were analyzed for statistical significance using Dunnett's two-tailed t-test. *, values that are significantly different from the vehicle at P<0.05. The results are representative of more than three separate experiments. 52 80 NA I LPS 60 4o- 20 k\\\\\4 . lng(ng/1O5 cells) NA LPS VH 3.0nM TCDD Figure 12. Effect of TCDD on LPS-induced IgM secretion in BCL-l cells. BCL-l cells (2x104 cells/ml) were treated with LPS (30 ug/ml) and 3.0 nM TCDD or vehicle (VH, 0.01% DMSO). Supernatants were harvested at 72 hr and analyzed for IgM by sandwich ELISA. Results from triplicate determinations are represented as mean IgM (ng/ml) :1: standard error (n=3). These results were analyzed for statistical significance using Dunnett's two-tailed t—test. *, values that are significantly different from the vehicle at P<0.05. The results are representative of more than three separate experiments. 53 10x1 07- 75x106 ‘ 50x1 06 - 25x1 06 - Ahr transcrlpts (mlcls/100 ng RNA) . .. ... z w .. .. O D ._ '1 4 .a O 2 4 6 8 12 24 Sl'muhtion (hr) Figure 13. Effect of LPS-induced differentiation on Ahr expression in the CH12.LX cells. CH12.LX cells (5x105 cells/ml) were treated with LPS (30 ug/ml) for selected time points. Quantitative RT-PCR analysis for Ahr was performed on RNA extracted from naive (NA) and LPS-stimulated cells at each time point. Ahr mRN A transcripts are represented on the y-axis as molecules(mlcls)/ 100 ng RNA. Bar, mean :l: standard error for two separate RNA isolations (n=2). The results are representative of more than two separate experiments. 54 QHl 2.LX EQL- jl 5 NA LPS Figure 14. Ahr expression in LPS-stimulated CH12.LX and BCL-l cells. CH12.LX (5x105 cells/ml) and BCL-l (5x105 cells/ml) cells were treated with LPS (30 pig/ml) for 6 hr. Qualitative RT-PCR analysis for Ahr was performed on RNA extracted from naive (NA) and LPS-stimulated cells. Results are representative of more than two separate experiments. 55 Vehicle LPS 014 812II4 812| kD 117— 4 AhR 78— Relative ,ntensity [1.00 3.88 1.94 3.89 1.26 24.58 25.98I Figure 15. AhR protein expression in LPS-stimulated CH12.LX cells. CH12.LX cells (1x105 cells/ml) were treated with LPS (30 ug/ml) for selected time points. Whole cell lysate was isolated from naive (NA) and LPS-stimulated cells at the selected time point. Cell lysate protein (12.5 pg) was loaded in each lane, resolved on a 7.5% SDS-PAGE gel and probed with 1 ug/ml of the AhR antibody. The relative intensity for each band was derived from the adjusted volume (OD times area). Results are representative of more than two separate experiments. 56 1.25x108 —- [2 NA I LPS 1.0008— ”,2 {1% 7.5007. $8 T C.- E a) cg 50007. E o “E 2.5x107_ 0.0 0 Time (hr) Figure 16. Amt expression in LPS-stimulated CH12.LX cells. CH12.LX cells were treated with LPS (30 gig/ml) for selected time points. Quantitative RT-PCR analysis for Amt was performed on RNA extracted from naive (NA) and LPS-stimulated cells (5x105 cells/ml) at each time point. Amt mRN A transcripts are represented on the y-axis as molecules (mlcls)/100 ng RNA. Bar, mean i standard error for two separate RNA isolations (n=2). The results are representative of more than two separate experiments. 57 1.0x105- A 75x105- I I] W *3? I 1 3.0nMTCDD § 3’ 5.0x105- .L 5:2 T ,.=, % 1 I v 25x105-. . .L *~ g _L é 0.0 — HZ- _ 0 2 4 6 8 12 24 Tlme (hr) Figure 17. Effect of TCDD on Ahr expression in the CH12.LX cells. CH12.LX cells (5x105 cells/ml) were treated with 3.0 nM TCDD or vehicle (VH, 0.01% DMSO) for selected time points. Quantitative RT-PCR analysis for Ahr was performed on RNA extracted from each treatment group at each time point. Ahr mRNA transcripts are represented on the y-axis as molecules (mlcls)/ 100 ng RNA. Bar, mean :I: standard error for two separate RNA isolations. The results are representative of more than two separate experiments. 58 1.5x107 _ I LPS + VH E LPS + 3.0 nM TCDD * 2 1.0x107 ,1 .- 9 z ./ ’ a. CC /’,. 