Part-I:Studies on the inhibitory effects of cannabinoids on LPS-stimulated murine J774 macrophages Part-II: Generation of baculovirus-expressed TR2 fusion protein and monoclonal antibodies

• Main Authors: Yinghsin Chang, 張櫻馨
• Other Authors: WanWan Lin
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/62155167898001552302

碩士 === 國立臺灣大學 === 藥理學研究所 === 87 === Part-I D9-THC is the major psychoactive component of marijuana. The behavioural and pharmacological actions of D9-THC have been intensively studied. Two subtypes of cannabinoid receptors have been identified and mediate the pharmacological effects of D9-THC; CB1 is expressed primarily although not exclusively in brain, and CB2 is found primarily in cells of myeloid lineage. Both receptors are involved in mediating the diverse biological action of cannabinoid compounds, including immune suppression and alterations in central nervous system function. Anandamide (AEA) and 2-arachidonyl-glycerol (2-AG) were recently identified as endogenous ligands for cannabinoid receptors and in vivo produce effects very similar to those of the classical agonists of the cannabinoid receptor. Furthermore, because of its structural similarities to polyunsaturated fatty acids, AEA and 2-AG could serve as substrate for lipoxygenases and cyclooxygenases that metabolize polyunsaturated fatty acids to potent bioactive molecules. The objective of present study was to investigate the immunomodulatory effects of cannabinoids on murine J774 macrophages. D9-THC (1-10mM) and AEA (3-30mM) were shown to diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibited the production of IL-6 but had little increased effect on NO production. In parallel, D9-THC, AEA and IMMA (a ligand for CB receptors) could inhibit iNOS induction in response to LPS, whereas 2-AG could not. D9-THC also could inhibit LPS-induced PGE2 production and COX-2 induction. Besides, D9-THC, AEA and 2-AG could inhibit LPS-induced IL-6 expression. Furthermore, in order to understand the possible involvement of ligand metabolites on these events, we explore the effects of PGE2-ethanolamide, arachidonic acid (AA) and PGE2, which might be the metabolites of AEA and 2-AG. We found that PGE2-ethanolamide, the reported AEA metabolite, neither influenced the LPS-induced NO nor IL-6 production. On the other hand, AA and PGE2 could increase NO production, but inhibit IL-6 production. COX inhibitors could reverse the effects of AA, suggesting the involvement of COX metabolites in AA response, while they cannot prevent the inhibitory effects of AEA and 2-AG. In addition, AM404, an AEA uptake inhibitor, slightly inhibited LPS-induced NO and IL-6 release, while potentiated the inhibition by AEA. To characterize CB2 expression in J774 macrophages, we found that only a weak CB2 mRNA expressed and that cannabinoids potentiate cAMP formation rather than inhibition, as predicted from the Gi-coupled CB2 receptor signaling. The CB2 antagonist, SR144528, could not antagonize the inhibitory effects of cannabinoids. Electrophoretic mobility shift assay shows the inhibitory effects of D9-THC, AEA and 2-AG on LPS-induced activation of AP-1 and NF-IL-6. D9-THC could also inhibit LPS-induced NF-kB activation, but AEA and 2-AG could not. All these results suggest that the cannabinoids, D9-THC, AEA and 2-AG, elicit the inhibitory effects on LPS-stimulated macrophages possibly via a cannabinoid receptor -independent pathway. Part-II The tumor necrosis factor receptor (TNFR) superfamily consists of approximately 10 characterized members of human proteins. A newly identified member, TR2 (or herpes virus entry mediator) from a search of an expressed sequence tag data base is encoded by a single gene, which maps to chromosome 1p36.22-36.3, and the TR2 open reading frame sequence encodes a 283-amino acid single transmembrane protein (30 kDa). TR2 is expressed mainly in hemopoietic tissues, particularly in lymphoid tissues such as spleen and thymus, and is involved in T cell activation. In this study, we have successfully expressed TR2-ECD-Fc fusion protein and generated seven monoclonal antibodies (4A12, 4D10, 3E11, 4D12, 4B12, 5F6 and 6A11). One antibody is IgG2a isotype and the others are IgG1 type. TR2 mRNAs, as detected by RT-PCR, are normaly expressed in human T-cell line CEM, B-cell line Ramos, peripheral blood mononuclear cells (PBMC), leukocyte cell line HL-60 and endothelial cell line ECV. TR2 mRNA expression could be induced by various stimuli in T-cell line CEM, monocyte cell line U937 leukocyte, cell line HL-60, endothelial cell line ECV and pulmonary A549 epithelial cells. ECV cells treated with IL-1b (10 ng/ml) for 8 hr and A549 cells treated with IFN-g (100 U/ml) for 8hr expressed greater amount of TR2 mRNA. In parallel, the immunostaining of TR2 in both cell lines was increased after cytokine stimulation. In conclusion, the antibodies for TR2 are useful tools to understand the role of TR2 in host immune response and to detect the presence of soluble TR2 during various inflammatory diseases.