Suppression of interleukin-2 by the putative endogenous cannabinoid 2-arachidonyl-glycerol is mediated through down-regulation of the nuclear factor of activated T cells.

ABSTRACT 2-Arachidonyl-glycerol (2-Ara-Gl) recently was identified as a putative endogenous ligand for cannabinoid receptor types CB1 and CB2 by competitive binding. More recent immune function assays demonstrated that 2-Ara-Gl possessed immunomodulatory activity. Because several plant-derived cannabinoids inhibit interleukin-2 (IL-2) expression, 2-Ara-Gl was investigated for its ability to modulate this cytokine. The direct addition of 2-Ara-Gl to mouse splenocyte cultures suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 secretion and steady state mRNA expression in a dose-dependent manner. 2-Ara-Gl also produced a marked inhibition of IL-2 promotor activity as determined by transient transfection of EL4.IL-2 cells with a pIL-2-CAT construct. 2-Ara-Gl at 5, 10, 20, and 50 M suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 promotor activity by 18%, 28%, 39%, and 54%, respectively. To further characterize the mechanism for the transcriptional regulation of IL-2 by 2-Ara-Gl, the DNA-binding activity of transcription factors, nuclear factor of activated T cells (NF-AT), nuclear factor for immunoglobulin chain in B cells (NF-B/Rel), activator protein-1(AP-1), octamer, and cAMP-response element binding protein was evaluated by electrophoretic mobility shift assay in mouse splenocytes. In addition, a reporter gene expression system for p(NF-B) 3 -CAT, p(NF-AT) 3 -CAT, and p(AP-1) 3 -CAT was used in transiently transfected EL4.IL-2 cells to determine the effect of 2-Ara-Gl on promoter activity for each of the specific transcription factors. 2-Ara-Gl reduced both the NF-AT-binding and promoter activity in a dose-dependent manner and, to a lesser degree, NF-B/Rel-binding and promoter activity. No significant effect was observed on octamer-and cAMP-response element-binding activity. AP-1 DNA-binding activity was not inhibited by 2-AraGl, but a modest inhibition of promoter activity was observed

-Tetrachlorodibenzo-p-dioxin-Mediated Impairment of B Cell Differentiation Involves Dysregulation of Paired Box 5 (Pax5) Isoform, Pax5a

