PNA受体阳性淋巴细胞在小鼠移植肿瘤前后的变化及其对GvHR抑制活性的研究

参照Reisner(1976)的方法,从花生果仁中提取的花生凝集素(PNA)能凝集小鼠PNA受体阳性的淋巴细胞(PNA~+细胞),并使之与PNA受体阴性的淋巴细胞(PNA~-细胞)分离。据Nakano(1980)报道,PNA~+淋巴细胞在功能上表现免疫抑制活性,因而推测它们可能是抑制性T淋巴细胞(Ts)。为了探讨带瘤宿主免疫调节功能的失常与Ts细胞的关系,我们应用PNA凝集试验和荧光标记技术分离并检测了小鼠(BALB/C)移植肿瘤(Ehrlich腹水瘤,EAC)前后,脾及肠系膜淋巴结中PNA~+细胞的变化,并     

甘草甜素抑制小鼠接触性超敏反应的实验研究

目的：探讨甘草甜素(GL)对二硝基氟苯(DNFB)所致小鼠接触性超敏反应(CHS)的抑制效果。 方法： BALB/c小鼠分为GL1(11 mg/kg)组、GL2(22 mg/kg)组、GL3(44 mg/kg)组、生理盐水(NS)组和地塞米松(DXM)组5组；利用DNFB诱发小鼠右耳CHS，各组于 -1 d 至5 d每天腹腔注射0.2 mL上述不同剂量药物(溶于生理盐水)。以耳厚度差、耳重量差及右耳病理变化，观察GL对小鼠CHS的抑制效果；同时计算胸腺指数、脾指数，了解GL对CHS小鼠免疫系统的影响。 结果： 3个剂量组GL和DXM均可明显抑制CHS小鼠右耳厚度及重量的增加，与NS组间差异显著(P<0.01)，同时亦可明显抑制右耳组织水肿和炎细胞浸润。3种剂量GL均可减低CHS小鼠胸腺重量，与NS组间有统计学差异(P<0.05)，而对脾脏重量无明显影响；DXM可显著降低CHS小鼠胸腺和脾脏重量。 结论： 甘草甜素可明显抑制DNFB诱发的小鼠接触性超敏反应。     

新生小鼠移植耐受的诱导及其机理探讨

本文用去T细胞的同种细胞及杂交一代(F_1)细胞成功地建立了新生小鼠移植耐受模型。通过耐受小鼠脾淋巴结细胞体内过继转移试验及移植物抗宿主反应试验(GVHR),对耐受的机理进行了探讨。实验结果表明,新生期诱导的移植耐受是特异性的,耐受的维持与T抑制细胞(Ts)参与的主动抑制机理有关。     

用组织相容性抗原局部免疫诱导Ts细胞

用同种异基因脾细胞皮下或静脉免疫小鼠可以诱导出迟发型过敏反应(DTH)。但是,也有报道指出静脉注射较多的同种异基因脾细胞,能诱导出抗原特异性Ts细胞,特异地抑制对该脾细胞的DTH,这种Ts细胞对DTH的抑制作用不受MHC限制。与此相反,另有报道用同种异基因脾细胞免疫小鼠后脚掌(HFI)能诱导出抗原非特异性Ts细胞,在体外非特异地抑制混合淋巴细胞     

白芍总甙对小鼠抑制性T细胞的作用

本文用过继性转移系统和McAb间接免疫荧光法等观察白芍总甙(TGP)对小鼠抑制性T细胞(Ts细胞)的作用。一、对超适量二硝基氟苯(DNFB)诱导Ts细胞的作用正常供体ICR小鼠腹部均匀涂以1%DNFB300μ1超适量免疫(SOI),诱导特异性Ts细胞。第5天将供体鼠脾细胞4×10~7经静脉转移给同系     

带瘤小鼠T细胞集落形成率的动态变化及其机理的初步探讨

本文应用微量甲基纤维素培养系统测定了正常与带瘤(EAC)小鼠的T细胞集落形成能力。发现小鼠带瘤后脾脏中的T细胞集落形成能力显著低于正常鼠脾细胞。用带瘤鼠的无细胞腹水、腹水瘤细胞培养上清、脾细胞条件培养液以及脾脏PNA~+细胞培养上清等对培养系统进行处理.这些提取物均能抑制正常小鼠的T细胞集落形成.据此认为,肿瘤相关的提取物能通过某种可溶性抑制因子对T细胞集落形成产生抑制效应。     