5 g ,. é o /’i a o , “ 5 3’?" K _. g g 5 0x106 of, E. f; ,. ,. / .1 / '( "//",- \ \‘f‘ \“ 0.0 Time (hr) Figure 18. Effect of TCDD on LPS-induced Ahr expression in the CH12.LX cells. CH12.LX cells (5x105 cells/ml) were treated with LPS (30 ug/ml) and 3.0 nM TCDD or vehicle (VH, 0.01% DMSO) for selected time points. Quantitative RT—PCR analysis for Ahr was performed on RNA extracted from each treatment group at each time point. Ahr mRN A transcripts are represented on the y-axis as molecules (mlcls)/ 100 ng RNA. Bar, mean i standard error for two separate RNA isolations (n=2). The results are representative of more than two separate experiments. 59 to an initial lag period after cell seeding followed by normal progression through the cell cycle. F. Ahr upregulation does not enhance the sensitivity of activated CH12.LX cells to TCDD If the AhR mediates the effects of TCDD, then perhaps an increase in AhR gene and protein expression following B-cell activation would enhance the sensitivity of B-cells to TCDD. To test this possibility, we evaluated the kinetics and magnitude of TCDD-induced CypI a1 induction in naive and LPS-stimulated CH12.LX cells. LPS cotreatment had little effect on TCDD-induced Cypl a1 expression over a 24 hr time course (Figure 19). However, LPS-stimulation caused a slight decrease at 8 hr and a slight increase at 24 hr as compared to unstimulated cells (Figure 19). Further evaluation at the 24 hr time point demonstrated no difference in TCDD-induced CypI a1 induction between unstimulated and LPS-stimulated CH12.LX cells (Figure 20). Since a 12 hr LPS treatment maximally increased AhR protein expression (Figure 15), CH12.LX cells were pretreated for 12 hr with LPS followed by an additional 12 hr treatment with 0.03 nM TCDD. A lower TCDD concentration (suboptimal for CypI a1 induction) was used in this experiment to avoid the possibility that 3 nM TCDD might produce a maximum induction of CypI a1 resulting in an inability to detect any additional LPS-induced changes. Again, no alteration in CypIaI induction was observed above and beyond that produced by TCDD (Figure 21). However, it is notable that the fold-induction of CypI a1 by 0.03 nM TCDD was much less than observed in previous experiments. 60 2.0x106 .4 I. I] 3.0 nM TCDD ‘ J. I LPS+TCDD 1.5x106 - :9. .O'A i c 0 g 2 1.0(1m .1 ‘ l a g T to _ 312, T 6 50(105 - 2 4 6 8 12 Time (hr) Figure 19. Time course of Cy 1a] induction in CH12.LX cells cotreated with LPS and TCDD. CH12.LX cells (5x1 cells/ml) were treated with LPS (30 [lg/ml) and 3.0 nM TCDD or vehicle (VH, 0.01% DMSO) for selected time points. Quantitative RT-PCR analysis for CypI al was performed on RNA extracted from each treatment group at each time point. Cypl a1 mRN A transcripts are represented on the y-axis as molecules (mlcls)/ 100 ng RNA. Bar, mean :l: standard error for two separate RNA isolations (n=2). The results are representative of more than two separate experiments. 61 El No stimulation 4Dx105 .. I LPS stimulation T 30x105 — 20x106 .. Cyp1a1 Transcripts (mlcls/100 ng RNA) 10x106 - r—_Il_—l 0.0 | I l NA VH 3.0 DM LPS VH 3.0 nM TCDD TCDD Figure 20. Effect of a 24 hr LPS cotreatment on TCDD-induced Cyp] a1 induction in CH12.LX cells. CH12.LX cells (5x105 cells/ml) were treated with LPS (30 rig/ml) and 3.0 nM TCDD or vehicle (VH, 0.01% DMSO) for 24 hr. Quantitative RT-PCR analysis for Cyp] al was performed on RNA extracted from each treatment group. Cyp] a1 mRN A transcripts are represented on the y-axis as molecules (mlcls)/ 100 ng RNA. Bar, mean :l: standard error for two separate RNA isolations (n=2). The results are representative of more than two separate experiments. 