苏木对体外人淋巴细胞增殖的抑制作用

作者观察了苏木水煎液体外对PHA或SAC诱导的人淋巴细胞增殖功能,以及诱生IL-2活性的影响.实验以雷公藤水煎液为对照.结果表明苏木体外对SAC诱导的人B淋巴细胞增殖有明显的抑制作用,对PHA诱导的人T淋巴细胞增殖和诱生的IL-2活性亦有明显的抑制作用(P<0.001),其结果与雷公藤相似.而苏木预育人淋巴细胞6h后,则可明显抑制T细胞增殖(P<0.001)和诱生IL-2活性(P<0.05),其抑制强度要明显大于雷公藤(P<0.001).实验提示苏木可作为免疫抑制性中药作进一步的研究和开发.     

在人扁桃体细胞中诱生γ干扰素

本文初步研究了用人扁桃体细胞诱生γ干扰素及其影响因素。试验结果显示:细胞浓度在5×10~7/ml时产生的γ干扰素效价最高;PHA、PWM、ConA等T细胞促分裂剂诱生γ干扰素的适宜浓度范围分别为50～400μg/ml,2.5～40μg/ml,5～80μg/ml;诱生时间均以72小时为宜;2-ME对人γ干扰素的产生没有影响。本文还将人扁桃体细胞和外周血白细胞对4种γ干扰素诱生利(PHA、PWM、ConA、SEA)和3种α干扰素诱生剂(NDV、SPA菌体、CP)的反应进行了比较。     

外源性IL-23对病毒性心肌炎小鼠Th17细胞增殖的影响

目的:观察小鼠重组IL-23(rIL-23)对病毒性心肌炎(VMC)小鼠Th17细胞增殖的影响。方法:BALB/c小鼠腹腔注射柯萨奇病毒B3(CVB3)建立VMC小鼠模型,1周后磁珠分离脾CD4+T淋巴细胞后进行体外培养,分别加入植物血凝素(PHA)与重组小鼠IL-23(rIL-23)或PHA单独刺激进行体外培养5天。流式细胞术测定培养后Th17细胞占CD4细胞的百分比、RT-PCR测定培养细胞IL-17 mRNA的表达、ELISA法测定培养物上清液中IL-17的水平。结果:与单纯PHA刺激比较,rIL-23刺激后Th17细胞明显增加(1.82%±0.17%比4.70%±1.29%),且培养细胞IL-17 mRNA及上清液中IL-17浓度也增加,两组比较有统计学差异(P均<0.05)。结论:外源性rIL-23可以增加并维持Th17细胞的增殖。     

雷公藤甲素对人扁桃体淋巴细胞表面IL—2受体表达的抑制作用

应用体外培养方法和APAAP 桥联酶标技术,观察了雷公藤甲素对人扁桃体淋巴细胞增殖、IL-2产生及其表面IL-2受体表达的影响。结果发现,雷公藤甲素(0.4～10ng/ml)可显著抑制PHA 诱导的人扁桃体细胞增殖及IL-2受体表达,且呈剂量依赖关系;而在相同浓度范围内,对PHA 诱导的IL-2产生无明显影响。提示雷公藤甲素的免疫抑制作用不是通过抑制IL-2产生,而可能是由于抑制了IL-2受体表达,以及干扰IL-2的信号传导系统所致。     

Con A诱生小鼠不同功能T调节细胞的简易方法

<正> 小鼠T调节细胞(即Th,Ts细胞)的诱生可采用体内方法和体外方法。但用不同浓度Con A进行体外诱生时,Con A浓度难以掌握,故有可能出现双向作用而影响实验结果的分析。吴易元等用Con A与人外周血淋巴细胞共同培养不同时间诱导人类T调节细胞。本文在此基础上,建立了一种同时诱生小鼠T调节细胞的简易方法。     

细胞因子的复合诱生及初步纯化

采用PHA和LPS对人胎脾脏单个核细胞进行细胞因子复合诱生,结果显示上清中同时含有淋巴因子IL-2、IL-6和单核因子IL-1、IL-6、TNF,而且比单刺激组含量有所提高。进一步采用超滤或盐析技术进行初步纯化,纯化倍数分别为650～730倍和64～78倍,收率分别为74.4～82.6％和36.00～40.5％,继而证明复合细胞因子在低剂量IL-2(100μ/ml)时即可诱导出LAK活性,仅为单纯基因重组IL-2用量的1/10。     