ABSTRACT The persistent environmental contaminant and immunotoxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), markedly suppresses humoral immune responses. We recently reported impaired down-regulation of paired box 5 (Pax5), a repressor of B cell differentiation and concomitant suppression of the IgM response by TCDD in the murine CH12.LX B cell line. The objectives of the current study were to determine the impact of TCDD treatment on molecular outcomes characteristic of terminal B cell differentiation and to assess the role that Pax5 isoforms plays in the suppression of B cell differentiation by TCDD. In this study, we show that the highly abundant fulllength Pax5 isoform, Pax5a, and at least two additional modestly expressed Pax5 isoforms were expressed in CH12.LX and splenic B cells. In lipopolysaccharide (LPS)-activated B cells, all of the identified Pax5 isoforms were synchronously down-regulated, and in the presence of TCDD cotreatment they were abnormally and synchronously elevated, suggesting a common mechanism of regulation. Furthermore, B cell differentiation markers X-box protein-1 and major histocompatibility complex class II showed that the levels to which Pax5 was derepressed by TCDD were sufficient to impair B cell differentiation and immunoglobulin gene expression. Confirming the involvement of Pax5, ectopic expression of Pax5a in the LPS-activated CH12.LX cells closely mimicked the suppression of the IgM response by TCDD. In summary, our results demonstrate that Pax5a has a critical role in both the TCDD-mediated impairment of B cell differentiation and the suppression of the humoral immune response. Suppression of primary humoral immune responses is one of the most sensitive sequela associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous environmental contaminant. This suppression is characterized by a striking reduction in plasma cell formation and IgM secretion, and it is mediated through a direct effect by TCDD on B cells Activation by antigen or via polyclonal activators (e.g., 463 lipopolysaccharide; LPS) induces B cells to undergo cycles of intense proliferation (i.e., clonal expansion) followed by terminal differentiation into plasma cells. Terminally differentiated plasma cells specialize in secretion of antigen-specific Ig. A number of phenotypic changes distinguish terminally differentiated plasma cells from the resting B cells Materials and Methods Chemicals. TCDD, in 100% dimethyl sulfoxide (DMSO), was purchased from AccuStandard (New Heaven, CT). DMSO and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Mice. Virus-free, female B6C3F1mice (six weeks old) were purchased from Charles River (Portage, MI). On arrival, mice were randomly grouped with five per plastic cage on sawdust bedding. Mice had free access to food (Purina Certified Laboratory Chow) and water at all times. The mouse holding rooms were maintained at 21 to 24°C with 40 to 60% relative humidity on a 12-h light/dark cycle. All of the experimental procedures and conditions were performed according to the guidelines of the All University Committee on Animal Use and Care at Michigan State University (East Lansing, MI). Cell Line. The CH12.LX B cell line was derived from the murine B cell lymphoma, CH12, which arose in B10.H-2aH-4bP/Wts mice (B10.A ϫ B10.129) and has been characterized previously Flow Cytometric Analysis. Cells were harvested from culture at the indicated times by centrifugation at 300g for 10 min at 4°C, washed twice in ice-cold 1ϫ Hanks' balanced-salt solution, and stained using the BD Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. In brief, cells were incubated with 1 g/10 6 cells of purified rat anti-mouse CD16/CD32 monoclonal antibody (BD Pharmingen) for 15 min at 4°C to prevent nonspecific binding and then stained with surface marker detection antibody [anti-fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse I-A P or anti-allophycocyanin-conjugated anti-mouse CD19] or a respective isotype control (BD Pharmingen). To exclude nonviable cells, 2 l of 7-aminoactinomycin D (7-AAD) solution (Sigma-Aldrich) containing 1 mg of 7-AAD, 50 l methanol, and 950 l of Hanks' balanced-salt solution were added simultaneously with detection antibodies to the cells in 50 l of staining buffer. The cells were then fixed, washed, and maintained in staining buffer containing 10 mM actinomycin D (Sigma-Aldrich) to prevent 7-AAD leakage from fixed cells. For the detection of nuclear Pax5 protein, cells were fixed and permeabilized before staining with anti-Pax5 antibody or isotype control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Fluorescence detection was performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) on 10,000 viable cells per sample. Data analysis was performed using BD CellQuest Pro software (BD Biosciences). In Vitro Polyclonal IgM Antibody-Forming Cell Response. Single-cell suspensions of splenocytes from naive mice were adjusted to 1 ϫ 10 6 cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 10% bovine calf serum (HyClone Laboratories), 100 units/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol. Spleen cells were transferred to 48-well culture plates in 500-l aliquots with four wells per treatment group. TCDD (3, 10, or 30 nM) and/or vehicle (0.01% DMSO) were added directly to each well in 5-l aliquots. The splenocytes were sensitized with 10 g/ml LPS and cultured for 3 days in a pressurized chamber at 5.0 psi containing 10% O 2 , 7% CO 2 , and 83% N 2 gas mixture with continuous rocking at 37°C. Enumeration of the IgM antibody-forming cell (AFC) response was performed using trinitrophenol-haptenated sheep red blood cells as described previously the cell mixture was poured onto a 100 ϫ 15-mm Petri dish and immediately covered with a 45 ϫ 50-mm glass coverslip. Upon solidification of the agar, the Petri dishes were placed in a humidified 37°C incubator overnight to allow for plaque formation. The number of splenocytes from each spleen was determined using a Coulter Counter (Beckman Coulter, Fullerton, CA). Results are expressed as AFC/10 6 viable splenocytes Ϯ S.E. Splenocyte viability was measured using pronase (EMD Biosciences, San Diego, CA) as described previously Purification of Splenic B Cells. Spleens were removed aseptically and made into a single-cell suspension. B cells were then isolated using the B Cell Isolation Kit (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's protocol. In brief, 40 l of MACS buffer (phosphate-buffered saline solution, pH 7.2, supplemented with 0.5% bovine serum albumin and 2 mM EDTA, 4°C) per 10 7 cells was used to suspend the splenocytes, and 10 l of Biotin-Antibody Cocktail (Miltenyi Biotec Inc.) per 10 7 cells was added to label non-B cells. After incubation at 4 to 8°C for 10 min, 30 l of buffer and 20 l of Anti-Biotin Microbeads (Miltenyi Biotec Inc.) per 10 7 cells were added. The cell suspension was incubated for another 15 min at 4 to 8°C, washed with 10 to 20 times the labeling volume, and then centrifuged at 300g for 5 min. The cell pellet was finally resuspended in 500 l of buffer per 1 ϫ 10 8 cells, and passed through the prerinsed LS column (Miltenyi Biotec Inc.), followed by four washes of the column with 3 ml of buffer. The entire effluent containing the purified B cell fraction was collected. The purity of