血吸虫感染宿主免疫抑制现象的研究 Ⅱ.日本血吸虫感染鼠血清的免疫抑制作用

在正常鼠脾淋巴细胞和刀豆素A(ConA)的培养物中,加入感染日本血吸虫的同系鼠血清,发现血清浓度在2.5,5和10％时,对ConA诱导的增生应答均有抑制作用,进一步实验是在脾淋巴细胞和ConA的培养物中加入10％的感染后1～10周的鼠血清,以观察对脾淋巴细胞增生应答的抑制作用有无显著变化。结果表明,正常鼠脾淋巴细胞对ConA的应答,加入感染后第5周血清时,其抑制作用明显减弱。然而,感染鼠脾淋巴细胞对ConA的增生应答,加入感染后第5周血清时,其抑制作用的减弱不明显。提示感染血清对ConA增生应答的免疫抑制作用可能受双重因素影响,包括感染后某时期血清本身抑制作用的强弱,以及这一时期的淋巴细胞对ConA应答能力大小。     

全反式维甲酸对Th2极化的调节作用

为了研究全反式维甲酸(all-trans retinoic acid,ATRA)对Th细胞分化的影响和可能的机制,无菌分离BALB/c小鼠脾细胞后,用2.5μg/ml的ConA刺激脾细胞,在不同时间加入不同剂量的ATRA,72 h后收集细胞,用3H-TdR掺入法测淋巴细胞增殖活性,用RT-PCR方法检测在Th细胞分化中起作用的细胞因子的mRNA表达水平。结果ATRA呈剂量依赖抑制ConA诱导的淋巴细胞的活化,10~(-5)mol/L的ATRA对ConA诱导的淋巴细胞活化的抑制作用最强;此抑制作用与作用时间呈相关性,ConA活化淋巴细胞24 h后加入ATRA对淋巴细胞活化的抑制作用最显著,并且淋巴细胞的活化程度越低,ATRA对其的抑制作用越强。ATRA可增强经ConA活化的淋巴细胞Th2型细胞因子(IL-4、IL-6)mRNA的表达水平,而Th1型细胞因子(IFN-γ、TNF-α)mRNA的表达水平显著性降低(P<0.05)。上述结果表明ATRA总体上可抑制ConA刺激的淋巴细胞增殖活性,但却可增强,Th2细胞的分化。这一研究为Th细胞免疫偏离的基础与临床研究提供了依据。     

青蒿素及其二种衍生物的免疫抑制作用比较

<正> 青蒿素是由中药黄花蒿中提纯的倍半萜内酯类化合物。它除了有特效的抗疟作用外,近来还曾在临床上试用于治疗自体免疫性疾病如盘形红斑狼疮,表现出一定的疗效。关于青蒿素类药物对机体免疫系统的影响,本实验室曾报道青蒿素的衍生物——青蒿素琥珀酸酯钠对分裂原刺激下的小鼠脾细胞及正常人外周血淋巴细胞的~3H-TdR掺入有显著抑制作用,也观察到青蒿素琥珀酸酯钠对小鼠体内脾细胞介导红细胞溶血反应有抑制作用。本文进一步比较了青蒿素及其二种衍生物蒿甲醚和青蒿素琥珀酸酯钠对小鼠体内脾细胞介导红细胞溶血反应的影响。     

青蒿素的免疫抑制作用

本文报道青蒿素水溶性衍生物——琥珀酸酯钠(简称QHS)对体外免疫反应的显著抑制及体内外反应的规律。 一、QHS的体外作用 (一)抑制作用及反应时间:用经典的掺入试验发现QHS对致分裂原(PHA及ConA)刺激的小鼠(C57BL或CFW)脾细胞或正常人外周血淋     

金银花提取物对小鼠淋巴细胞体外活化与增殖的影响

目的 研究金银花(Flos lonicerae,FL)提取物对小鼠淋巴细胞体外活化和增殖的影响,并初探其免疫抑制作用机制.方法 分离小鼠淋巴结细胞,以荧光标记的单克隆抗体染色结合流式细胞术,检测FL对刀豆蛋白A(Concanavalin A,ConA)刺激的小鼠淋巴细胞活化的影响;利用羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA-SE)染色,分析FL对淋巴细胞增殖情况.结果 细胞活化分析表明,金银花提取物对ConA诱导的小鼠淋巴细胞CD69、CD25和CD71表达均有明显的抑制作用,且这种抑制程度与其药物浓度呈正相关(P＜0.05).CFDA-SE染色分析显示,不同浓度的金银花提取物均能明显抑制淋巴细胞的增殖(P＜0.05).结论 金银花提取物对ConA诱导下的小鼠淋巴细胞体外活化和增殖均有明显抑制作用,是一种有潜在能力的免疫抑制剂.     

