Norbert E. Kaminski

Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors.

In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2--the two cannabinoid receptors identified thus far--with Ki values of 472 +/- 55 and 1400 +/- 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of delta 9-tetrahydrocannabinol (delta 9-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of delta 9-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than delta 9-THC.

Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism

Cannabinoid compounds, including the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (delta 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that delta 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a Gi-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of delta 9-THC (22 microM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 microM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 microM). Inhibition of the sRBC AFC response by both delta 9-THC (22 microM) and CP-55940 (5.2 microM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation of intracellular cAMP. These results indicate that inhibition of the sRBC AFC response by cannabinoids is mediated, at least in part, by inhibition of adenylate cyclase through a pertussis toxin-sensitive Gi-protein coupled cannabinoid receptor. Additionally, these studies further support the premise that cAMP is an important mediator of lymphocyte activation.

Interleukin-2 Suppression by 2-Arachidonyl Glycerol Is Mediated through Peroxisome Proliferator-Activated Receptor γ Independently of Cannabinoid Receptors 1 and 2

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative that binds cannabinoid receptors CB1 and CB2 and is hence termed an endocannabinoid. 2-AG also modulates a variety of immunological responses, including expression of the autocrine/paracrine T cell growth factor interleukin (IL)-2. The objective of the present studies was to determine the mechanism responsible for IL-2 suppression by 2-AG. Because of the labile properties of 2-AG, 2-AG ether, a nonhydrolyzable analog of 2-AG, was also used. Both 2-AG and 2-AG ether suppressed IL-2 expression independently of CB1 and CB2, as demonstrated in leukocytes derived from CB1/CB2-null mice. Moreover, we demonstrated that both 2-AG and 2-AG ether treatment activated peroxisome proliferator-activated receptor γ (PPARγ), as evidenced by forced differentiation of 3T3-L1 cells into adipocytes, induction of aP2 mRNA levels, and activation of a PPARγ-specific luciferase reporter in transiently transfected 3T3-L1 cells. Consequently, the putative role of PPARγ in IL-2 suppression by 2-AG and 2-AG ether was examined in Jurkat T cells. Concordant with PPARγ involvement, the PPARγ-specific antagonist 2-chloro-5-nitro-N-(4-pyridyl)-benzamide (T0070907) blocked 2-AG- and 2-AG ether-mediated IL-2 suppression. Likewise, 2-AG suppressed the transcriptional activity of two transcription factors crucial for IL-2 expression, nuclear factor of activated T cells and nuclear factor κB, in the absence but not in the presence of T0070907. 2-AG treatment also induced PPARγ binding to a PPAR response element in activated Jurkat T cells. Together, the aforementioned studies identify PPARγ as a novel intracellular target of 2-AG, which mediates the suppression of IL-2 by 2-AG in a manner that is independent of CB1 and/or CB2.

Smad3 Is Essential for TGF-β1 to Suppress IL-2 Production and TCR-Induced Proliferation, but Not IL-2-Induced Proliferation

Transforming growth factor-β1 is essential to maintain T cell homeostasis, as illustrated by multiorgan inflammation in mice deficient in TGF-β1 signaling. Despite the physiological importance, the mechanisms that TGF-β1 uses to regulate T cell expansion remain poorly understood. TGF-β1 signals through transmembrane receptor serine/threonine kinases to activate multiple intracellular effector molecules, including the cytosolic signaling transducers of the Smad protein family. We used Smad3−/− mice to investigate a role for Smad3 in IL-2 production and proliferation in T cells. Targeted disruption of Smad3 abrogated TGF-β1-mediated inhibition of anti-CD3 plus anti-CD28-induced steady state IL-2 mRNA and IL-2 protein production. CFSE labeling demonstrated that TGF-β1 inhibited entry of wild-type anti-CD3 plus anti-CD28-stimulated cells into cycle cell, and this inhibition was greatly attenuated in Smad3−/− T cells. In contrast, disruption of Smad3 did not affect TGF-β1-mediated inhibition of IL-2-induced proliferation. These results demonstrate that TGF-β1 signals through Smad3-dependent and -independent pathways to inhibit T cell proliferation. The inability of TGF-β1 to inhibit TCR-induced proliferation of Smad3−/− T cells suggests that IL-2 is not the primary stimulus driving expansion of anti-CD3 plus anti-CD28-stimulated T cells. Thus, we establish that TGF-β1 signals through multiple pathways to suppress T cell proliferation.