正常和荷瘤小鼠脾中VV~+细胞的反抑制功能

应用长柔毛野豌豆凝集素(VVL)粘附法,从小鼠脾中分离出对VVL有结合特异性的细胞(VV~+细胞)。VV~+细胞的表型确定为Thy1,2~+,Lyt2~-和L_3T~-_4。功能检测结果表明,VV~+细胞可消除或削弱Ts细胞介导的抑制作用。据此判定,VV~+细胞中富含反抑制T细胞(Tcs细胞)。小鼠在移植肿瘤后,其脾中VV~+细胞水平及其反抑制活性,均显著降低。结果提示,抑制和反抑制调节平衡的失常,可能是导致肿瘤宿主低免疫状态的原因之一。     

5-羟色胺对断奶仔鼠应激腹泻的影响

目的探讨断奶仔鼠应激性腹泻发生与5-羟色胺(5-HT)的相关性及断奶仔鼠应激性腹泻的发生机制。方法将72只雄性21 d断奶ICR小鼠随机分为6组,分别为正常对照组、氢溴酸西酞普兰(CH)对照组、对氯苯丙氨酸(PCPA)对照组、应激腹泻组、CH+应激腹泻组及PCPA+应激腹泻组。腹腔注射给药组(CH 10 mg/kg或PCPA 300mg/kg)处理4h后进行应激腹泻处理,即给予番泻叶(0.4 kg/L)按15ml/kg体重灌胃+后肢束缚应激处理(持续4h),其对照组分别给予相同剂量的生理盐水。处理第5天,检测血糖值、小鼠血浆皮质酮(Cort)、5-HT含量和分布的变化。结果与对照组相比,应激腹泻组和CH对照组小鼠血浆和肠道5-HT含量、血糖值和血浆Cort含量升高,腹泻评分增加,但是增重显著减小;CH+应激腹泻组较应激腹泻组相比5-HT含量、血糖值和Cort含量和腹泻评分显著增加,增重显著减小。PCPA对照组5-HT含量显著降低,与正常对照组相比除血浆Cort含量增加以外无显著变化;PCPA+应激腹泻组与应激腹泻小鼠相比肠道5-HT含量显著降低,增重升高,血糖值降低,但未达到正常水平。结论断奶仔鼠应激性腹泻伴随5-HT增加,CH引起的肠道5-HT含量升高直接导致并加重断奶仔鼠应激性腹泻,PCPA诱导的5-HT含量降低可减弱小鼠腹泻程度。     

异基因骨髓移植小鼠免疫功能缺损机制的探讨

异基因骨髓移植(Allogeneic Bone Marrow Transplantation,ABMT)后,受体的免疫功能长期缺损,是患者术后极易感染死亡的重要原因之一.本文对ABMT小鼠(C57BL/6→BALB/c)免疫功能缺损的机制进行了探讨,发现ABMT小鼠IL-2产生明显受损;其脾细胞与(C57BL/6小鼠脾细胞一起过继转移到致死量照射的BALB/C小鼠体内,能抑制移植物抗宿主病(GVHD)的发生.去除其脾T细胞后,这种抑制作用丧失,ABMT小鼠脾细胞上清中发现一种非特异的抑制因子,能抑制正常小鼠脾细胞产生混合淋巴细胞反应的能力;能抑制正常小鼠的脾细胞产生IL-2;抑制正常小鼠脾细胞毒T淋巴细胞(CTL)的杀伤活性.用抗Thy-1.2单抗和补体去除ABMT小鼠脾T细胞后,其脾细胞培养上(?)的上述抑制活性丧失.这说明ABMT小鼠脾T(?)细胞活性增强是其免疫功能缺损的重要原因之一,它通过释放非特异的抑制因子执行其免疫抑制功能.     

