灵芝孢子粉碱提多糖对小鼠巨噬细胞的免疫调节作用

目的 :研究灵芝孢子粉碱提多糖 (LZSBS)对小鼠巨噬细胞的激活作用。方法 :用灵芝孢子粉碱提多糖刺激体外培养的小鼠巨噬细胞 ,用ELISA法检测巨噬细胞分泌至培养基中的TNF α和IL 1β的含量 ;用Griess法检测培养上清中NO的含量。小鼠巨噬细胞对乳胶颗粒的吞噬率用显微镜计数。结果 :经灵芝孢子粉碱提多糖刺激后 ,小鼠巨噬细胞变大 ,颜色加深 ,并能显著刺激巨噬细胞分泌TNF α和IL 1β ,并产生大量的NO。小鼠巨噬细胞对乳胶颗粒的吞噬功能也明显的增强。结论 :灵芝孢子粉碱提多糖对小鼠巨噬细胞具有明显的激活作用     

激活素A对RAW264.7巨噬细胞活性的调节作用

目的探讨激活素A对参与炎症反应的小鼠巨噬细胞活性调节作用。方法以LPS刺激活化的小鼠巨噬细胞系RAW264．7细胞作为阳性参照，ELISA法检测激活素A及LPS刺激的小鼠腹腔巨噬细胞系RAW264．7细胞IL-1β分泌水平，还原酶法分析NO分泌水平，RT-PCR检测IL-1β和iNOS mRNA的表达，瑞氏染色检测RAW264．7细胞吞噬活性。结果在激活素A刺激下RAW264．7细胞IL-1β和NO分泌水平均明显升高，IL-1β和iNOS mRNA表达亦增加，巨噬细胞吞噬活性增强；激活素A和LPS共刺激RAW264．7细胞时，激活素A明显抑制LPS刺激的RAW264．7细胞IL-1β和NO产生水平，以及IL-1β和iNOS mRNA表达，巨噬细胞吞噬活性也明显低于LPS单独刺激组。结论激活素对巨噬细胞的活性调节具有双重作用，这种作用与巨噬细胞的激活状态有关。     

转移程度不同的小鼠肝癌细胞株对腹腔巨噬细胞免疫功能的影响

目的 研究转移程度不同的小鼠肝癌细胞株对腹腔巨噬细胞功能的影响.方法 在两株转移程度不同的小鼠腹水型肝癌模型中,取荷瘤小鼠腹腔巨噬细胞,检测其在干扰素γ(IFN-γ)和脂多糖(LPS)刺激下产生一氧化碳(NO)和肿瘤坏死因子α(TNF-α)的水平,并检测其活化后的杀伤能力.进一步采用夹心酶联免疫吸附测定(ELISA)方法,研究不同荷瘤小鼠腹水中IFN-γ与转化生长因子β1(TGF-β1)的水平,并用相应抗体封闭TGF-β1后,检测活化巨噬细胞产生NO能力及杀伤活性.结果 经IFN-γ和LPS活化后,荷瘤小鼠腹腔巨噬细胞分泌NO和TNF-α能力明显低于正常巨噬细胞,杀伤能力下降.高转移性肝癌小鼠巨噬细胞分泌NO水平和杀伤能力均低于低转移性小鼠,但其分泌TNF-α量较高.此外,荷瘤小鼠腹水含较高水平IFN-γ与TGF-β1,不同转移程度荷瘤小鼠IFN-γ水平接近,但高转移性肝癌小鼠腹水含更多TGF-β1,而且TGF-β1的封闭可导致与肿瘤细胞共培养的巨噬细胞分泌NO的能力部分恢复.结论 肿瘤细胞可以通过分泌TGF-β1等抑制性因子下调巨噬细胞的活性和免疫功能.肿瘤的转移程度可能与其分泌免疫抑制因子的能力相关.     