人参三醇皂甙对人淋巴结细胞IL—5基因表达的促进效应

应用mRNA麦胚无细胞体外转译体系,动态地观察了人参三醇皂甙(PTGS)对人淋巴结细胞IL-5基因表达的促进效应。结果表明,PTGS可以明显促进PHA活化人淋巴结细胞分泌IL-5,最大促进效应可达66.67％。PTGS+PHA共刺激后人淋巴结细胞浆IL-5 mRNA转译IL-5的量明显高于单纯PHA组,最大促进效应40％。上述结果首次证明PTGS对IL-5的促诱生效应是通过调节IL-5基因表达而实现的。     

用经氢化可的松处理的小鼠胸腺细胞测定白细胞介素2

白细胞介素2(IL-2)是重要的免疫调节因子。对IL-2的检测,国外一般采用依赖IL-2的T细胞株CTLL细胞进行测定。该细胞在国内来源困难,不易维持。国内亦有用鼠胸腺细胞增殖试验来测定IL-2者,但由于不能消除IL-1的非特异影响,因此,该方法受一定局限。本文参照Christine的报道,用经Con-A(4μg/ml)诱导及氢化可的松(0.04μg/ml)处理的鼠胸腺细胞(HCIT)进行IL-2测定,并同时用CTLL-2细胞测定方法进行比较,结果证实:用HCIT细胞测定IL-2与用CTLL-2细胞测定两种方法有很好的相关性、相关系数r=0.94。但用CTLL细胞比用HCIT细胞较敏感。在本试验条件下,前者敏感量为0.4IL-2活性单位,后者为1.3IL-2活性单位。因此用HCIT细胞进行IL-2测定,也可以达到特异(对IL-1不发生反应),灵敏、重复性好的效果。且由于胸腺细胞容易获取,操作技术简便、易于掌握,是一种在没有CTLL细胞情况下测定IL-2的较好的方法。     

两种多克隆刺激条件下人诱导性Treg表型的多样性变化

目的:研究人诱导性调节性T细胞(iTreg)细胞表型的多样性变化,并比较PMA/Ionomycin和PHA两种多克隆刺激对人iTreg的诱导的异同。方法:用Ficoll密度梯度离心分离出人PBMC,空白对照组直接检测,其余则在细胞培养液(非刺激对照组)、PMA/Ionomycin溶液(PMA/Ionomycin刺激组)或PHA溶液(PHA刺激组)中培养16小时,以流式细胞仪检测CD4+CD25+FoxP3+CD127-、CD4+CD25+FoxP3+IL-2-、CD4+CD25+FoxP3+IL-10+、CD4+CD25+FoxP3+TGF-β+和CD4+CD25+FoxP3+IFN-γ+的表达。结果:空白对照组显示,约4%的CD4+细胞表达CD4+CD25+FoxP3+CD127-,即nTreg。PBMC在细胞培养液培养16个小时,iTreg的表达无明显变化,但经多克隆刺激后,各不同细胞表型的iTreg的表达明显上升(均P<0.01),表明多克隆刺激可以明显诱导人iTreg的产生。PHA对IL-2-和TGF-β+iTreg的诱导比PMA/Ionomycin强(P<0.01),但仅PMA/Ionomycin可以诱导产生CD4+CD25+FoxP3+IFN-γ+iTreg,而PHA不能诱导。刺激后产生的CD4+FoxP3+IFN-γ+PBL表达与CD25水平呈逆相关。结论:多克隆刺激可以诱导产生人iTreg,PMA/Ionomycin和PHA对人iTreg诱导的机制和程度不一样。多克隆刺激后产生的不同细胞表型反映了人iTreg的多样性变化。     

骨髓间充质干细胞体外对T淋巴细胞分泌IL-2、IL-4功能的影响

目的探讨间充质干细胞(Mesenchymal stem cells,MSCs)对T淋巴细胞分泌功能的调节作用.方法体外分离培养、扩增人骨髓MSCs,并通过形态学特征及流式细胞术检测其表面标志加以鉴定.将不同数量的MSCs(5×103、1×104、5×104个细胞/孔)分别与PHA激活的T细胞和混合淋巴细胞反应(MLR)体系共培养,并将不同浓度(25%、50%、75%)的MSCs培养上清和MLR体系共培养.应用ELISA分别检测各培养上清液中T细胞分泌IL-2、IL-4的水平.结果骨髓MSCs能抑制PHA作用下的T细胞分泌IL-2、IL-4(P＜0.05),并呈MSCs数量相关性(P＜0.05),且对IL-2的抑制作用更显著;也可抑制MLC体系中T细胞分泌IL-2、IL-4(P＜0.05),但各数量组的MSCs的抑制作用无显著性差异(P＞0.05).不同浓度的MSCs培养上清均可抑制MLC体系中T细胞分泌IL-2、IL-4(P＜0.05),但不同浓度的MSCs培养上清组对IL-4抑制作用无显著性差异(P＞0.05).结论骨髓MSCs及其培养上清均可抑制PHA或异体抗原作用下的T细胞分泌IL-2、IL-4,提示MSCs可能是直接作用于T细胞或通过分泌可溶性因子调节 Th1/Th2反应平衡而发挥免疫调节作用的.     