胃癌细胞总RNA修饰树突状细胞的肿瘤疫苗效果

目的应用肿瘤细胞RNA修饰小鼠树突状细胞(dendrticcell,DC)的肿瘤疫苗免疫小鼠,研究其抗肿瘤免疫作用.方法CD11c磁珠抗体标记小鼠脾单个核细胞悬液,磁性细胞分选器(MACS)分选出CD11c+的DC,短期培养,以肿瘤细胞总RNA脉冲DC,以此瘤苗皮下注射免疫615小鼠两次,间隔1周,最后1次免疫后7d,实验鼠皮下接种5×105小鼠前胃癌细胞(MFC).结果经免疫后的小鼠具有较强抗肿瘤免疫,RNA脉冲DC的瘤苗免疫后肿瘤生长受到明显抑制,其外周血和脾细胞中NK活性均明显升高,肿瘤特异性CTL也明显升高.结论以肿瘤RNA作为抗原脉冲DC可诱导较强的抗肿瘤免疫.     

Blockade of CD40-CD40 ligand pathway induces tolerance in murine contact hypersensitivity

Interactions between CD40 on antigen-presenting cells and its ligand (CD40L) on T cells has been implicated in T cell-mediated immune responses. Previously, we have shown that contact hypersensitivity (CHS), a cell-mediated cutaneous immune response in reaction to haptens, could be subclassified based on whether the hapten primed for Th1 or Th2 cytokines in cells isolated from draining lymph nodes. We also found that tolerance to a Th2-priming hapten could be induced only by simultane blockade of the CD40-CD40L and B7-CD28 at the time of sensitization. Here we demonstrate that blockade of CD40-CD40L signaling alone induces long-lasting unresponsiveness to the Th1 hapten 2,4-dinitrofluorobenzene (DNFB), and inhibits antigen-specific T cell proliferation in vitro. We find that CD40-CD40L signaling is required in the sensitization but not elicitation phase of DNFB-induced CHS, as treatment of mice with anti-CD40L monoclonal antibody (mAb) does not affect the response to hapten challenge in previously sensitized and untreated animals. Examination of cytokine production shows that anti-CD40L mAb decreases interferon-γ production by draining lymph node cells from DNFB-sensitized mice, and reciprocally increases interleukin (IL)-4 production. Consistent with this Th1 to Th2 immune deviation, anti-CD40L mAb prevents the induction of IL-12 mRNA in regional lymph nodes, an event which is normally seen within 12 h following hapten sensitization. In contrast, suppression of CHS by CTLA4Ig decreased the production of all cytokines by draining lymph node cells. Together, these data show that blockade of the CD40-CD40L pathway by itself is sufficient to induce tolerance to DNFB-induced CHS, and that this is associated with blockade of IL-12 induction and Th1 to Th2 immune deviation.     

Cross-reactivity of TNP immune effector T cells that mediate contact hypersensitivity and inflammatory bowel disease in the mouse

BACKGROUND: Experiments were aimed to test the cross-reactivity of immune Th1 cells that mediate contact hypersensitivity (CHS) or inflammatory bowel disease (IBD) to TNP in the mouse. METHODS: CBA/J mice were immunized either epicutaneously or intrarectally with TNP and after appropriate time intervals were challenged with antigen in a crossed manner. The CHS reaction was measured by the ear swelling test. IBD was quantified by increase of colon weight and myeloperoxidase level. Both reactions were confirmed histologically. In passive-transfer experiments, mesenteric lymph node cells of animals sensitized intrarectally and peripheral lymph node and spleen cells of mice immunized epicutaneously were used. In some experiments, before being immunized mice were made either unresponsive to the TNP hapten by induction of suppressor T cells, or resistant to suppression after induction of upregulatory T cells. RESULTS: Irrespective of the mode of sensitization upon appropriate challenge with antigen all mice developed a good CHS reaction as well as significant IBD. This cross-reactivity could be passively transferred by immune cells. In mice in which antigen-specific down- or upregulatory cells were induced before sensitization both CHS and IBD to TNP were modulated accordingly. CONCLUSION: TNP hapten deposited on skin or on mucosal surfaces induces effector cells that recognize antigen independent of its tissue localization, and produce a local inflammatory reaction. TNP-specific up- and downregulatory cells, shown before to regulate the CHS reaction, similarly modulate the generation and development of hapten-induced IBD.     