黄芪多糖通过NF-κB诱导巨噬细胞产生NO和TNF-α

目的 通过体外试验研究黄芪多糖(Astragalus mongholicus polysaccharides,ASP)激活巨噬细胞产生NO和TNF-α的分子机制和细胞内信号转导机制.方法 黄芪多糖刺激RAW264.7细胞,用Western blot方法 检测细胞核内核转录因子-κB(NF-κB)的变化.用Griess还原法观察黄芪多糖对巨噬细胞释放NO的作用的影响以及NF-κB抑制剂对黄芪多糖诱导巨噬细胞释放NO作用和分泌TNF-α的影响.ELISA法检测黄芪多糖对巨噬细胞分泌肿瘤坏死因子-α(TNF-α)的变化.结果 100μg/ml黄芪多糖刺激RAW264.7细胞,4 h后可引起细胞核内NF-κB含量显著增加,6 h达到顶峰.16 h后可显著诱导NO[(18.9±1.5)μmol/L;P<0.01]释放和TNF-α分泌[(81.2±16.7)pg/ml,P<0.01]的增加,以及诱导型一氧化氮合酶(iNOS)[(23.54±2.41)U/mg蛋白质,P<0.01]活性的增加.NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)可明显抑制黄芪多糖诱导RAW264.7生成NO和分泌TNF-α.结论 NF-κB在黄芪多糖诱导巨噬细胞生成NO和TNF-α过程中发挥重要作用.     

Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响

探讨viili胞外多糖(Viili exopolysaccharides,VEPS)对小鼠巨噬细胞RAW264.7激活和增殖的影响。噻唑蓝(MTT)比色法检测细胞的生长与增殖;中性红吞噬实验检测吞噬活性;Griess试剂盒检测培养上清液中NO分泌量,ELISA法检测VEPS不同浓度及不同作用时间培养上清中IL-6,IL-1β含量;扫描电子显微镜观察VEPS对细胞形态的影响;碘化丙啶(PI)染色检测VEPS对细胞周期的影响。结果显示,VEPS对RAW264.7细胞的增殖、吞噬能力、分泌NO、IL-6、IL-1β等都有显著的促进作用,VEPS为100μg/ml时促进作用最明显,呈剂量相关,作用72h时细胞因子分泌量达到最大,72h后下降。VEPS激活巨噬细胞并使其变得扁平伸展且形成伪足。VEPS促进G1期细胞增多,提高细胞的增殖能力。VEPS免疫调节作用与其激活RAW264.7细胞,促进NO、IL-6、IL-1β等分泌有关,且VEPS与LPS对RAW264.7细胞有相似的作用规律。以上结果证明VEPS能激活巨噬细胞,也可能最终激活淋巴细胞,达到增强非特异性和特异性免疫的作用。     

硫辛酸抑制脂多糖诱导的星形胶质细胞细胞因子及趋化因子的表达

目的探讨硫辛酸(LA)对脂多糖(LPS)激活的星形胶质细胞分泌TNF-α、IL-1β、IL-6和IL-10及相关趋化因子的影响。方法分离并鉴定新生C57BL/6小鼠大脑皮质星形胶质细胞,1μg/mL LPS刺激第2代星形胶质细胞,100μg/mL LA进行干预,Griess法检测NO的分泌,ELISA检测TNF-α、IL-1β、IL-6和IL-10炎性因子的含量,反转录PCR检测炎症趋化因子CC亚族趋化因子配体20(CCL20),单核细胞趋化蛋白1(MCP-1)和巨噬细胞炎性蛋白1α(MIP-1α)mRNA的表达。结果与正常组比较,LPS刺激星形胶质细胞后,NO、TNF-α、IL-1β、IL-6分泌显著升高,IL-10分泌下调(P0.05);LA能抑制LPS诱导的NO、TNF-α、IL-1β、IL-6的分泌,增加IL-10的分泌,与LPS组相比差异有统计学意义(P0.05)。LA能显著下调LPS所致的CCL20、MIP-1α、MCP-1 mRNA的分泌。结论 LA能抑制LPS激活星形胶质细胞所致的炎性反应,其作用与抑制炎性因子及趋化因子的分泌有关。     

