Stephanthraniline A inhibits the proliferation and activation of T cells in vitro and in vivo

Stephanthraniline A (STA) isolated from the stems of Stephanotis mucronata (Blanco) Merr. was evaluated for their suppression on T cells' immune responses in vitro and in vivo. In vivo, oral administration of STA significantly inhibited T cell-mediated delayed-type hypersensitivity (DTH) response. In vitro, STA has inhibitory effects on T cell proliferation induced by CD3/CD28 cross-linking or Con A; additionally, CD4(+) T cells are more sensitive to this inhibition than CD8(+) T cells. STA also suppressed the production of cytokines (IL-2, IFN-γ, IL-4 and IL-17) and mRNA expression of the genes associated with T cell activation, proliferation and differentiation. Our data indicate that STA inhibits the proliferation of T cells by inducing cell cycle arrest but not inducing apoptosis. The inhibitory mechanism of STA on T cells was correlated with the gene change related to multi-signal transduction pathways. Furthermore, we also provided lines of evidence that STA, distinct from glucocorticoids, did not activate the glucocorticoid receptor. These findings would be beneficial for further understanding the therapeutic effects of S. mucronata in the treatment of autoimmune diseases. It also suggested the potential of the natural steroid STA as the effective candidate compounds for use in the treatment of inflammatory and autoimmune diseases.     

雷公藤治疗系统性红斑狼疮免疫机制的研究

目的：探讨雷公藤治疗系统性红斑狼疮的免疫作用机制。方法：用系统性红斑狼疮（ＳＬＥ）的外周血单个核细胞（ＰＢＭＣ）体外培养观察雷公藤对于ＳＬＥ的Ｔ,Ｂ细胞功能的影响。结果;雷公藤不仅能抑制ＳＬＥ病人ＰＢＭＣ对ＰＨＡ诱导的增殖反应,也能抑制ＳＬＥ病人活化Ｂ细胞的自发增殖以及ＳＡＣ诱导的静止期Ｂ细胞的增殖反应。另外雷公藤还能明显的抑制ＳＬＥ病人ＰＢＭＣ的自发性ＩｇＧ分泌以及ｒ－ＩＬ２诱导的ＰＢＭＣ的Ｉｇ     

In vitro and in vivo effects of mercuric chloride on thymic endocrine activity, NK and NKT cell cytotoxicity, cytokine profiles (IL-2, IFN-gamma, IL-6): role of the nitric oxide-L-arginine pathway

Mercury (Hg2+) affects cell-mediated immunity, including thymulin production. Thymulin, a zinc-dependent thymic hormone synthesized by thymic epithelial cells (TECs), is involved in NK cell cytotoxicity and Th1 cytokine production (IL-2 and IFN-gamma), which in turn affect both NKT and classic NK spleen cell cytotoxicity. High doses of Hg2+ induce an inflammatory status, increased production of IL-6 and consequent Th1/Th2 imbalance as well as cell-mediated immune depression. The mechanisms by which Hg+ affects the cell-mediated immune response are still unclear. The nitric oxide (NO) pathway may be implicated. The aim of this work was to further explore its noxious role in innate and adaptive immunity and to study the possible role played by the NO pathway. Young Balb/c mice treated in vivo for 1 month with 1.0 mg HgCl2/kg b.w. showed low thymulin activity, depressed NO production (as measured by nitrite and nitrate plasma levels), impaired classic NK spleen cell cytotoxicity, decreased Th1 (IL-2 and IFN-gamma) cytokine profiles, and increased IL-6 production. In vitro, 10(-6) M of HgCl2 inhibited active thymulin kinetics, TEC proliferation, NKT cell cytotoxicity and Th1 cytokine production, whereas IL-6 increased. L-arginine restored thymulin activity, TEC proliferation, NKT cytotoxicity, cytokine profiles and nitrite and nitrate plasma levels both in vivo and in vitro. Since L-arginine is the substrate for NO production, it may compensate for the cell-mediated immune defect induced by HgCl2, via the arginine-NO-pathway. L-arginine is also able to reduce glomerular kidney IgG antibodies deposits induced by higher dose of HgCl2 administration.     