the isolated B cells was evaluated using flow cytometry to enumerate the percentage of CD19 ϩ cells through directly labeling with FITC-conjugated anti-CD19 antibody (BD Biosciences). Real-Time Polymerase Chain Reaction. Total RNA was isolated from naive or LPS-activated cells using a SV Total RNA Isolation kit (Promega, Madison, WI). To synthesize cDNA, 1000 ng/ sample of total RNA was incubated with 600 ng of random primer (Invitrogen) in 10 l of endonuclease-free water at 70°C for 10 min, cooled on ice for 10 min, and reverse transcribed in 20 l of 1ϫ First-Strand Synthesis buffer (Invitrogen), containing 0.2 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, and 200 units of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42°C for 60 min, and the reaction was stopped by incubation at 75°C for 15 min. Real-time polymerase chain reaction (PCR) detection was performed using TaqMan primers and probes Isolation of Pax5a cDNA. cDNA was obtained by PCR amplification of total RNA isolated from CH12.LX cells. Total RNA was extracted with the SV Total RNA Isolation kit (Promega), and firststrand cDNA was synthesized by reverse transcription (RT) using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). PCR was carried out as follows: one initial 2-min denaturing step at 94°C followed by denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and extension for 90 s at 72°C during 30 cycles in the presence of Pfu polymerase (Stratagene, La Jolla, CA) under the conditions suggested by the supplier (at a final primer concentration of 0.5 M). Sequences of the primers designed for this PCR reaction were as follows: 5Ј-CTGTCCATTTCATCAAGTCCTGA-3Ј and 5Ј-ACC-GTCACTACCCTCAGAG-3Ј. The resulting PCR product of 1.2 kilobase was separated in 1% agarose gel electrophoresis in 1ϫ TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3) and stained with ethidium bromide at 1 g/ml. The PCR fragment was eluted from agarose using the QIAquick gel extraction kit (QIAGEN, Valencia, CA). Cloning and Ectopic Expression for Pax5a cDNA. Pax5a cDNA, generated as described above, was inserted into the pcDNA 3.1 V5-His-TOPO vector (Invitrogen) according to the manufacturer's protocol. The new Pax5a-V5-His tag vector sequence was confirmed by sequencing. Additional PCR reactions, in which Pax5a-V5-His vector was used as template, were performed to generate a new Pax5a expression plasmid, Pax5a-V5-His-GFP. The primer used for 5Ј-end cloning was as follows: 5Ј-CTC ACT ATA GGG AGA TCT AAG CTG GCT AGT-3Ј (BglII restriction site is underlined); and the primer used for the 3Јend cloning was as follows: 5Ј-TGA TCA GCG GGT TTA AAA GCT TTG GGA TGG TG-3Ј (HindIII restriction site is underlined). PCR products of 1.38 kilobase were obtained and isolated as described above. The Pax-5a-V5-His PCR fragment was then cloned into the vector phCMV-C-GFP (Genlantis, San Diego, CA) by a standard ligation reaction using T4-DNA ligase (New England Biolabs, Ipswich, MA). The phCMV vector from Genlantis contains an optimized cytomegalovirus promoter and intron sequences and a kanamycin/neomycin resistance gene for generation of stable cell lines. Transfection of Pax5a-V5-His-GFP. CH12.LX cells (2.5 ϫ 10 6 ) were transfected with 5 g of each plasmid using Amaxa Nucleofector (Amaxa Inc., Gaithersburg, MD). Cells, DNA, and 100 l of 90:20 (solution V supplement) were mixed and electroporated using program A-20 following the manufacturer's recommendations. A total of TCDD Impairment of B Cell Differentiation by Altered Pax5 465 2.5 ϫ 10 7 cells was used in each experiment. The backbone plasmid, phCMV-C-GFP, was used as a control in all of the transfection experiments. After electroporation, cells were incubated at 37°C in an atmosphere of 5% CO 2 for 8 h. Eight hours after transfection, cells were collected and sorted by flow cytometry for green fluorescent protein (GFP)-expressing cells. Cells that showed fluorescence above the level of fluorescence of naive CH12.LX cells were isolated using a BD FACSCalibur. The isolated cells were counted and LPS-activated for assessments of IgM secretion as determined by enzymelinked immunosorbent assay (ELISA). An aliquot of the sorted cells was also used to prepare protein extracts to determine endogenous/ ectopic Pax5a levels by Western blotting and by flow cytometry. IgM ELISA. IgM ELISA was performed as described previously Western Blotting. Proteins were extracted from transfected cells before and after sorting by flow cytometry. Protein extracts were resolved on 4 to 12% Nu-Page gradient gel (Invitrogen) and then transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Immunoblotting was performed using anti-␤-actin and anti-Pax5 antibodies (Santa Cruz Biotechnology, Inc.). SuperSignal West Pico reagent (Pierce, Rockford, IL) was used for protein detection. Statistical Analysis of Data. Mean Ϯ S.E. was determined for each treatment group of a given PCR or ELISA experiment. Statistical differences between groups in each experiment were determined by a two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for PCR experiments and by a one-way ANOVA followed by Bonferroni's post hoc test for ELISA experiments. Results TCDD Decreases the Levels of XBP-1 in LPS-Activated B Cells. Previous studies in LPS-activated CH12.LX cells demonstrated robust suppression of the humoral IgM response by TCDD, which correlated with a marked reduction in XBP-1 protein levels Additional studies were performed to determine whether LPS and/or TCDD treatment affects the post-transcriptional modification of XBP-1. Transcriptional activity of XBP-1 is known to be enhanced by a splicing event, which removes a 26-nucleotide-long fragment from XBP-1 mRNA, resulting in an open reading frame shift and yielding a larger XBP-1 466 Schneider et al. protein that is more potent as a transcription factor TCDD Attenuates Cell Surface MHC Class II DownRegulation in LPS-Activated CH12.LX Cells. An additional event closely associated with terminal differentiation of B cells is the down-regulation of MHC class II on the cell membrane. Levels of surface MHC class II in LPS-activated CH12.LX cells and in splenic B cells (i.e., CD19 ϩ ), in the presence and absence of TCDD, were monitored by flow cytometry. LPS activation induced a down-regulation of MHC class II on the CH12.LX cell surface between 24 and 72 h of culture compared with the time-matched naive control TCDD Attenuates the LPS-Induced Down-Regulation of Pax5 Protein in LPS-Activated CH12.LX Cells. We also examined the impact of TCDD on Pax5, a repressor of B cell differentiation. Expression of Pax5 protein was characterized in splenic B cells and CH12.LX cells by flow cytometry facilitating the evaluation of individual cells in contrast to prior Western blot studies that we performed exclusively in CH12.LX cells Characterization of Pax5 Isoforms in CH12.LX Cells. In light of the altered Pax5 expression observed by flow cytometry and by real-time PCR, studies were undertaken to further characterize the effect of TCDD on Pax5 regulation. In particular, we examined the role of alternative splicing in the deregulation of Pax5 by TCDD Additional studies performed in CH12.LX cells showed that the levels of amplicons I, II, and III were down-regulated by LPS between 24 and 72 h, as determined by real-time quantitative PC