雷公藤对IL2的产生和IL2受体表达的抑制作用

观察雷公藤对T淋巴细胞免疫应答的抑制作用,发现雷公藤水煎液抑制小鼠脾细胞和胸腺细胞用ConA诱导的增殖反应,抑制作用与雷公藤剂量呈正比例。5mg/ml雷公藤完全抑制脾细胞的ConA增殖反应;该剂量雷公藤与脾细胞共育2小时不影响脾细胞的活率,细胞经洗涤后对CouA的增殖反应与正常细胞相同。进一步研究,在ConA诱导脾细胞72小时培养的早期加入雷公藤,完全抑制细胞的增殖反应;而在培养后期加入仅见部份抑制作用,推测雷公藤是抑制细胞的活化而不是对细胞的直接毒性作用和对DNA合成的抑制。ConA诱导脾细胞表达IL2受体和分泌IL2。雷公藤的加入完全抑制受体的表达,也抑制但不完全抑制细胞分泌IL2。外源性IL2的加入也不能逆转雷公藤对脾细胞ConA增殖反应的抑制作用,提示IL2分泌的减少与细胞DNA合成的抑制无直接联系。此外,IL2受体表达的受阻与DNA合成受抑制之间的联系还有待深入研究。     

双黄连粉针剂对小鼠T淋巴细胞体外活化与增殖的影响

目的:探讨双黄连粉针剂(以下简称SHL)对小鼠T淋巴细胞的体外活化和增殖的影响.方法:MTT法检测SHL对小鼠淋巴细胞的毒副作用;双色荧光抗体染色技术结合流式细胞术分析SHL对小鼠T淋巴细胞在多克隆刺激剂(ConA)刺激下体外活化抗原CD69表达的影响;MTT法以及羧基荧光素乙酰乙酸(CFDA-SE)标记技术结合流式细胞术分析SHL对小鼠T淋巴细胞在ConA诱导下体外增殖的影响.结果:SHL对小鼠淋巴结来源的淋巴细胞毒副作用非常小;终质量浓度为60、 80、 100、 120 mg/L的SHL对ConA刺激下的T淋巴细胞体外活化抗原CD69表达具有抑制作用(P<0.01);MTT法和CFDA-SE染色法都显示, 终质量浓度为60、 80、 100、 120 mg/L的SHL对ConA诱导的T淋巴细胞增殖作用具有明显的抑制作用(P<0.01).结论:SHL对小鼠T淋巴细胞的体外活化和增殖具有明显的抑制作用.     

低剂量Con A预刺激抑制足剂量Con A诱导的肝炎发生

目的:探讨低剂量刀豆蛋白A(ConA)预刺激对后续足剂量ConA诱导的小鼠肝炎的抑制作用。方法:足剂量ConA(15μg/g体重)静脉注射小鼠以诱导ConA肝炎模型。低剂量ConA预刺激组,在诱导ConA肝炎模型前12小时静脉注射低剂量ConA(3μg/g体重);PBS预处理对照组,在诱导ConA肝炎模型前12小时静脉注射PBS。观察两组血清转氨酶(ALT/AST)及肝脏病理变化,同时用流式细胞仪检测肝脏淋巴细胞亚群的比例及绝对数目变化。结果:与PBS预处理相比,低剂量ConA预刺激导致血清转氨酶(ALT/AST)水平显著降低(ALT由3433±788U/L降低到334±68U/L,AST由1358±297U/L降低到167±33U/L),肝脏病理切片显示由ConA引起的肝脏损伤明显减轻,同时伴随着肝脏内CD3+T淋巴细胞比例及绝对数目减少,并且其活化受到明显抑制。结论:低剂量ConA预刺激可以使小鼠抵抗后续的足剂量ConA诱导的肝脏损伤。这种现象与肝脏回流的T淋巴细胞减少及活化程度有关,其细胞与分子机制正在进一步探讨之中。     

连翘提取物对小鼠T淋巴细胞体外活化与增殖的影响

目的:分析连翘(FS)提取物对小鼠淋巴结T细胞的体外活化与增殖的影响,初步探讨其免疫抑制作用机制.方法:无菌分离小鼠淋巴结细胞,加入多克隆刺激剂刀豆蛋白A(ConA)进行刺激,利用荧光标记的单克隆抗体(mAb)染色结合流式细胞术(FCM),检测小鼠T淋巴细胞的表达的活化抗原CD69、CD25、CD71的表达情况;以羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA-SE)染色,以FCM分析FS对淋巴细胞体外增殖的影响.结果:终浓度为40、80、160 mg/L的FS均对ConA刺激诱导的T细胞CD69、CD25和CD71的表达有降低作用(P＜0.05).CFDA-SE染色分析显示,上述浓度的FS对ConA诱导的小鼠T淋巴细胞增殖具有抑制作用(P＜0.05).结论:FS对ConA诱导的T细胞早、中、后期活化和体外增殖有抑制作用.     