白藜芦醇对脂多糖诱导的人肺上皮细胞焦亡的保护机制研究

目的:探究白藜芦醇对脂多糖(LPS)诱导的人肺上皮细胞(又称BEAS-2B)增殖、炎症因子释放和焦亡的影响。方法:用CCK-8法检测LPS和LPS与白藜芦醇联用对BEAS-2B细胞增殖的影响;采用ELISA法检测细胞上清炎症因子肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)和白介素-18(IL-18)表达以及qPCR和Western blot检测焦亡基因NLRP3、Gasdermin D、Caspase-1、ELAVL1的表达;分析其对细胞活力、BEAS-2B细胞中细胞因子的含量及其焦亡相关基因、相关蛋白表达的影响。结果:LPS可以抑制BEAS-2B细胞的增殖,同时可以促进炎症因子的表达,经qPCR和Western blot检测结果表明LPS可以促进NLRP3、Gasdermin D、Caspase-1、ELAVL1基因的表达;其经用白藜芦醇处理48 h后,可以有效逆转LPS所引起的细胞增殖受抑制和炎症因子高表达的现象;此外,NLRP3、Gasdermin D、Caspase-1、ELAVL1的表达也部分受抑制。结论:白藜芦醇通过减少炎症因子的释放和NLRP3、Gasdermin D、Caspase-1、ELAVL1表达对LPS诱导的BEAS-2B焦亡起到一定的保护作用。     

YM-58483, a selective CRAC channel inhibitor, prevents antigen-induced airway eosinophilia and late phase asthmatic responses via Th2 cytokine inhibition in animal models

T cells play a regulatory role in the pathogenesis of various immune and allergic diseases, including human asthma. Recently, it was reported that a pyrazole derivative, YM-58483 (BTP2), potently inhibits Ca²⁺ release-activated Ca²⁺ (CRAC) channels and interleukin (IL)-2 production in T cells. We investigated the effects of YM-58483 on T helper type 2 (Th2) cytokine production in vitro and antigen-induced airway asthmatic responses in vivo. YM-58483 inhibited IL-4 and IL-5 production in a conalbumine-stimulated murine Th2 T cell clone (D10.G4.1), and IL-5 production in phytohemagglutinin-stimulated human whole blood cells with IC₅₀ values comparable to those reported for its CRAC channel inhibition (around 100 nM). YM-58483 inhibited antigen-induced eosinophil infiltration into airways, and decreased IL-4 and cysteinyl-leukotrienes content in inflammatory airways induced in actively sensitized Brown Norway rats. Furthermore, orally administered YM-58483 prevented antigen-induced late phase asthmatic broncoconstriction and eosinophil infiltration in actively sensitized guinea pigs. These data suggest that the inhibition of Ca²⁺ influx through CRAC channel leads to the prevention of antigen-induced airway inflammation, probably via the inhibition of Th2 cytokine production and inflammatory mediators release. YM-58483 may therefore be useful for treating airway inflammation in bronchial asthma.     

In vitro immunosuppressive properties of spergualins to murine T cell response

Deoxyspergualin has strong immunosuppressive activity in animals. However, it shows less in vitro immunosuppressive activity at the therapeutic concentration used for in vivo administration. Recently, we reported that there are some technical problems with in vitro experiments. In this report, the effects of spergualins were examined under in vitro conditions which excluded these problems, and compared with cyclosporine A (CYA). Spergualins have suppressive effects on mixed lymphocyte response (MLR) and cytotoxic T lymphocyte induction. Furthermore, interleukin-2 (IL-2) induced proliferation of concanavalin A blasts and CTLL-2 were inhibited at low concentration. However, spergualins have little effect in the early stage of MLR or the mitogen response. These results suggest that spergualins act on the proliferation and differentiation of T cells which respond to growth factors, such as IL-2.     