Anandamide-Induced Depressor Effect in Spontaneously Hypertensive Rats. Role of the Vanilloid Receptor

Abstract—To test the hypothesis that activation of the vanilloid receptor (VR1) contributes to the anandamide-induced depressor effect in spontaneously hypertensive rats (SHR), we used a selective VR1 antagonist capsazepine (CAPZ) and a selective cannabinoid type 1 receptor antagonist SR141716A in conjunction with a VR1 agonist capsaicin in both SHR and Wistar-Kyoto rats (WKY). Mean arterial pressure was increased in SHR compared with WKY (P <0.05). Intravenous administration of capsaicin caused a greater depressor response in SHR compared with WKY (P <0.05), which was blocked by ≈60% by CAPZ (P <0.05) in SHR only. Methanandamide caused a similar greater depressor response (P <0.05), which was blocked by ≈50% and 60% by CAPZ and SR141716A, respectively, in SHR (P <0.05) but not in WKY. Radioimmunoassay showed that methanandamide increased plasma calcitonin gene-related peptide (CGRP) levels from baseline in both SHR and WKY (P <0.05), with no difference between 2 strains. Western blot showed that protein expression for the calcitonin receptor–like receptor—but not receptor activity modifying protein 1, VR1, and cannabinoid type 1 receptors—was increased in mesenteric resistance arteries in SHR compared with WKY (P <0.05). These data indicate that in addition to activation of cannabinoid type 1, anandamide may serve as an endogenous compound to stimulate VR1, leading to a decrease in blood pressure via CGRP release from sensory nerve terminals. Increased mesenteric CGRP receptor expression in SHR may account for increased sensitivity of blood pressure to anandamide and may serve as a compensatory response to buffer the increase in blood pressure in SHR.

Δ9-tetrahydrocannabinol selectivity inhibits T-cell dependent humoral immune responses through direct inhibition of accessory T-cell function

Abstract The major psychoactive and immunosuppressive component of marihuana, Δ 9 -tetrahydrocannabinol ( Δ 9 -THC), was investigated for its effects on primary humoral immune responses in the B6C3F1 mouse strain. Oral administration of 50–200 mg/kg Δ 9 -THC produced a selective and dose related inhibition of primary humoral immune responses to the T-cell dependent antigen, sRBC, as measured by the antibody forming cell (AFC) response with no inhibitory effect on humoral responses to the T-cell independent antigen, DNP-Ficoll. A similar profile of immune inhibition was observed following in vitro direct addition of Δ 9 -THC to naive spleen cell cultures sensitized with defined antigens. Δ 9 -THC produced a marked and dose related inhibitio n of the in vitro sRBC AFC response while having no inhibitory effects on T-cell independent responses to either DNP-Ficoll or the polyclonal B-cell activator, lipopolysaccharide. This selective inhibition of the sRBC response was not due to a shift in the peak day of response or a direct cytotoxic effect on spleen cells. In vivo kinetic studies demonstrated that inhibition by Δ 9 -THC of the sRBC response was most pronounced when drug administration occured at times surrounding antigen sensitation. To further evaluate the direct effect of Δ 9 -THC on T-cell function, T-cell proliferative responses to stimulation by anti-CD3 monoclonal antibodies were measured. Δ 9 -THC was found to produce a marked and dose related inhibition of anti-CD3 mAb-induced T-cell proliferation which was cell density dependent. These results suggest that suppression of humoral responses by Δ 9 -THC may be mediated through early selective inhibition of T-cell accessory function.

Inhibition of the Cyclic AMP Signaling Cascade and Nuclear Factor Binding to CRE and κB Elements by Cannabinol, a Minimally CNS-Active Cannabinoid

Immune suppression by cannabinoids has been widely demonstrated in a variety of experimental models. The identification of two major types of G-protein-coupled cannabinoid receptors expressed on leukocytes, CB1 and CB2, has provided a putative mechanism of action for immune modulation by cannabinoid compounds. Ligand binding to both receptors negatively regulates adenylate cyclase, thereby lowering intracellular cyclic AMP (cAMP) levels. In the present studies, we demonstrated that cannabinol (CBN), a ligand that exhibits higher binding affinity for CB2, modulates immune responses and cAMP-mediated signal transduction in mouse lymphoid cells. Direct addition of CBN to naive cultured splenocytes produced a concentration-dependent inhibition of lymphoproliferative responses to anti-CD3, lipopolysaccharide, and phorbol-12-myristate-13-acetate/ionomycin stimulation. Similarly, a concentration-related inhibition of the in vitro anti-sheep red blood cell IgM antibody forming cell response was also observed by CBN. Evaluation of cAMP signaling in the presence of CBN showed a rapid and concentration-related inhibition of adenylate cyclase activity in both splenocytes and thymocytes. This decrease in intracellular cAMP levels produced by CBN resulted in a reduction of protein kinase A activity, consequently leading to an inhibition of transcription factor binding to the cAMP response element and kappaB motifs in both cell preparations. Collectively, these results demonstrate that CBN, a cannabinoid with minimal CNS activity, inhibited both cAMP signal transduction and immune function, further supporting the involvement of CB2 receptors in immune modulation by cannabimimetic agents.