Effect of formaldehyde gas exposure in a murine allergic contact hypersensitivity model

To clarify the effect of formaldehyde (FA) gas exposure on contact hypersensitivity (CHS), CHS reactions against 2,4,6-trinitrochlorobenzene (TNCB) was studied in BALB/c mice with a low dose of FA gas exposure. The TNCB-induced CHS reactions were slightly suppressed by the FA gas exposure immediately after sensitization, whereas they were significantly enhanced and prolonged in mice continuously exposed to FA gas before and after sensitization. We showed that exposure to FA gas enhanced the Th2 dominant responses in draining lymph node (LN) in early stage of CHS. In contrast, T cell subsets and their intracellular cytokine production in the draining LN were similar during the early stage of CHS by FA gas exposure during the sensitization phase. The percentage of CD8+ T cells was increased, and the percentage of CD4+CD25+ T cells was decreased in the FA gas-exposed group at 72 hr after elicitation. These results indicate that FA gas-exposed might influence regulatory T cells. Furthermore, in the chronic CHS model that was repetitively elicited with TNCB, more intensive and prolonged CHS reactions, and increased numbers of mast cells were found in the FA gas-exposed group at 4 hr after elicitation than in the control group, FA gas exposure may alter the intensity of allergic CHS.     

Epicutaneous immunization induces alphabeta T-cell receptor CD4 CD8 double-positive non-specific suppressor T cells that inhibit contact sensitivity via transforming growth factor-beta

Since it was previously shown that protein antigens applied epicutaneously in mice induce allergic dermatitis mediated by production of T helper 2 (Th2) cytokines we postulated that this might induce suppression of Th1 immunity. Here we show that epicutaneous immunization of normal mice with a different protein antigen applied on the skin in the form of a patch induces a state of subsequent antigen-non-specific unresponsiveness caused by suppressor T cells (Ts) that inhibit sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by alphabeta-T-cell receptor CD4(+) CD8(+) double positive lymphocytes harvested from lymphoid organs of skin patched animals and are not major histocompatibility complex-restricted nor antigen specific. Both CD25(+) and CD25(-) CD4(+) CD8(+) T cells are able to suppress adoptive transfer of Th1 effector cells mediating cutaneous contact sensitivity. In vivo treatment with monoclonal antibodies showed that the cytokines interleukin (IL)-4, IL-10 and transforming growth factor-beta (TGF-beta) are involved in the induction of the Ts cells. Additionally, using IL-10(-/-) mice we found that IL-10 is involved in skin induced tolerance. Further in vitro experiments showed that lymph node cells of skin tolerized mice non-specifically suppress [(3)H]thymidine incorporation by antigen-stimulated immune cells and this effect can be abolished by adding anti-TGF-beta, but not anti-IL-4 nor anti-IL-10 antibodies. These studies indicate the crucial role of TGF-beta in skin induced tolerance due to non-antigen-specific Ts cells and also show that IL-4, IL-10 and TGF-beta play an important role in the induction of epicutaneously induced Ts cell suppression.     

Lymphokine profile and activation pattern of two unrelated antigen- or idiotype-specific T suppressor cell clones.

Two T suppressor (Ts) clones of different specificity have been analyzed for their lymphokine spectrum. BVI/5 is an I-Ek-restricted bovine serum albumin (BSA)-specific Ts cell clone from a CBA/J mouse tolerized by low doses of BSA. It affects directly or indirectly the function of BSA-specific T helper (Th) cells. The Ts cell clone 178-4 from a BALB/c mouse is I-Ed restricted and recognizes the public J558 Id on B cells. It prevents alpha(1----3)dextran B 1355S (Dex)-specific IgG antibody production and drives Dex-specific J558 idiotype-bearing B cells into an anergic B IgG memory cell state. Both Ts cell clones thus cause specific suppression, yet in different experimental systems using different effector mechanisms. Upon stimulation with concanavalin A or fixed CD3-specific monoclonal antibody, both clones produce high levels of interferon (IFN)-gamma and tumor necrosis factor (TNF) but in contrast to Th1 cells no interleukin (IL)-2. Both clones produce low levels of IL-3 and IL-6 but no IL-4, IL-5 and IL-9. Furthermore, unlike Th2 cells, both clones do not respond to IL-1. The mechanism of the idiotype-specific induction of anergy in Dex-specific B IgG memory cells by 178-4 Ts cells is not yet understood. BVI/5 Ts cells suppress in vitro the BSA-specific proliferation of the BSA-specific Th cell clone 83/1, as well as the response of BSA-primed CBA/LN cells. Whereas the suppressive effect on 83/1 cells is due to IFN-gamma alone the suppression of BSA-specific lymph node cells can be simulated neither by IFN-gamma nor the combination of IFN-gamma and TNF. Thus these mediators cannot account for the antigen-specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from lymph node cells and probably not for the induction of in vivo unresponsiveness.     