Dimethoxycurcumin, a metabolically stable analogue of curcumin, exhibits anti-inflammatory activities in murine and human lymphocytes

The aim of this study was to investigate whether dimethoxycurcumin (DiMC), a synthetic curcumin analogue having higher metabolic stability over curcumin, could exhibit anti-inflammatory activity in murine and human lymphocytes. Both curcumin and DiMC suppressed mitogen as well as antigen driven proliferation of murine splenic lymphocytes. Further, mitogen and antigen-stimulated cytokine (IL-2, IL-4, IL-6 and IFN-γ) secretion by T cells was also abrogated by curcumin and DiMC. Interestingly, curcumin and DiMC suppressed B cell proliferation induced by lipopolysaccharide. Curcumin and DiMC also inhibited Con A-induced activation of early and late T cell activation markers. They scavenged basal reactive oxygen species and depleted GSH levels in lymphocytes. The suppression of mitogen-induced T cell proliferation and cytokine secretion by curcumin and DiMC was significantly abrogated by thiol containing antioxidants suggesting a role for redox in their anti-inflammatory activity. Further, the possibility of curcumin and DiMC directly interacting with thiol-containing antioxidant GSH was monitored by changes in absorbance. Both curcumin and DiMC inhibited Con A induced activation of NF-κB and MAPK. More importantly, curcumin and DiMC inhibited phytohaemagglutinin induced proliferation and cytokine secretion by human peripheral blood mononuclear cells. To explore their therapeutic efficacy, they were added to lymphocytes post-Con A stimulation and we observed a significant suppression of IL-2, IL-6 and IFN-γ. The present study for the first time demonstrates the potent anti-inflammatory activity of DiMC. Further, DiMC could find application as an alternative to curcumin, which is currently used in several clinical studies, due to its superior bioavailability and comparable efficacy.     

白藜芦醇抑制HepG2细胞生长及诱导细胞凋亡的初步研究

目的　研究白藜芦醇 (Res)对人肝癌HepG2细胞生长增殖的影响 ,进而探讨Res诱导HepG2细胞凋亡的作用。方法　用不同浓度的Res处理HepG2细胞 ,MTT法检测Res对HepG2细胞生长增殖的抑制作用 ,通过HE染色、透射电子显微镜、原位末端标记法 (TUNEL)和流式细胞术观察凋亡细胞的形态结构变化以及定性、定量检测细胞凋亡。结果　Res能抑制HepG2细胞的生长增殖 ,并呈浓度依赖性 ;Res能诱导HepG2细胞凋亡。 结论　Res能够通过诱导HepG2细胞凋亡抑制HepG2细胞的增殖。     

Inhibition of lymphocyte activation and function by the prenylation inhibitor L-778,123

Prenylated Ras GTPases transduce signals from the T cell receptor, CD28 costimulatory receptor and IL-2 receptor. Since signals from these receptors mediate T cell activation, proliferation and survival, we hypothesized that the prenylation inhibitor L-778,123 would impart immunomodulation.The effect of L-778,123 on T cell activation (CD71 or CD25 surface expression) was determined by flow cytometry. Peripheral blood mononuclear cell (PBMC) proliferation in the presence of L-778,123 and/or cyclosporine (CsA) was determined by [3H]thymidine incorporation. The ability of L-778,123 to inhibit IL-2 receptor signaling was investigated by measuring IL-2 induced proliferation in CTLL-2 cells and IL-2 prevention of apoptosis in activated human PBMC. L-778,123 inhibited lectin induced expression of CD71 and CD25 with IC50's of 6.48 +/- 1.31 microM and 84.1 +/- 50.0 microM, respectively. PBMC proliferation was inhibited by L-778,123 with an IC50 of 0.92 +/- 0.23 microM, and addition of CsA did not increase the potency. L-778,123 did not inhibit IL-2 and IFN-gamma production by T cells. L-778,123 abrogated IL-2 induced proliferation of CTLL-2 cells with an IC50 of 0.81 +/- 0.44 microM. However, L-778,123 minimally reversed the prosurvival effect of IL-2 in activated lymphocytes. IL-2 ligand and receptor production during T cell activation are relatively unaffected by L-778,123. However, the activation and proliferative effects of IL-2 on T cells are potently blocked by L-778,123. These results reveal a selective blockade of the IL-2 cytokine axis distal to the IL-2 receptor by the L-778,123 and warrant evaluation of prenylation inhibitors in treating transplant rejection and autoimmune diseases.     