驱虫斑鸠菊注射液对小鼠免疫功能的影响

目的:探讨驱虫斑鸠菊对小鼠免疫功能的影响。方法:采用[3H]-TdR掺入法和脾细胞介导羊红细胞定量溶血分光光度法以及迟发型超敏反应试验等观察了驱虫斑鸠菊对小鼠兔疫功能的作用。结果:驱虫斑鸠菊在体内外均可以明显抑制ConA刺激的小鼠T淋巴细胞的增殖反应和LPS刺激的小鼠B淋巴细胞的增殖反应（P<0.01）;对小鼠胸腺指数和脾脏指数、绵羊红细胞（SRBC）诱导的正常小鼠脾抗体细胞生成反应以及小鼠皮肤迟发型超敏反应都显示出明显的抑制作用,而且上述抑制作用与药物浓度有一定的剂量效应关系。结论:驱虫斑鸠菊对机体体液免疫、细胞免疫都有明显的抑制作用。     

磷酸萘酚喹与青蒿素伍用增效和延缓疟原虫抗药性的研究

本文采用鼠疟模型进行磷酸萘酚喹与青蒿素伍用的药效学研究。用正交设计和4天抑制试验法，研究两药伍用增效的最适配比。采用药物剂量递增培育抗性的方法，连续血传100代，分别培育鼠疟原虫对磷酸萘酚喹、青蒿素及两药伍用的抗药性。结果显示磷酸萘酚喹和青蒿素最适配比为1：50，对鼠疟敏感株和抗氯喹株的增效指数分别为4．2和8．2，杀虫速度和治愈率均优于单药。培育至100代时，磷酸萘酚喹、青蒿素和两药伍用抗性指数分别为200．3、5．6和4．4。结果表明，磷酸萘酚喹与青蒿素配伍具有增效作用，并能明显延缓疟原虫对单药抗性产生的速度，降低抗性水平。     

Suppression of contact hypersensitivity by short-term ultraviolet irradiation: I. Immunosuppression by serum from irradiated mice.

Serum from UV-irradiated mice was shown to be immunosuppressive in vitro and in vivo. It suppressed leucocyte adherence inhibition reactions of cells from sensitized syngeneic and allogeneic mice, and suppressed the development of contact hypersensitivity after passive transfer to mice. Supernatants of cultures of spleen cells from irradiated mice were also suppressive. The suppressive factors in sera and culture supernatants were non-dialysable. The suppressive effect of UV irradiation was abrogated by cyclophosphamide, but this restored reactivity was still inhibited by serum from irradiated donors; this suggests that the serum factor requires T suppressor cells for its production but not for its action. The level of interleukin 1 (IL-1) was not raised in serum from UV-irradiated mice; thus the serum factor appears not to be IL-1.     

Testosterone-induced abrogation of self-healing of Plasmodium chabaudi malaria in B10 mice: mediation by spleen cells.

This study investigates the suppressive effect of testosterone (Te) on the self-healing of Plasmodium chabaudi malaria in female mice of the strain C57BL/10, and, in particular, the possible role of spleen cells in mediating this Te effect. Our data show the following. (i) About 80% of B10 mice infected with 10(6) P. chabaudi-infected erythrocytes are capable of self-healing the infections. This capability is progressively impaired and finally abrogated after pretreating the B10 mice with Te for 3 weeks. (ii) The spleen is Te responsive. This becomes evident in a reduction of total spleen cells from 1.05 x 10(8) to 0.54 x 10(8) on average after Te treatment for 3 weeks. Moreover, Te treatment causes an increase in the relative proportion of CD8+ cells by about 4% and a decrease of Ig+ cells by about 4.5%, as revealed by flow cytometry. (iii) Spleen cells mediate the suppressive Te effect as revealed by adoptive transfer experiments. The percentage of self-healing mice dramatically decreases to about 8% when they receive, just prior to infection, nucleated spleen cells isolated from mice treated with Te for 3 weeks. This suppressive effect can be transferred by T cells in particular but also by non-T cells, though to a lesser extent. (iv) The adoptively transferred cells mediate their suppressive effect on self-healing only if the recipient mice receive Te during infection. Our data suggest that spleen cells become functionally changed by the Te treatment for 3 weeks. Particularly T cells, but also non-T cells, gain P. chabaudi-specific suppressive activities, and the cells require a Te-induced factor(s) to mediate these activities.     