全蝎水提物对巨噬细胞活化作用研究

目的:观察全蝎水提物(Buthus martensii water extract,BMWE)在体外对小鼠巨噬细胞株RAW264.7的活化作用。方法:MTT法测定细胞活性;采用FITC标记的酵母测定吞噬活性;利用Griess试剂和ELISA方法测定细胞上清NO、TNF-α和IL-6释放量。结果:BMWE在不影响细胞活力的剂量范围内可增强正常及免疫抑制的RAW264.7细胞吞噬荧光酵母能力(P0.01),增加RAW264.7细胞NO释放量(P0.01),促进RAW264.7细胞TNF-α和IL-6产生(P0.01),均具有良好的量效关系。结论:BMWE可明显提高巨噬细胞吞噬功能与分泌能力,活化巨噬细胞,此作用与全蝎传统用于治疗疮疡、瘰疬,现代用于肿瘤的临床应用相关。     

Vilon(L-Lys-L-Glu)对小鼠腹腔巨噬细胞分泌IL-1β、NO的影响

目的 探讨Vilon(L-Lys-L-Glu)对参与炎症反应的小鼠腹腔巨噬细胞分泌IL-1β、NO的影响.方法 以小鼠原代培养腹腔巨噬细胞为阳性参照,ELISA法检测Vilon及脂多糖(LPS)共刺激的小鼠腹腔巨噬细胞IL-1β的分泌水平;还原酶法分析NO分泌水平;RT-PCR法检测IL-1β和iNOS mRNA的表达.结果 Vilon和LPS共刺激小鼠腹腔巨噬细胞时,Vilon对LPS活化的小鼠腹腔巨噬细胞分泌IL-1β及NO具有明显的促进作用,并且呈剂量依赖关系;同时也促进了IL-1β和iNOS mRNA表达.结论 Vilon对活化的小鼠腹腔巨噬细胞分泌IL-1β、NO具有明显的促进作用.     

Fractalkine对脂多糖激活的小胶质细胞炎症相关因子表达的影响

目的:探讨趋化因子Fractalkine对脂多糖(LPS)诱导的小鼠小胶质细胞(N9)激活时所分泌的TNF-α、IL-1β和一氧化氮(NO)表达的影响.方法:用Fractalkine处理经LPS激活的小鼠小胶质细胞24 h,以ELISA法检测细胞培养上清中TNF-α和IL-1β的浓度,以NO试剂盒检测培养上清中NO的浓度.结果:LPS能够激活小胶质细胞,使IL-1 β、TNF-α和NO的表达量与对照组相比明显升高;Fractalkine能够降低LPS激活的小胶质细胞IL-1β、TNF-α和NO的表达.结论:Fractalkine可能通过抑制炎症相关因子的产生而在中枢神经系统中发挥神经保护作用.     

双歧杆菌的完整肽聚糖对大鼠腹腔巨噬细胞NF-κB的调控

双歧杆菌定植于人体肠道内,对维持肠道的微生态平衡和人体的健康起重要作用。完整肽聚糖(whole pepfidogly- can,WPG)是双歧杆菌细胞壁中的多糖,能激活巨噬细胞,增强其吞噬能力,提高其能量代谢水平以及细胞毒功能。此外,WPG也能促使巨噬细胞分泌多量的IL-1、IL-6、IL-12、IL-18、TNF-α和NO。本文以Western blot和凝胶迁移电动分析(dectrophoretic mobility shift assay,EMSA)检测了分叉双歧杆菌的WPG对大鼠腹腔巨噬细胞PKC、I_κB_α和NF-_κB的影响,旨在探讨WPG激活巨噬细胞的信号途径。     

Interleukin-12 secretion by Mycobacterium tuberculosis-infected macrophages.

Infection with Mycobacterium tuberculosis or phagocytosis of large latex beads induced interleukin-12 (IL-12) production in macrophages. In contrast, tumor necrosis factor (TNF) was produced only in response to M. tuberculosis infection, not after phagocytosis of latex beads. Comparable results were obtained with cells from immunocompetent C57BL/6 and gamma interferon receptor-deficient mutant mice. Thus, phagocytosis by mechanisms not specific for M. tuberculosis was a sufficient trigger for IL-12 secretion, emphasizing the central role of this cytokine in the initiation of anti-infective immunity.     