Human lymphocyte reactivity after in vitro exposure to technical and analytical grade pentachlorophenol

To investigate the potential effects of technical pentachlorophenol (PCP-T, contaminated with polychlorinated dioxins and furans) and of analytical grade pentachlorophenol (PCP-A) on the human immune system, in vitro assays with freshly prepared human peripheral blood lymphocytes were used as an alternative to experimental animals. Both cell-mediated and humoral immune functions were examined after direct lymphocyte exposure to PCP-T or PCP-A at concentrations ranging from 0–200 μM. In each case the viability of the treated cells remained within the control value range. T lymphocyte blastogenesis after 3 days incubation with PCP was measured using both optimal and suboptimal mitogen (PHA) concentration. Interleukin-2 activity of 24.5-h supernatants of lymphocytes in response to PHA, pretreated with PCP for 20–24 h, was examined in a bioassay using the mouse the IL-2-dependent CTLL-6 cell line. The synthesis of immunoglobulins was determined after stimulation with T-dependent (PWM) and T-independent (KlebsM) polyclonal B cell activators. In the proliferation assay the effects of PCP-T became more evident after suboptimal mitogen stimulation. Whereas after optimal mitogen stimulation blastogenesis was affected only at the highest concentration of 200 μM PCP-T, cell reactivity after suboptimal PHA stimulation was altered by all PCP-T doses. In the lower concentration range PCP-T caused enhanced proliferative responses, but at the two highest PCP-T concentrations cell reactivity was significantly suppressed as compared to the medium controls. Significant differences between the effects of PCP-T and PCP-A could be demonstrated only after optimal mitogen stimulation at the highest PCP concentration (200 μM). In contrast, lymphokine production as well as Ig secretion showed severe dose-dependent suppression after exposure to both PCP-T and PCP-A. The humoral immune response appeared to be more suppressed when cultures were stimulated with T-dependent rather than T-independent mitogens. The two different PCP preparations caused immunosuppression of both lymphocyte functions to the same extent. To summarize, the results of our studies indicate that PCP itself is directly immunotoxic to human immunocompetent cells and the T helper subset appears to be especially sensitive to PCP exposure. Furthermore, the observation of a direct effect on humoral immunity is similiar to previous results showing considerable alterations of antigen specific antibody production in experimental animals after in vivo exposure.     

Effects of brazilin on induction of immunological tolerance by sheep red blood cells in C57BL/6 female mice

Brazilin was examined for its effects on the induction of immunological tolerance. Brazilin was administered to C57BL/6 female mice for 2 consecutive days before the immunization with high dose SRBC (10⁹ cells) which can produce immunological tolerance. Delayed type hypersensitivity, IgM plaque forming cells, ConA induced IL-2 production and mitogen- or antigen-induced proliferation of lymphocytes were measured as evaluation parameters. Administration of brazilin prior to immunization could keep the DTH and IL-2 production almost optimaly immunized levels. Brazilin also inhibited the elevation of non-specific suppressor cell activity. ConA induced proliferation of splenocytes in high dose SRBC immunized mice was significantly decreased by pretreatment of brazilin. And this might be one of the reason for augmentation of DTH by brazilin. However, IgM plaque forming cells were not affected by the treatment of brazilin. These results indicate that brazilin prevents the induction of immunological tolerance caused by high dose SRBC by suppressing the elevation of suppressor cell activity and by inhibiting the decrease in IL-2 production in C57BL/6 female mice.     