62 El No stimulation 1 0x107 I LPS stimulation 7 T _ T £9 7.5x105 _ .gA :- C o ‘2 O 5.0x106 _ +— 2 _|_ a 2 .9 g E AV 0 2.5x106 _ 0.0 I I I NA VH 0.03 nM LPS VH 0.03 nM TCDD TCDD Figure 21. TCDD-induced Cyp] a1 induction in LPS pretreated CH12.LX cells. CH12.LX cells (5x105 cells/ml) were pretreated for 12 hr with LPS (30 pig/ml) followed by an additional 12 hr incubation with 0.03 nM TCDD or vehicle (VH, 0.01% DMSO). Quantitative RT-PCR analysis for Cyp] al was performed on RNA extracted from each treatment group. Cyp] a1 mRN A transcripts are represented on the y-axis as molecules (mlcls)/100 ng RNA. Bar, mean 1 standard error for two separate RNA isolations (n=2). The results are representative of more than two separate experiments. 63 III. The SAR of several PCDD-mediated endpoints and AhR binding affinity A. PCDD-mediated inhibition of LPS-induced IgM secretion in CH12.LX B-cells follows a SAR which is concordant with AhR ligand binding affinity and CypIaI induction As discussed previously, transcriptional regulation through an AhR/DRE mechanism of TCDD-induced Cyplal expression is well established (76, 167-169). PCDD congeners that bind to the AhR and upregulate metabolic enzymes require halogen atoms in at least three of the lateral ring positions (positions 2,3,7, and 8) and require at least one unsubstituted ring position (170). For the following specific PCDD congeners, previous reports have determined the rank order for AhR binding affinity and AhR- dependent induction of Cyp] a1 , as TCDD > HxCDD > TriCDD >> MCDD with the MCDD congener having no affinity for the AhR and unable to induce 7-ethoxyresorufm O- deethylase (EROD) (171) which is a measure of CYP1A1 activity. The binding affinities (kd) for TCDD, HxCDD and TriCDD are 0.034, 0.77 and 1.92 nM, respectively (171 and personal communication, A. Poland). Likewise, induction of Cyp] al in the CH12.LX cells following a 24 hr incubation with the PCDD congeners was concentration-dependent, as determined by quantitative RT-PCR, and correlated with AhR binding affinity (Figure 22). The rank order potency was TCDD > HxCDD > TriCDD >> MCDD; MCDD had no effect on Cyp] a1 expression (Table 2). To further characterize the AhR-dependency of TCDD-induced IgM secretion, the effect of these congeners on LPS-induced IgM protein secretion from the CH12.LX B-cell line was examined by ELISA. Inhibition of IgM secretion was concentration-dependent and correlated with AhR binding affinity (Figure 23). Similar to the results for Cyp] a1 induction, the rank order potency was TCDD > HxCDD > TriCDD >> MCDD; again, MCDD had no affect on IgM secretion (Table 2). 5.0x107 - 7 .. ' 30"“) ------ o ----- TriCDD 4.0x107 4 —-— TCDD 1i I, 2.0x107 - 1.0x107 - Cyp1a1 transcripts (mlcls/100 ng total RNA) . . . .r V. \"J 1 tom" ' @ NA 0.0 0.1 1.0 10.0 0.0 - nM Congener Figure 22. Concentration-dependent effect of selected chlorinated dibenzo-p-dioxin congeners on Cyp] a1 expression in CH12.LX cells. CH12.LX cells (1)1105 cells/ml) were treated with TCDD, HxCDD, TriCDD and MCDD at various concentrations. The vehicle control (0.0 nM congener) was 0.01% DMSO. Quantitative RT-PCR analysis for Cyp] al was performed on RNA extracted at 24 hr from each treatment group. Cyplal mRN A transcripts are represented on the y-axis as molecules (mlcls)/ 100 ng RNA. Symbol, mean :l: standard error for three separate RNA isolations (n=3). These results were analyzed for statistical significance using Dunnett's two—tailed t-test. *, # and ‘1, values that are significantly different from the VH control within the TCDD, HxCDD and TriCDD experiments, respectively, at P<0.05. The results are representative of more than two separate experiments. 65 .88 he 82 :3 s Bias “agendas .895 as one... é 22 2m: 9 8388 8§£§$ douse. Ea name 8.. 32 Sea 9 Baas academia» .QQUH e8 cmom 335 on. 8 gmfioo ooqaofiewmmu .805. he Rum 3.5 e BEES Saosfimu .QDUH new 3503.5 550 Eu 3 33958 oofiouimmm... ...v88§o 8a.. 88:3 .9 . Z .anwEwmm 38:38.0. 