The suppressive effect of carrier priming on the response to a hapten-carrier conjugate

Carrier-primed cells, having some of the properties of T lymphocytes, have been found to inhibit the primary anti-hapten response of spleen cells to challenge with a hapten carrier conjugate. The antibody response of CBA/Ig^b spleen cells in irradiated CBA (Ig^a) recipients was measured by means of a radioimmunoassay using ¹²⁵I-labeled anti-Ig^b antibody. It was found that the primary anti-2,4,6-trinitrophenyl(TNP) response to TNP-KLH (keyhole limpet hemocyanin) was suppressed in recipient mice primed with KLH as compared with similarly treated unprimed or bovine serum albumin-primed recipients. This suppression was transferred by injection of 10⁸ spleen cells from CBA mice primed with KLH seven days beforehand, but not by injection of serum from such mice. The suppressive effect was abolished by treating the carrier-primed spleen cells with anti-T cell sera and complement before transfer. Priming with KLH seven days beforehand had only a small suppressive effect on the response to an unrelated antigen, DNP-ovalbumin, but there was marked suppression if KLH was again administered with the unrelated antigen. It is considered that the suppressive effect is specific in induction, but nonspecific in expression and that it is a manifestation of a homeostatic mechanism limiting the extent of the immune response.     

Inhibition of mixed leukocyte culture by anti-idiotypic antibodies

(CBA x C57BL/6)F1 antisera prepared by injecting F1 mice with CBA T lymphocytes or CBA anti-C57BL/6 alloantibodies were tested for their ability to inhibit the mixed leukocyte culture (MLC) response. Presence of antiserum throughout the culture period in the absence of complement did not have any effect on the MLC response. Treatment of CBA responder cells with F1 antiserum and complement prior to the culture specifically inhibited the MLC response. Specificity of the suppression was ascertained; absorption of the F1 sera with F1 or C57BL/6 spleen cells did not remove the suppressive factor, whereas absorption with CBA spleen cells did so. F1 antiserum treatment left intact the response to third party alloantigens (DBA/2). Immunabsorbent columns with alloantibody of corresponding specificity removed the suppressive factor from anti-T cell sera as well as from antisera to alloantibody. The data suggest that the circulating alloantibody population contains molecules which share idiotypic determinants with surface receptors on T cells recognizing the same alloantigens.     

Genetic resistance in the graft-versus-host reaction

Genetic resistance (GR) of (B10 x A/Ph)F1 hybrid mice to parental B10 grafts limits the 'performance' of both hemopoietic and lymphoid cells. In comparison with A/Ph spleen cells, B10 cells induce only a slight regional graft-versus-host (GVH) reaction and a delayed systemic GVH reaction. The GR to lymphoid cells is not sensitive to total body irradiation, to cyclophosphamide, or to various xenogeneic sera (e.g. antilymphocyte, antimacrophage, antithymocyte). A high dose of antimarrow serum was partially effective, but the most suppressive effect was obtained by treatment with silica. Besides some similar features, GR action on hemopoietic and lymphoid cells revealed several differences and the nature of these is discussed.     

Adenosine 5'-triphosphate (ATP)-mediated stimulation and suppression of DNA synthesis in lymphoid cells. II. Suppressive effect of ATP on murine T-cell functions

The suppressive effects of ATP on murine T-cell functions were studied. The suppressive effects of ATP as well as adenosine on the DNA synthesis of spleen cells are due to the presence of mature T-cells, because ATP has no suppressive effect on athymic nu/nu spleen cells. Further characterization of the cells which are responsible for ATP-mediated suppression of DNA synthesis revealed that the cells are nylon wool-adherent T-cells and PHA-reactive T-cells. In addition, the suppressive effects of ATP on both spontaneous and mitogen-induced proliferative responses are stronger than that of adenosine, and T-cells are more sensitive to ATP than B-cells. The observation that both ATP and adenosine have unique effects on T-cells compared to B-cells may contribute toward explaining why patients with severe combined immunodeficiency (SCID) associated with adenosine deaminase (ADA) deficiency have greater T-cell than B-cell abnormalities.     