Depression of lymph node cell proliferation induced by oxazolone

The influence of topical exposure to two sensitizing chemical on draining lymph node cell proliferative responses in BALB/c mice has been examined. Conventional contact sensitization with 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone) has been shown to induce a rapid and systemic suppression of subsequent proliferative responses to topically applied chemical which can be adoptively transferred to recipient mice with immune lymph node cells. In contrast to some previous reports in which such suppression was found to be largely antigen-specific in nature, we report that, at least initially, the inhibition of lymphocyte proliferation induced by skin sensitization is hapten-non-specific. The relevance of this phenomenon to the regulation of contact sensitization is discussed.     

Sensitization of mice to chemical allergens modulates the responsiveness of isolated mast cells to IgE-dependent activation.

It is known that the release of granule-associated inflammatory amines by isolated mouse tissue-type mast cells is subject to regulation in vitro by a number of cytokines that are produced during the immune response. In this study we investigated whether mast cell secretory function might also be subject to regulation in vivo during active sensitization. Mice were sensitized with one of three chemical allergens (trimellitic anhydride, TMA; 2,4-dinitrochlorobenzene, DNCB; or oxazolone) all of which induce contact sensitization and one of which (TMA) in addition causes immediate hypersensitivity. Peritoneal mast cells isolated from treated mice and sensitized passively with IgE released a greater proportion of cellular serotonin (5-HT) on stimulation either by anti-IgE or by specific antigen than did cells isolated from vehicle-treated controls. These results show that the function of mast cells is susceptible in vivo to regulatory influences that result from induction of an immune response.     

Contact allergic response to dinitrochlorobenzene (DNCB) in rats: insight from sensitization phase

Contact hypersensitivity (CHS) is a T-cell-mediated skin inflammatory reaction to cutaneous exposure to small sensitizing chemicals, haptens. Majority of CHS studies were conducted in mice and there is paucity of data in other experimental animals. In the present study, characteristics of contact hypersensitivity reaction to dinitrochlorobenzene (DNCB) were determined in Th1-prone Dark Agouti (DA) rats by evaluating sensitization phase as a function of time-dependent changes in draining lymph nodes (DLN). Apart from basic indices of DLN activity (cellularity and proliferation), the production of cytokines relevant for CHS induction, interleukin-6 (IL-6), interferon-γ (IFN-γ), interleukin-17 (IL-17) and interleukin-4 (IL-4) was analyzed. Anti-inflammatory cytokine interleukin-10 (IL-10) production by DLN cells was determined as well. Highest production of IL-6, IFN-γ and IL-17 in sensitized animals was observed at day 3 after DNCB application, with a decrease at day 5. Increased messages for IFN-γ and IL-17 were noted at this time point. In contrast to inflammatory cytokines, anti-inflammatory cytokine interleukin-4 (IL-4) was undetectable during the entire sensitization phase. Differential pattern (IL-6 and IFN-γ) and level (IFN-γ and IL-17) of inflammatory cytokine production was noted in sensitized Th2-prone Albino Oxford (AO) rats. Similarly to DA rats, no changes in IL-4 were noted in AO rats. Strain-dependent differences in inflammatory cytokine production seem to be based on anti-inflammatory cytokine interleukin-10 (IL-10). Production of IFN-γ concomitantly with undetectable IL-4 in both strains classify rat CHS to DNCB as Th1/type 1 reaction. Detection of IL-17 in sensitized DLN cells points to the involvement of T_IL-17 cells in rat contact hypersensitivity.     

