Effects Of TMB-8 On Mice Immune Response In Vitro And Delayed Type Hypersensitivity

Objective:To investigate effects of [8-(Diethylamino)Octyl-3, 4, 5-Trimethoxybenzoate] (TMB-8), an intracellular Ca²⁺ antagonist, on mice immune response in vitro and delayed type hypersensitivity in vivo, in order to elucidate the immunosuppressive effect of TMB-8.Methods:1. Using Concanavalin A (Con A) and / or Phorbol 12, 13-dibutyrate (PDB) plus Ionomycin (Ion) as the mitogen to stimulate the lymphocytes, in order to study the effect of TMB-8 on the behavior of lymphocytes. Using the flow cytometry to analyze the ratio of CD69 expressed on activated T cells by fluorescence conjugated monoclonal antibodies labeling, the proliferation of T lymphocytes by Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) labeling, the cell cycle by propidium iodide (PI) staining. The proliferation of B lymphocytes induced by Lipopolysaccharide (LPS) was dectected by Monotetrazolium (MTT) colorimetry.2. Using the cytometric bead array (CBA) to investigate the effect of TMB-8 on the lymphocyte secretion of Thl/Th2 cytokines (IL-2, IFN-γ, TNF-α, IL-4, IL-5) after treated with Con A.3. The effect of TMB-8 on the intracellular Ca²⁺ mobilization of lymphocytes treated with Con A and / or Ion was examined by loading with Fluo-4/AM and analyzed by flow cytometry.4. A mouse delayed type hypersensitivity (DTH) model was established by using the DNFB to sensitize the abdomen and challenge the mouse left ear for 24 h. The effect of TMB-8 was explored according to the thickness and weight difference of ear, and the pathological change of challenged ear section. Thymus index and spleen index were calculated to evaluate the effect of TMB-8 on mice lymphoid organs in DTH. Lymphocytes were isolated from spleen and lymphoid nodes to detect cell proliferation and cell cycle, thymocytes were isolated from thymus to detect apoptosis.Results:1. TMB-8 had no effect on resting lymphocytes. After stimulated with Con A, TMB-8 significantly down-regulated the expression rate of CD69⁺ on CD3⁺ lymphocytes(P＜0.05) as well as CD69⁺ on CD3^- lymphocytes(P＜0.01). TMB-8 could exert an evident inhibiting effect on the proliferation induced by Con A and / or PDB plus Ion, block the transition of cells from G0/G1 phase to S phase (P＜0.01). MTT colorimetry indicated the inhibitory effect of TMB-8 on proliferation of B lymphocytes.2. TMB-8 showed suppression on secretion of Thl/Th2 cytokines (IL-2, IFN-γ, TNF-α, IL-4, IL-5) by activated lymphocytes stimulated with Con A(P＜0.05).3. TMB-8 could down-regulate the elevated Ca²⁺ concentration in lymphocytes induced by Con A or Ion. The effect was more evident as time prolonged.4. Compared with DTH group, the ear thickness difference(P＜0.01), ear weight difference (P＜0.05), spleen index and thymus index (P＜0.05) were both significantly reduced in TMB-8 treated group. Meanwhile the infiltration of inflammatory cells and tissue edema of ear were also inhibited by TMB-8. TMB-8 inhibited lymphocyte proliferation in vitro, mainly blocked the cells from G0/G1 phase to S phase, and decreased the thymocyte apoptosis in DTH model (P＜0.01).Conclusion:TMB-8 can significantly inhibit activation and proliferation of lymphocytes in vitro and thesecretion of Th1/Th2 cytokines in response to Con A, the results demonstrated the immunoregulation effect of TMB-8, which maybe associated with its downregulation of elevated Ca2+ concentration of lymphocytes induced by Con A or Ion. Moreover, the mouse DTH indicated that TMB-8 could inhibit T lymphocyte-mediated skin inflammatory reaction. Key words: TMB-8; lymphocytes; activation; proliferation; cell cycle; calcium ion; Fluo-4/AM; cytokine; cytometric bead array (CBA); delayed type hypersensitivity (DTH)...