Impact of Interleukin-17 on Macrophage Phagocytosis of Apoptotic Neutrophils and Particles

There is now substantial evidence that the cytokine interleukin-17 orchestrates the accumulation of neutrophils in mammals and thereby contributes to host defense. However, the role of IL-17 in controlling neutrophil turnover is not fully understood. Here, we demonstrate that IL-17 stimulates the apoptosis of mouse neutrophils and, simultaneously, the release of the microbicidal compound, myeloperoxidase. IL-17 also stimulates mouse macrophages to phagocytose aged neutrophils and latex beads, and it induces an increase in a soluble form of the phagocytic receptor, lectin-like oxidized low-density lipoprotein receptor-1 as well. In contrast, IL-17 does not markedly increase the release of the archetype neutrophil-recruiting cytokine, macrophage inflammatory protein-2 in mouse macrophages. Importantly, IL-17 also stimulates the phagocytosis of latex beads in human monocyte-derived macrophages. Thus, IL-17 bears the potential to control both phagocytosis and neutrophil turnover during activation of host defense.     

Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Macrophage Nitric Oxide Synthase Associates with Cortical Actin but Is Not Recruited to Phagosomes

Nitric oxide (NO) produced from inducible NO synthase (iNOS) is an important component of host defense against intracellular pathogens. To understand how phagocytes deliver NO to ingested microorganisms while avoiding cytotoxicity, we set out to study the subcellular localization of iNOS within macrophages following phagocytosis. Confocal microscopy of immunostained cells showed that iNOS was located not only diffusely within cytoplasm but also in vesicles, as well as immediately adjacent to the peripheral cell membrane. This peripheral iNOS colocalized with the cortical actin cytoskeleton and was removed by the actin-depolymerizing drug cytochalasin B. Biochemical fractionation of RAW 264 macrophages showed that 32.75% (+/-5.11%; n = 3) of iNOS was present in a particulate fraction, which cosedimented with low-density cellular vesicles. Following phagocytosis of latex beads, zymosan, immunoglobulin G-coated beads, or complement-coated zymosan, submembranous cortical iNOS was not recruited to phagosomes, nor was there any relocalization of intracellular iNOS. Similarly, following phagocytosis of Salmonella enterica serovar Typhimurium there was no recruitment of iNOS to the Salmonella vacuole at any stage after internalization. NO mediated significant killing of intracellular S. enterica serovar Typhimurium in RAW macrophages treated with lipopolysaccharide and gamma interferon; this was evident 4 h after infection. Although not recruited to phagosomes, iNOS association with the submembranous cortical actin cytoskeleton is ideally suited to deliver NO to microbes in contact with the cell surface and may contribute to early killing of ingested Salmonella.     

Nitric oxide up-regulates the release of inflammatory mediators by mouse macrophages

Nitric oxide (NO) plays a key role in mediating macrophage cytotoxicity towards different targets, including tumoral cells and intracellular pathogens. However, its role in macrophage immunoregulation is less well defined. In this study, we have investigated the effect of altering NO levels on the production by mouse macrophages of cytokines, and reactive oxygen intermediates as measured by luminol-dependent chemiluminescence. Our results demonstrate that NO can enhance the release of both tumor necrosis factor-α and interleukin-1α, and chemiluminescence. Thus, in addition to acting as a powerful effector molecule in mediating cytotoxic activities of mouse macrophages, NO can play a role in enhancing the production of a variety of other inflammatory mediators, and thus can contribute both directly and indirectly to the immunopathology of macrophage-dependent inflammation.     