Effects of first-order Cryptococcus-specific T-suppressor cells on induction of cells responsible for delayed-type hypersensitivity.

Cell-mediated immunity is an important aspect of host resistance against Cryptococcus neoformans. Using a CBA/J murine model, we demonstrated that injection of cryptococcal antigen (CneF) at dosages sufficient to stimulate the antigenemia observed in cryptococcosis patients induces specific T-cell-mediated suppression of the cryptococcal delayed-type hypersensitivity response. The purpose of this study was to establish whether Lyt 1+, first-order T-suppressor (Ts1) cells block the induction of T cells responsible for delayed-type hypersensitivity (TDH cells) or whether they function by inducing Lyt 2+, efferent suppressor (Ts2) cells. In one set of experiments, suppression was observed when Ts1 cells were adoptively transferred to recipient animals the day before, the day of, or the day after immunization; however, when Ts1 cells were transferred after TDH cells were present, no suppression occurred. In other experiments, putative TDH cells from lymph nodes (LN) or spleens were adoptively transferred from mice after immunization or after a suppressive dose of CneF or adoptive transfer of Ts1 cells and immunization. Delayed-type hypersensitivity could not be transferred with LN or spleen cells from mice receiving the suppressive dose of CneF or the Ts1 cells, even when the LN or spleen cells were treated with anti-Lyt 2.1 antibody and complement to remove any Ts2 cells. Delayed-type hypersensitivity was readily transferred with LN or spleen cells from immunized mice whether the cells were or were not treated with anti-Lyt 2 and complement. Furthermore, the cells in the tolerized LN cell pools responsible for suppression of TDH cell induction were Lyt 1+ 2-, I-J+ cells, which is the phenotype of the Ts1 cells. Taken together, these data indicate that Ts1 cells inhibit the induction of TDH cells. This finding, coupled with the previous demonstration that Ts1 cells or a Ts1 cell-derived soluble factor (TsF1) induces Ts2 cells, establishes that the cryptococcal Ts1 cells are bifunctional in the suppressive pathway.     

Impact of the magnitude of sensitization dose on the incidence and intensity of CHS to dinitrochlorobenzene (DNCB): Insight from ear swelling and challenged-skin draining lymph node response in rats

Abstract

Allergic contact dermatitis (ACD) is a common skin inflammatory disease that develops in hosts sensitized with contact allergens. Elucidation of dose–response relationships represents one of the approaches in studying the type of ACD in humans/animal models, termed as contact hyper-sensitivity reaction (CHS). Such studies have demonstrated that the intensity of sensitization determines the response to elicitation with a contact allergen, but underlying mechanisms are unclear. The aim of this study is to explore the impact of the sensitization on contact hypersensitivity to dinitrochlorobenzene (DNCB) in rats by measuring the incidence and intensity of a challenge response in hosts sensitized with two different doses (i.e. low and high) of this hapten. Assumptions concerning the contribution from the magnitude of sensitization doses were drawn on the basis of effects from the two doses on the measured reaction parameters. Ear swelling and activity of lymph nodes that drain challenged skin (cdLN), including cellularity, proliferation, and effector cytokine IFNγ and IL-17 production was measured in rats sensitized with 0.4% or 4% DNCB and challenged with a non-irritant (0.13%) dose. Sensitization with 4% DNCB resulted in a greater proportion of rats who responded more intensely (than unsensitized challenged rats) to challenge in terms of ear swelling and increases in cdLN activity (except for IFNγ). The intensity of cdLN responses was higher in these hosts as well. Among the high-dose-sensitized rats, greater cellularity/proliferation of cells from lymph nodes (sdLN) that drain the high-dose-sensitized skin, as well as higher IL-17 production, was noted compared to what was seen in rats that received low-dose sensitization. In contrast, unchanged spontaneous and even decreased hapten-stimulated IFNγ production after the high DNCB dose was observed. Based on the data, it seems the impact of magnitude of sensitization dose on CHS might be related to the rise in the proportion of rats that responded to challenge with an increase of dLN activity. Coincidental higher production of IL-17 by dLN cells from the high-dose-sensitized rats and following challenge of these hosts underscored the significance of IL-17 for a CHS to DNCB.