83238 83 modvm .32 8:80am“. .fioumnmmm ammo— u an 830:8 <>Oz< ~83; a 3 85.58% 33 888 89 Ba Sum 5033 8:285. Ragga .955 05%“: “32523 38 a an v85 203 35 mezzo 8.8%“: 535588 032038 Soc Bfioaow 203 v5 Aamgoé 2: mm @858qu 8a 825%». BEEN—9:3 mats—05w 8955258 $88 was “renown Bmdflmoflfi memofiovod .9 .z mwodfimwmd QQUE. H cue as fine _. Gus omodflmvwo hoodflmwvd mmvdfitcd ownofimmwd annex: a cue Sue Gus a Qua Sodiirmood Sodfivmfio ammoficmmd Eodflmbod QQUH Qua Anna * fine a $1.5 82 30H 32 omum Monomeou nausea 2mm £43 5&1 33.6 .3156 .«o 830:2: Sm omom .8 53808 £8on 98 commmoaxo 1 coon—“Emma: mo 95335 8m 32 Shauna Hoaownou ”N 2an 6 80.0 -1 25.0 _ :5 ......................... _- M;- g 20.0 - 8 . --« choo ID a 15 0 _ ------ TriCDD : . ‘2" o ---- *r """" MCDD 9 10.0 - 5.0— A 0'0 l l "I l l l I "I l NA LPS [0.0 0.1 1.0 10.0 30.0. nM Congener Figure 23. Concentration-dependent effect of selected chlorinated dibenzo—p-dioxin congeners on LPS-induced IgM secretion from CH12.LX cells. CH12.LX cells (1x104 cells/ml) were treated with 3 ug/ml LPS (filled symbols) and selected concentrations of TCDD, HxCDD, TriCDD or MCDD. The vehicle control (0.0 nM congener) was 0.01% DMSO. Supernatants were harvested at 72 hr and analyzed for IgM by sandwich ELISA. IgM is represented on the y-axis as ng/lOS cells. Symbol, mean :l: standard error for triplicate determinations (n=3). These results were analyzed for statistical significance using Dunnett's two-tailed t-test. *, #, ‘1, and S, values that are significantly different from the VB control within the TCDD, HxCDD, TriCDD and MCDD experiments, respectively, at P<0.05. The results are representative of more than three separate experiments. 67 B. Specific PCDD congeners have no affect on CyplaI expression or LPS-induced IgM secretion from the AhR-deficient BCL-l B-cells In agreement with a lack of AhR expression, TCDD, HxCDD, TriCDD and MCDD had no affect on Cyp] a1 expression in BCL-l cells as determined by qualitative RT—PCR (Figure 24). To confirm the AhR-dependency of congener-induced inhibition of IgM secretion, the effect of specific PCDD congeners on LPS-induced IgM secretion from the AhR-deficient BCL-l cell line was evaluated by ELISA. As expected, TCDD, HxCDD, TriCDD and MCDD had no affect on IgM secretion (Figure 25). C. LPS-induced [.1 expression in CH12.LX cells is inhibited by PCDD congeners and follows an SAR for AhR binding Since IgM is composed of two heavy chains and two light chains, the genes encoding these proteins are potential transcriptional targets modulated by TCDD. To determine if transcriptional regulation of the 11 gene underlies the inhibition of IgM secretion by TCDD, expression of the 11 gene in LPS-stimulated CH12.LX cells was analyzed by quantitative RT-PCR analysis following a 24 hr treatment with TCDD, HxCDD and TriCDD at various concentrations. Although TCDD and HxCDD inhibited )1 gene expression in a concentration-dependent manner with TCDD being more potent than HxCDD, TriCDD which induces Cyp] a1 and inhibits IgM secretion did not affect [.1 gene expression (Figure 26, Table 2). In addition the effect of HxCDD exhibited a rather flat concentration- response which is in contrast with its effect on Cyp] a1 induction (compare Figure 22 and Figure 26). The lack of effect by TriCDD and the blunted response of HxCDD may be due to slower kinetics of AhR activation by lower affinity AhR ligands. This potential effect on kinetics might be more pronounced in the shorter, 24 hr [.1 assay as opposed to the longer, 72 hr IgM protein secretion assay. In contrast to [.1 expression, CypIaI induction, 68 BCL— l X -4 Al I U ['1 c: c: L) '— NA VH MCDD Tri CDD TCDD HxCDD Figure 24. Effect of selected chlorinated dibenzo-p—dioxin congeners on Cyp] a1 expression in BCL-l cells. BCL-l cells (3x105 cells/ml) were treated with TCDD, HxCDD, TriCDD or MCDD at 30 nM. CH12.LX cells (1x105 cells/ml) served as a positive control and were treated with 3.0 nM TCDD. The vehicle (VH) control was 0.01% DMSO. Qualitative RT-PCR analysis for CypIaI was performed on RNA extracted at 48 hr from each treatment group. Results are representative of more than two separate experiments. 