Macrophages from inflamed but not normal glomeruli are unresponsive to anti-inflammatory cytokines

This study examined the properties and responsiveness to cytokines of macrophages purified from normal and nephritic glomeruli to ascertain whether macrophages activated in vivo develop programmed unresponsiveness to cytokines as do bone marrow-derived macrophages in vitro when activated by interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), interleukin-4 (IL-4), or transforming growth factor-beta (TGF-beta). Macrophages from normal glomeruli did not generate nitric oxide (NO) spontaneously but only after treatment with IFN-gamma and TNF-alpha. NO generation by these macrophages was abrogated by administering IL-4, TGF-beta, or TNF-alpha before but not after IFN-gamma treatment. Glomerular macrophages also expressed beta-glucuronidase, which was increased by TGF-beta and decreased by IFN-gamma and TNF. By contrast, glomerular macrophages from rats with nephrotoxic nephritis did not express beta-glucuronidase even after exposure to TGF-beta. Furthermore, they generated NO spontaneously, and this spontaneous generation of NO was not suppressed by IL-4, TGF-beta, or TNF-alpha. Systemic treatment of nephritic rats with IL-4 reduced NO generation by 40% but did not prevent activation, which is similar to the effect of IL-4 on bone marrow-derived macrophages in vitro when given simultaneously with IFN-gamma. We conclude that macrophages infiltrating inflamed glomeruli have developed programmed unresponsiveness to activating cytokines. This may enable them to function appropriately in the complex conditions within an inflammatory focus.     

Vanillic acid inhibits inflammatory mediators by suppressing NF-κB in lipopolysaccharide-stimulated mouse peritoneal macrophages

Vanillic acid is a benzoic acid derivative that is used as a flavoring agent. It is an oxidized form of vanillin. At present, the mechanisms by which vanillic acid exerts its anti-inflammatory effects are incompletely understood. In this study, we attempted to determine the effects of vanillic acid on lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages. Our findings indicate that vanillic acid inhibits LPS-induced production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. During the inflammatory process, the levels of cyclooxygenase (COX)-2 and nitric oxide (NO) increased in mouse peritoneal macrophages, but vanillic acid suppressed both the enhanced levels of COX-2 and the production of prostaglandin E(2) and NO. Moreover, vanillic acid suppressed the activation of nuclear factor-kappa B (NF-κB) and caspase-1. These results provide novel insights into the pharmacological actions of vanillic acid and are indicative of the potential use of this molecule in the treatment of inflammatory diseases.     

Vanillic acid inhibits inflammatory mediators by suppressing NF-κB in lipopolysaccharide-stimulated mouse peritoneal macrophages

Vanillic acid is a benzoic acid derivative that is used as a flavoring agent. It is an oxidized form of vanillin. At present, the mechanisms by which vanillic acid exerts its anti-inflammatory effects are incompletely understood. In this study, we attempted to determine the effects of vanillic acid on lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages. Our findings indicate that vanillic acid inhibits LPS-induced production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. During the inflammatory process, the levels of cyclooxygenase (COX)-2 and nitric oxide (NO) increased in mouse peritoneal macrophages, but vanillic acid suppressed both the enhanced levels of COX-2 and the production of prostaglandin E₂ and NO. Moreover, vanillic acid suppressed the activation of nuclear factor-kappa B (NF-κB) and caspase-1. These results provide novel insights into the pharmacological actions of vanillic acid and are indicative of the potential use of this molecule in the treatment of inflammatory diseases.     

Anticancer drugs plus cytokines: immunodulation based therapies of mouse tumors

As demonstrated in this laboratory, several cytotoxic anticancer agents have immunomodulating effects at relatively low doses and, in combination with non-toxic doses of certain cytokines, can exert immunity-dependent curative effects in mouse tumor models. Thus, adriamycin (Dox) has been shown to enhance the activation of macrophages with associated increases of IL1 and TNF production, to stimulate the production of IL2 and the development and action of CTLs. In the EL4 lymphoma C57BL/6 mouse model, combinations of appropriate regimens of Dox plus IL2 or TNF induce cures and the long-term survivors exhibit life-long immunological memory. Combinations of cyclophosphamide plus TNF have analogous effects. In the E0771 breast tumor C57BL/6 mouse model, Dox plus TNF at doses which are without antitumor activity when given alone, cause complete cures of established tumors with concomitant stimulation of CTL and NK cells responses. The mechanisms involved in these therapeutic responses are discussed. In conclusion, the results obtained substantiate the possibility of establishing antitumor curative combination regimens based on the utilization of low non-toxic immunomodulating doses of certain anticancer drugs and specific cytokines.