The effect of experimental haemocarbofiltration upon activity of mononuclear cells from normal and autoimmune patients

We examined the functional activity of peripheral blood mononuclears (PBM) of 18 healthy subjects, 18 patients with pemphigus vulgaris, 12 with bullous pemphigoid and nine with discoid lupus erythematosus, after haemofiltration on carbon haemo-adsorbents of 'SKN' type. Proliferation in the response to PHA and Con A, for IL-1 and IL-2 production, and exogenous IL-2 absorption were assayed. The presence of IL-1 and IL-2 inhibitors in haemocarbo-adsorbent eluants was shown. We also investigated the natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities. We found significant increases of both lectin-dependent proliferation and of interleukin production by PBM of autoimmune patients after two to four perfusions. The ability of PBM to absorb IL-2 displayed a steady growth after each perfusion, whereas increase of NK and ADCC activities was observed after not less than six passes. The enhancement of PBM functional activity in autoimmune patients was accompanied by accumulation of IL-1 and IL-2 inhibitors in the sorbent. It was concluded that therapeutic effects of haemofiltration in autoimmune diseases involve improvement of immunocompetent cell function due to their deligandization by activated charcoal.     

Effect of phorbol myristate acetate on T cell colony formation, interleukin-2 (IL-2) receptor expression and IL-2 production by cells from patients at all stages of HIV infection.

We and others have shown that several T cell responses induced by the mitogen phytohaemagglutinin (PHA), including T cell colony formation, IL-2 receptor (IL-2R) expression, and IL-2 production are impaired in patients with AIDS and lymphadenopathy syndrome (LAS). We investigated whether phorbol myristate acetate (PMA) could act in synergy with PHA (as it does in healthy subjects) to enhance in vitro T cell responses of patients at all stages of infection by HIV. In AIDS patients with opportunistic infections (AIDS/OI), PHA + IL-2 + PMA led to a total disappearance of T cell colonies in 10/11 patients, among whom six already displayed very low numbers of colonies induced by PHA + IL-2 (less than 50 colonies/5 x 10(4) cells). In contrast, T cell colony formation induced by PHA + IL-2 + PMA was maintained or increased, compared with that induced by PHA + IL-2, in five out of six AIDS patients with Kaposi's sarcoma (AIDS/KS), 10/14 LAS and six out of seven HIV-seropositive asymptomatic (HIV+/AS) homosexuals. In these three groups of patients, a low percentage of colony cells induced by PHA + IL-2 + PMA expressed CD3 and CD4 molecules, but 50-89% of cells were IL-2R (Tac) positive, as in healthy controls. Studies on T cell activation and IL-2 production were performed on a selected group of 12 HIV-infected patients for whom sufficient numbers of lymphocytes could be obtained. PMA induced CD4 down-modulation in controls and in HIV-infected patients. However, CD3 down-modulation and induction of the Tac chain of IL-2R by PMA were significantly impaired in patients, compared with controls, and these two parameters were correlated. Although PHA alone induced virtually normal levels of Tac antigen on patients' cells, Tac induction by PHA + PMA was significantly decreased in patients versus controls. Cells from five out of 10 patients tested failed to produce detectable amounts of IL-2 after PHA stimulation, whereas IL-2 production increased significantly in all patients tested (n = 9) after PHA + PMA, with a level of IL-2 activity significantly higher than in controls. No correlation was found in this group of patients between the effects of PMA + PHA on T cell colony formation, Tac expression, or IL-2 production, as compared with PHA alone. Taken together, our results indicate that in vitro T cell functional studies with PMA may be useful to evaluate better the defects of T cell activation in HIV-infected patients.     

In vitro correction of the interleukin-2 and interferon-gamma defect in multiple sclerosis.