69 1.0x109 - -200 # 2 7.5x108 - -150 “E a as 8 z 3 l2) :2, a, 5.0x108- ”100 '- E S P d—l o v 11- E :. 5 2 2.5x108 - .. .50 L5, 0.0 . -0.0 NA LPS VH IMCDD Tn’CDD TCDD HxCDDl llgM protein 30 nM Congener I ll transcripts Figure 25. Effect of selected chlorinated dibenzo-p-dioxin congeners on LPS-induced u expression and IgM secretion in BCL—l cells. For analysis of 11 RNA expression, BCL-l cells (3x105 cells/ml) were treated with 30 ug/ml LPS (filled bars) and TCDD, HxCDD, TriCDD or MCDD at 30 nM. Quantitative RT-PCR analysis for u was performed on RNA extracted at 48 hr from each treatment group. Transcripts for u are identified on the left y- axis as molecules (mlcls)/100 ng RNA. Bar, mean :l: standard error for three separate RNA isolations (n=3). For analysis of IgM secretion, BCL—l cells (2x105 cells/ml) were treated with 30 [Lg/ml LPS (filled bars) and TCDD, HxCDD, TriCDD or MCDD at 30 nM. Supernatants were harvested at 72 hr and analde for IgM by sandwich ELISA. IgM is represented on the right y-axis as ng/105 cells. Bar, mean 3: standard error for triplicate determinations (n=3). These results were analyzed for statistical significance using Dunnett's two-tailed t-test. * and #, values that are significantly different, at P<0.05, from the vehicle (VH; 0.01% DMSO) controls for the u expression and IgM secretion experiments, respectively. The results are representative of more than two separate experiments. 70 25x107 - .,. 1 r ..... I c ........ "'. ------ J} 20x107~ 1 we": a- 2 T .. """"" ' Z 9 mm 7 .I. - 53-3. 15x10 ' 1' +1.01) 2 O 8 3 ES 10x107- ' 22. ' E 2 6 g 5.0x10 7,0 5 0.0 | l #T l I I NA LPS 0.0 0.1 1.0 10.0 nM Congener Figure 26. Concentration-dependent effect of a 24 hr incubation with selected chlorinated dibenzo-p—dioxin congeners on 1; expression in CH12.LX cells. CH12.LX cells (1x105 cells/ml) were treated with Bug/ml of LPS (filled symbols) and TCDD, HxCDD or TriCDD at various concentrations. The vehicle control (0.0 nM congener) was 0.01% DMSO. Quantitative RT-PCR analysis for 1). expression was performed on RNA extracted at 24 hr from each treatment group. Transcripts for p. are identified on the y-axis as molecules/100 ng RNA. Symbol, mean :l: standard error for three separate RNA isolations (n=3). These results were analyzed for statistical significance using Dunnett's two-tailed t-test. *, # and ‘1, values that are significantly different from the VH control within the TCDD, HxCDD and TriCDD experiments, respectively, at P<0.05. The results are representative of more than two separate experiments. 71 in a 24 hr assay, is extremely sensitive to TCDD, HxCDD and TriCDD (Figure 22). This sensitivity is likely due to the presence of six DRES in the Cyp] a1 promoter; five of which are capable of positively regulating transcription as demonstrated by reporter gene assays (172). Furthermore, the presence and functionality of DRES within critical regulatory regions of u is unclear. To explore the possibility of slower kinetics for the effects of TriCDD and HxCDD on u expression, LPS-stimulated CH12.LX cells were incubated with the PCDD congeners for 48 hr followed by RT-PCR analysis. In contrast to the 24 hr results, u expression was inhibited in a concentration-dependent manner by TriCDD and the concentration—response for HxCDD was sigmoidal (Figure 27). The rank order potency was TCDD > HxCDD > TriCDD >> MCDD; MCDD had no affect on u expression (Table 2, Figure 27). The effect of the PCDD congeners on LPS-induced p. expression in the AhR-deficient BCL—l cells was also analyzed at 48 hr by quantitative RT- PCR. All of the congeners had no effect on u gene expression from the BCL-l cells (Figure 25). D. Inhibition of IgM protein secretion and )1 expression is AhR dependent IC50 and EC50 values were generated from extensive concentration response curves (i.e., at least nine concentrations per congener) for