Activated carbons of varying pore structure eliminate the bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a mammalian (mouse) model

The use of activated carbon (AC) as an in situ sorbent amendment to sequester polychlorinated-dibenzo-p-dioxins and furans (PCDD/Fs) present in contaminated soils and sediments has recently gained attention as a novel remedial approach. This remedy could be implemented at much lower cost while minimizing habitat destruction as compared to traditional remediation technologies that rely on dredging/excavation and landfilling. Several prior studies have demonstrated the ability of AC amendments to reduce pore water concentrations and hence bioaccumulation of PCDD/Fs in invertebrate species. However, our recent study was the first to show that AC had the ability to sequester 2,3,7,8‑tetrachlorodibenzo‑p‑dioxin (TCDD) in a form that eliminated bioavailability to a mammalian (mouse) model. Here we show that three commercially available ACs, representing a wide range of pore size distributions, were equally effective in eliminating the bioavailability of TCDD based upon two sensitive bioassays, hepatic induction of cyp1A1 mRNA and immunoglobulin M antibody-forming cell response. These results provide direct evidence that a wide range of structurally diverse commercially available ACs may be suitable for use as in situ sorbent amendments to provide a cost-effective remedy for PCDD/F contaminated soils and sediments. Potentially, adaption of this technology would minimize habitat destruction and be protective of ecosystem and human health

Stochastic Modeling of B Lymphocyte Terminal Differentiation and Its Suppression by Dioxin