Effects of resveratrol on number and activity of endothelial progenitor cells from human peripheral blood

1. It has been well established that oestrogens can increase the number of endothelial progenitor cells (EPC) by anti-apoptotic effects. Resveratrol, a polyphenolic phytoalexin extracted from grapes and wine, has been reported to act as an oestrogen receptor agonist. We hypothesize that putative phyto-oestrogen may promote EPC proliferation and survival in vitro. 2. Endothelial progenitor cells were isolated from human peripheral blood and identified immunocytochemically. Endothelial progenitor cells were incubated with resveratrol (1, 10, 25 and 50 mmol/L) or control for specified times. Cell proliferation, migration and in vitro vasculogenesis were assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, modified Boyden chamber assay and in vitro vasculogenesis detection, respectively. 3. Resveratrol increased the number of EPC and promoted EPC proliferation, adhesion and migration in a dose- and time-dependent manner. Cell number peaked at 50 mmol/L resveratrol after incubation for 24 h compared with vehicle control (61.3 +/- 5.8 vs 112.8 +/- 7.2, respectively; P < 0.01). 4. Furthermore, cell cycle analysis showed that 50 mmol/L resveratrol significantly increased the S phase and decreased the G(0)/G(1) phase of EPC. In addition, resveratrol increased vascular endothelial growth factor production and further induced vasculogenesis in vitro. 5. In conclusion, resveratrol significantly induces EPC proliferation, migration and further promotes angiogenesis in vitro.     

Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the<Emphasis Type="Italic">In Vitro</Emphasis> immune response in pristane-induced lupus mice

Systemic lupus erythematosus (SLE) is characterized by overactive B cells that differentiate into autoantibody-forming cells, aberrant T cell function that provides helping B cells produce autoantibodies, and overproduction of proinflammatory cytokines. However, immunodysregulation in lupus pathogenensis remains incomplete. We examined mitogen-stimulated production of proinflammatory cytokines, cell proliferation, T cell activation, and T cell apoptosis in vitro in pristane-induced lupus BALB/c mice compared to normal mice. LPS-stimulated production of IL-6 and IL-10 by splenocytes and macrophages from pristane-induced lupus mice were remarkably up-regulated compared to normal mice, whereas production of macrophage TNF-alpha was significantly down-regulated. Moreover, in vitro production of IL-2, IL-6, IL-10 and IFN-gamma by Con A-stimulated splenocytes, cell proliferation in LPS- or Con A-stimulated- thymocytes and splenocytes, and expression of CD69+CD4+ T cells in Con A-stimulated splenocytes were greatly increased in cells derived from pristane-induced lupus mice compared to normal mice. In addition, splenic T cells and CD4+ T cells in thymocytes from pristane-induced lupus mice were more resistant than nonautoimmune normal cells to Con A-induced apoptosis. Our findings indicate that immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune responses in pristane-induced lupus mice may explain some of lupus pathogenesis.     

Fumigaclavine C improves concanavalin A-induced liver injury in mice mainly via inhibiting TNF-alpha production and lymphocyte adhesion to extracellular matrices

Fumigaclavine C, an alkaloidal metabolite, was produced by Aspergillus fumigatus (strain No. CY018). This study examined the effect of this compound on concanavalin A (Con A)-induced liver injury in mice, a T cell-dependent model of liver damage. Con A administration resulted in severe liver injury, T lymphocyte activation and a strong increment in spleen cell adhesion, as well as in tumour necrosis factor-alpha (TNF-alpha) production. Against this liver injury, the intraperitoneal administration of fumigaclavine C dose-dependently inhibited the elevation in transaminase activity, TNF-alpha production in serum and the histological changes, including inflammatory infiltration, hepatocyte necrosis and degeneration and Kupffer cell hyperplasia. In addition, this compound in-vitro also inhibited the proliferation of spleen cells induced by Con A, and reduced their IL-2 and TNF-alpha production. Moreover, the intraperitoneal administration of fumigaclavine C inhibited the potential of spleen cells isolated from the liver-injured mice to adhere to fibronectin, laminin and type IV collagen. These results suggest that the improvement of this T cell-mediated liver injury by fumigaclavine C may be related to the inhibition of lymphocyte activation, proliferation and adhesion to extracellular matrices as well as the reduction in TNF-alpha production.     