Time-Dependent Airway Epithelial and Inflammatory Cell Responses Induced by Influenza Virus A/PR/8/34 in C57BL/6 Mice

The present study examines the kinetics of airway epithelial remodeling and inflammation in the airways of C57BL/6J mice infected with influenza virus A/PR/8/34 (PR8). Mice were intranasally instilled with 50 plaque forming units (pfu) of virus or its respective vehicle, saline, and then were sacrificed at 3, 7, 10, 15, or 21 days postinfection (dpi). PR8 treatment resulted in airway epithelial cell regeneration as suggested by proliferating cell nuclear antigen (PCNA) positive staining at 7 and 10 dpi and mucous cell metaplasia (MCM) evident at 10, 15, and 21 dpi. PR8 treatment resulted in a classic pattern of inflammation observed in bronchoalveolar lavage fluid (BALF), in which neutrophils peaked at 3 and 7 dpi and monocytes, lymphocytes, and eosinophils peaked at 10 dpi before returning to background levels of detection. Chemokine (MCP-1) and cytokine (IL-6, TNF-α, IFN-γ, IL-5, IL-4, and IL-9) levels peaked at 7 dpi in BALF. IL-13 levels were unaffected by PR8 treatment. Concurrent with inflammation, MUC5AC gene expression was markedly increased by PR8 treatment at 7 dpi. Collectively, the results of this study indicate that the onset of MCM in airway epithelium occurs during the remodeling process and persists after the inflammatory response has diminished.

Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study

Abstract Dioxins and dioxin-like compounds are tumor promoters that cause liver cancer in rats and mice. The aryl hydrocarbon receptor (AHR) has been implicated as a key component in this tumor promotion response. Despite extensive knowledge of the toxicology of dioxins, no mode of action (MOA) hypothesis for their tumorigenicity has been formally documented using the Human Relevance MOA framework developed by the International Programme on Chemical Safety (IPCS). To address this information gap, an expert panel was convened as part of a workshop on receptor-mediated liver tumorigenicity. Liver tumors induced by ligands of the AHR were assessed using data for dioxins and related chemicals as a case study. The panel proposed a MOA beginning with sustained AHR activation, eventually leading to liver tumors via a number of other processes, including increased cell proliferation of previously initiated altered hepatic foci, inhibition of intrafocal apoptosis and proliferation of oval cells. These processes have been identified and grouped as three key events within the hepatocarcinogenic MOA: (1) sustained AHR activation, (2) alterations in cellular growth and homeostasis and (3) pre-neoplastic tissue changes. These key events were identified through application of the Bradford-Hill considerations in terms of both their necessity for the apical event/adverse outcome and their human relevance. The panel identified data supporting the identification and dose–response behavior of key events, alteration of the dose–response by numerous modulating factors and data gaps that potentially impact the MOA. The current effort of applying the systematic frameworks for identifying key events and assessing human relevance to the AHR activation in the tumorigenicity of dioxins and related chemicals is novel at this time. The results should help direct future regulatory efforts and research activities aimed at better understanding the potential human cancer risks associated with dioxin exposure.

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

Suppression of humoral immune responses by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was first reported in the mid-1970s. Since this initial observation, much effort has been devoted by many laboratories toward elucidation of the cellular and molecular mechanisms responsible for the profound impairment of humoral immune responses by TCDD, which is characterized by decreased B cell to plasma cell differentiation and suppression of immunoglobulin production. These efforts have led to a significant body of research demonstrating a direct effect of TCDD on B-cell maturation and function as well as a requisite but as yet undefined role of the aryl hydrocarbon receptor (AhR) in these effects. Likewise, a number of molecular targets putatively involved in mediating B-cell dysfunction by TCDD, and other AhR ligands, have been identified. However, our current understanding has primarily relied on findings from mouse models, and the translation of this knowledge to effects on human B cells and humoral immunity in humans is less clear. Therefore, a current challenge is to determine how TCDD and the AhR affect human B cells. Efforts have been made in this direction but continued progress in developing adequate human models is needed. An in-depth discussion of these advances and limitations in elucidating the cellular and molecular mechanisms putatively involved in the suppression of B-cell function by TCDD as well as the implications on human diseases associated in epidemiological studies with exposure to TCDD and dioxin-like compounds is the primary focus of this review.