each congener (Table 2). An abbreviated version of these curves is represented in Figures 22-27. For a given congener, statistical comparisons of the IC505 for 1; expression (48 h) and IgM protein secretion and the EC50 for induction of Cypl a1 expression were not significantly different with the exception of a slight difference between Cyp] a1 induction and u (48 h) inhibition with the TriCDD congener (Table 2). These results suggest a common mechanism of action and since induction of Cyp] a1 is an established AhR-mediated event, these results continue to support AhR-mediated inhibition of u expression and IgM protein secretion. In addition, the 1C5os and EC503 for a given endpoint among the PCDD congeners tended towards a 72 5.0x108 - 2 g 1.0x108 .. I - ” MI .9 a } ‘ fl , -4 " " ” .I. Q. o-l T M“... § 9 ‘WsT c 8’ . —l—TCDD .8 o " 1% 5.0X1O7 - ........ § ........ .. HXCDD T5 9 E ""9 """ TriCDD #43 -__-, ----- moon 0-0 I I '7 I I I I "5"— M LPS 0.0 0.1 1.0 10.0 100.0 I nM Congener Figure 27. Concentration-dependent effect of a 48 hr incubation with selected chlorinated dibenzo-p-dioxin congeners on II expression in CH12.LX cells. CH12.LX cells (1x10S cells/ml) were treated with 3 [lg/ml LPS (filled symbols) and TCDD, HxCDD, TriCDD or MCDD at various concentrations. The vehicle control (0.0 nM congener) was 0.01% DMSO. Quantitative RT-PCR analysis for u expression was performed on RNA extracted at 48 hr from each treatment group. Transcripts for u are represented on the y-axis as molecules/ 100 ng RNA. Symbol, mean :l: standard error for three separate RNA isolations (n=3). These results were analyzed for statistical significance using Dunnett's two-tailed t- test. *, #, ‘I and S, values that are significantly different from the VB control within the TCDD, HxCDD, TriCDD and MCDD experiments, respectively, at P<0.05. The results are representative of more than two separate experiments. 73 structure activity relationship which was concordant with the AhR binding affinity for the respective PCDD congeners, again supporting AhR-mediation of these three responses (Table 2). IV. Alteration of protein binding at the 3'01 Ig heavy chain enhancer by TCDD A. TCDD induces binding to a DRE-like site located within the 3'0tE(hs1,2) and 3'a-hs4 enhancers We have identified several DRE-like sequences in the 3'01 enhancer of the mouse Ig heavy chain gene. Our studies focused on two of the DRE-like sites; one of which is located in the 3'aE(hsl,2) enhancer and the other in the 3'a—hs4 enhancer (Figure 28). In the CH12.LX cells, EMSA analysis demonstrated TCDD-inducible binding, which migrated similarly to the DRE3 positive control, to both the 3'aE(hsl,2) and the 3'0t-hs4 oligomers (Figs. 29A and 30A, lanes 2, 4 and 5). Binding to these oligomers was also reduced with the addition of unlabeled DRE3, though not as effectively as with the unlabeled oligomers themselves (Figure 29A and 30A, lanes 5-7). These results suggest that the AhR-nuclear complex binds to both DRE-like sites identified within the 3'01E(hsl,2) and 3'01-hs4 oligomers, which was confirmed by EMSA- Westem analysis. Antibodies specific for the AhR and ARNT identified these proteins as components of the TCDD-inducible complex in both the 3'01.E(hsl,2) and 3'a-hs4 oligomers, as well as in the DRE3 positive control (Figure 29B and C, lanes 2 and 4 and Figure 30B and C, lanes 2 and 4). The AhR and ARNT migrated identically among the oligomers in the EMSA-Western (Figs. 29 and 30, B and C, compare lanes 2 and 4), as well as with the TCDD-inducible protein complexes formed with both oligomers in the EMSA (Figs. 29 and 30, compare lane 2 and 5 of A to lanes 2 and 4 of B and C). However, it is notable that in the EMSA, the TCDD-inducible complex formed with the 74 .0020: 03 00:0 my 00313:: 0:: 83020:: man 95:00:00 53:00:00.. .vmnéh :5 3,3590% 05 5 000000— 86 m: :3 0:0 3:00: a mm =03 m: 8:0 002-me 05 3820:: 0000002: 0:0 00:080.