Modulation of mite antigen-induced immune responses by lecithin-bound iodine in peripheral blood lymphocytes from patients with bronchial asthma.

1. Dermatophagoides farinae (Df) mite antigen induced IgE synthesis associated with an imbalance of cytokine production in mite-sensitive patients with bronchial asthma; increased production of interleukin 4 (IL-4), and decreased production of interferon-gamma (IFN-gamma) was specifically induced in these patients'' lymphocytes. 2. Lecithin-bound iodine (LBI), with which children with bronchial asthma have been successfully treated in the range of 0.5 to 5 microM, concentrations comparable to LBI blood levels in medicated individuals, modified mite antigen-induced immune responses, thereby decreasing abnormal lymphocyte functions. 3. In Df antigen-driven immune responses, inhibition of IgE generation accompanied by suppression of IL-4 and the recovery of IFN-gamma production was successful when LBI was used in vitro. 4. LBI also acted on normal PBMCs by downregulating the IL-4-induced IgE synthesis, phytohaemagglutin (PHA)- and phorbol myristate acetate (PMA) plus calcium ionophore (CaI)-induced IL-4 secretion, and by upregulating purified protein derivatives (PPD)-induced IFN-gamma production. Therefore, LBI was capable of inhibiting the IgE and IL-4 responses and of enhancing IFN-gamma production both from allergen-stimulated atopic cells and from non-atopic cells appropriately stimulated. 5. The expression of human histocompatibility leukocyte antigen (HLA), class II antigens and intercellular adhesion molecule 1 (ICAM-1) on monocytes, crucial molecules for T cell-monocyte interactions, was not altered by LBI. 6. LBI probably acts as an immunomodulator to ameliorate mite antigen-induced abnormal cell-mediated immune responses in patients with bronchial asthma caused by Df antigen thereby leading to improvement of their clinical status.     

Immunosuppressive effects of vermiculine in vitro and in allotransplantation system in vivo

Vermiculine, a macrocyclic aglycosidic dilactone isolated from Penicillium vermiculatum, has been shown to have immunomodulatory properties. Here, we tested the effects of vermiculine on selected parameters of cell-mediated immunity in vitro and on skin allograft survival in vivo. Vermiculine inhibited in a dose-dependent manner the proliferation of mouse spleen cells stimulated with Concanavalin A ((Con A), i.e. T-cell mitogen), bacterial lipopolysaccharide ((LPS), B-cell mitogen) or with irradiated allogeneic cells. In addition, vermiculine dose-dependently inhibited the production of Thl (IL-2, IFN-gamma) and Th2 (IL-4, IL-10) cytokines and suppressed the production of nitric oxide (NO) by activated macrophages. When compared with cyclosporine (CsA), vermiculine was less inhibitory for IL-2 gene expression and IL-2 synthesis, comparably suppressive on IL-10 production and even more inhibitory for NO synthesis. These observations suggest that vermiculine and CsA inhibit immune reactions by different mechanisms. Treatment of graft recipients with vermiculine or CsA prolonged survival of skin allografts in a mouse model. The combination of both drugs enhanced the survival of allografts significantly more than either drug alone. The results thus suggest that vermiculine is a potential immunosuppressive drug acting by a mechanism distinct from that of CsA, and thus it may be used alone or in combination with other drugs for immunoregulatory purposes.