<p>Abstract</p> <p>Background</p> <p>Upon antigen encounter, naïve B lymphocytes differentiate into antibody-secreting plasma cells. This humoral immune response is suppressed by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other dioxin-like compounds, which belong to the family of aryl hydrocarbon receptor (AhR) agonists.</p> <p>Results</p> <p>To achieve a better understanding of the immunotoxicity of AhR agonists and their associated health risks, we have used computer simulations to study the behavior of the gene regulatory network underlying B cell terminal differentiation. The core of this network consists of two coupled double-negative feedback loops involving transcriptional repressors Bcl-6, Blimp-1, and Pax5. Bifurcation analysis indicates that the feedback network can constitute a bistable system with two mutually exclusive transcriptional profiles corresponding to naïve B cells and plasma cells. Although individual B cells switch to the plasma cell state in an all-or-none fashion when stimulated by the polyclonal activator lipopolysaccharide (LPS), stochastic fluctuations in gene expression make the switching event probabilistic, leading to heterogeneous differentiation response among individual B cells. Moreover, stochastic gene expression renders the dose-response behavior of a population of B cells substantially graded, a result that is consistent with experimental observations. The steepness of the dose response curve for the number of plasma cells formed vs. LPS dose, as evaluated by the apparent Hill coefficient, is found to be inversely correlated to the noise level in Blimp-1 gene expression. Simulations illustrate how, through AhR-mediated repression of the AP-1 protein, TCDD reduces the probability of LPS-stimulated B cell differentiation. Interestingly, stochastic simulations predict that TCDD may destabilize the plasma cell state, possibly leading to a reversal to the B cell phenotype.</p> <p>Conclusion</p> <p>Our results suggest that stochasticity in gene expression, which renders a graded response at the cell population level, may have been exploited by the immune system to launch humoral immune response of a magnitude appropriately tuned to the antigen dose. In addition to suppressing the initiation of the humoral immune response, dioxin-like compounds may also disrupt the maintenance of the acquired immunity.</p

Perceived Conflict of Interest in Health Science Partnerships

University scientists conducting research on topics of potential health concern often want to partner with a range of actors, including government entities, non-governmental organizations, and private enterprises. Such partnerships can provide access to needed resources, including funding. However, those who observe the results of such partnerships may judge those results based on who is involved. This set of studies seeks to assess how people perceive two hypothetical health science research collaborations. In doing so, it also tests the utility of using procedural justice concepts to assess perceptions of research legitimacy as a theoretical way to investigate conflict of interest perceptions. Findings show that including an industry collaborator has clear negative repercussions for how people see a research partnership and that these perceptions shape people’s willingness to see the research as a legitimate source of knowledge. Additional research aimed at further communicating procedures that might mitigate the impact of industry collaboration is suggested

Reception Test of Petals for the End Cap TEC+ of the CMS Silicon Strip Tracker

The silicon strip tracker of the CMS experiment has been completed and was inserted into the CMS detector in late 2007. The largest sub system of the tracker are its end caps, comprising two large end caps (TEC) each containing 3200 silicon strip modules. To ease construction, the end caps feature a modular design: groups of about 20 silicon modules are placed on sub-assemblies called petals and these self-contained elements are then mounted onto the TEC support structures. Each end cap consists of 144 such petals, which were built and fully qualified by several institutes across Europe. Fro

Integration of the End Cap TEC+ of the CMS Silicon Strip Tracker

The silicon strip tracker of the CMS experiment has been completed and inserted into the CMS detector in late 2007. The largest sub-system of the tracker is its end cap system, comprising two large end caps (TEC) each containing 3200 silicon strip modules. To ease construction, the end caps feature a modular design: groups of about 20 silicon modules are placed on sub-assemblies called petals and these self-contained elements are then mounted into the TEC support structures. Each end cap consists of 144 petals, and the insertion of these petals into the end cap structure is referred to as TEC integration. The two end caps were integrated independently in Aachen (TEC+) and at CERN (TEC--). This note deals with the integration of TEC+, describing procedures for end cap integration and for quality control during testing of integrated sections of the end cap and presenting results from the testing

Cannabinoid-mediated elevation of intracellular calcium: A structure-activity relationship

This laboratory has reported previously that 9-tetrahydrocan-nabinol (9-THC) and cannabinol (CBN) robustly elevate intra-cellular calcium ([Ca2]i) in resting human and murine T cells, whereas CP55,940 [5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol], a high-affinity ligand for CB1 and CB2, does not. In light of our previous studies, the objec-tive of the present investigation was to examine the ability of various cannabinoid compounds to elevate [Ca2]i in the CB2 receptor-expressing human peripheral blood acute lymphoid leukemia T cell line and the dependence of structural similarity to 9-THC therein. The present studies demonstrate that CBN and HU-210 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tet-rahydro-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol], both tricyclic and in that respect structurally similar to 9-THC, ele