Cyclophosphamide-mediated enhancement of antitumor immune potential of immunosuppressed spleen cells from mice bearing a large MOPC-315 tumor

We have utilized 4-hydroperoxycyclophosphamide (4-HP-CY) as a probe for the immunomodulatory activity of the metabolites of cyclophosphamide (CY) since 4-HP-CY hydrolyzes spontaneously in aqueous solution to the same metabolites as those formed after in vivo conversion of CY by microsomal enzymes. Exposure of immunosuppressed MOPC-315 tumor bearer spleens to a low concentration of 4-HP-CY (0.1-3.0 micron) resulted in augmented antitumor immune potential. The level of antitumor immune potential exhibited by 4-HP-CY-treated tumor bearer spleen cells was not further augmented but was actually reduced by depletion of glass-adherent cells, a procedure which is effective in removing the cells known to have immunosuppressive activity (i.e. metastatic tumor cells and macrophages) from the spleen of untreated MOPC-315 tumor-bearing mice. In fac, 4-HP-CY was superior to depletion of glass-adherent cells in augmenting the antitumor immune potential of immunosuppressed tumor bearer spleen cells. When cells from the primary tumor nodule were incubated with a low concentration of 4-HP-CY which only marginally inhibited their proliferation, the drug completely abolished the suppressive activity of the cells for in vitro generation of antitumor cytotoxicity by normal spleen cells. Moreover, a high level of antitumor cytotoxicity developed when normal spleen cells were cultured in vitro with 4-HP-CY-treated tumor cells at a wide range of ratios of spleen cells to tumor cells. Thus, in the MOPC-315 tumor model, metabolites of CY eliminate the inhibitory effectiveness of splenic suppressor cells and induce the appearance of immunopotentiating activity. The results obtained with 4-HP-CY in vitro provide support for the hypothesis that low-dose CY therapy of mice bearing a large MOPC-315 tumor leads to the appearance of augmented antitumor immune potential in their hitherto immunosuppressed spleen cells as a result of the in situ immunomodulatory effect of the drug on cells in the spleen.     

The Expression of Histocompatibility-2 Antigens on Hemopoietic Stem Cells

Antisera directed against histocompatibility-2 antigens of the mouse suppress the formation of spleen colonies by pluripotent hemopoietic stem cells. Of antisera which are specific for subregions of the H-2 complex, only those which contain anti-H-2K or anti-H-2D activity are effective. Specific anti-Ia sera do not react with the stem cell. If the titer of the CFU-s suppressive effect of the antisera is compared to their toxicity to spleen lymphocytes, it can be concluded that CFU-s express antigens in the same density as spleen lymphocytes. The amount of H-2 antigens expressed on the surface of CFU-s is independent of its rate of proliferation.     

Allosuppression of B cells in vitro by graft-vs.-host reaction-derived T cells is caused by cytotoxic T lymphocytes

An acute graft-vs.-host reaction (GVHR) was induced by i.v. injection of 10(8) lymphoid cells from C57BL/10 (B10) donors (H-2b/b) into adult non-irradiated (B10 X DBA/2)F1 mice (H-2b/d). Previous experiments have established that spleen cells obtained from such GVHF1 mice suppress the primary antibody response of normal F1 spleen cells to sheep red blood cells. This type of suppression was termed "allosuppression", and it was shown to be mediated by Ly-2+ T cells of donor origin that react against H-2 antigens of the host. It was unclear, however, whether the actual mechanism of allosuppression was due to suppressive factors generated by donor T cells or whether the latter killed the F1 B cells. Here, we show that for their suppressive effect GVHF1 spleen cells need direct cell contact with F1 spleen cells; no suppressive effect was measured in a double-chamber culture system in which the GVHF1 spleen cells were separated from the responding normal F1 spleen cells by a cell-tight membrane filter. The suppressive effect only affected cells expressing the appropriate H-2 class I alloantigen; in mixed cultures of irradiated F1 spleen cells and GVHF1 spleen cells with third-party B cells the antibody response of the third-party B cells was not suppressed. GVHF1 spleen cells showed cytotoxic T lymphocyte (CTL) activity specific for the allogeneic F1 H-2 antigen. The suppressive effect of the GVHF1 spleen cells did not differ from that exerted by cloned CTL specific for MHC class I alloantigens; cloned CTL suppressed the primary antibody response of murine spleen cells without affecting the response of third-party B cells added to the cultures. The combined findings show that "allosuppression" in vitro is not due to suppression of F1B cells, but to a direct killing of these cells by alloreactive CTL.