The effect of phytohaemagglutinin (PHA) and/or phorbol myristic acetate (PMA) on human interferon-gamma (HuIFN-gamma) and interleukin 2 (IL-2) production was measured in peripheral blood leucocytes (PBL) from multiple sclerosis (MS) patients. Seven out of 12 MS patients studied had PBLs which were unable to produce any detectable HuIFN-gamma after stimulation with PHA. The PBL cultures of the same seven patients were also defective for IL-2 production after PHA stimulation. Addition of PMA before and during PHA stimulation resulted in restored IL-2 and HuIFN-gamma production in all otherwise non-responsive cultures. In MS cultures responsive to PHA alone, the addition of PMA resulted in a 10-fold increase of HuIFN-gamma and IL-2 production. Analysis of interleukin 1 (IL-1) production by peripheral blood monocytes (PBM) isolated from the same cultures, revealed that both spontaneous and PMA-induced IL-1 yield was the same for all cultures tested, regardless whether they produced IL-2 and HuIFN-gamma after PHA stimulation or not.     

Effects of isoprinosine and NPT 15392 on interleukin-2 (IL-2) production

Two purine immunoenhancing drugs, isoprinosine and NPT 15392, were evaluated as inducers of IL-2 or enhancers of IL-2 induction by phytohemagglutinin (PHA). Both drugs were found to have significant enhancing activity on PHA induction of IL-2 by human lymphocytes over a wide range of concentrations. Alone they were unable to consistently induce IL-2 activity. NPT 15392 was able to enhance PHA-induced IL-2 production at 10-100 times lower concentrations than isoprinosine. The actions of these compounds to enhance IL-2 production may explain their effects on PHA-induced lymphocyte proliferation.     

Modulation of anti-Candida activity of human alveolar macrophages by interferon-gamma or interleukin-1-alpha

The fungicidal and bactericidal activities of human alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 18 healthy volunteers were evaluated. The results showed that AM were able to phagocytize and kill Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. However, killing of the bacteria was already complete in 2 h, whereas killing of Candida required 4 to 6 h despite an early phagocytosis of yeast cells. The fungicidal activity of freshly collected AM and PBM was also tested after effector cell exposure to interferon-gamma (IFN-gamma), interleukin-1-alpha (IL-1 alpha), endotoxin lipopolysaccharide (LPS), or interleukin 2 (IL-2). It was found that treatment with IFN-gamma, IL-1 alpha, or LPS significantly augmented macrophage and PBM candidacidal activity, whereas the addition of IL-2 was ineffective. We also evaluated killing of C. albicans by AM cultured in vitro for different times. While phagocytosis was apparently unaffected, the candidacidal activity progressively decreased over the in vitro culture period, an effect that was largely reversed by cell exposure to IFN-gamma, IL-1 alpha, or LPS. In an experimental model in which mice infected with an agerminative C. albicans strain (PCA-2) resisted lethal microbial challenge, freshly harvested AM showed increased cytotoxic activity to Aspergillus fumigatus in vitro as well as enhanced IL-1 production. In conclusion, present data confirm the crucial role of AM in the surveillance of bacterial and fungal infections and indicate that treatment of these cells with IFN-gamma or IL-1 alpha is able to enhance their antimicrobial capability.     

Combined effects of in vitro penicillin and sickle cell disease sera on normal lymphocyte functions

Previously published work has shown that sera from healthy sickle cell disease (SCD) patients inhibits normal lymphocyte response to phytohemagglutinin (PHA) in vitro. The objective of the current study is to ascertain what the combined effects of SCD sera plus penicillin have on normal lymphocyte cytokine production and mitogenic response to PHA. Steady state sera from 20 SCD patients not on penicillin prophylaxis and 20 comparable healthy controls were used in all experiments. Four normal healthy individuals were used as donors for obtaining peripheral blood mononuclear cells (PBMC), by density gradient. PBMC with or without penicillin were PHA stimulated by standard in vitro culture for mitogenic response and cytokine production. Supernatant cytokine levels for interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)2 were quantified by ELISA technique. Results revealed suppression of mitogenic response in the SCD group with or without penicillin, compared to control sera (P < .001). Cytokine production in the SCD sera group showed increased production of IFN-gamma and TNF-alpha in the absence of penicillin, but suppression at all doses of penicillin. The control group results were as follows: no significant difference in IFN-gamma production with or without penicillin, mean TNF-alpha levels were the opposite of SCD sera with lower levels in the absence of penicillin. IL-2 production demonstrated a similar pattern for both groups of sera. IL-2 production was low without penicillin, but there was increased production with penicillin, which appeared dose related. The data suggests that sera from healthy SCD patients and in vitro added penicillin may have a combined suppressive effect on normal lymphocyte in vitro production of IFN-gamma and TNF-alpha. The current study results suggest that penicillin has the beneficial effect of decreasing TNF-alpha production and increasing IL-2 production when combined with SCD steady state sera. However, this in vitro benefit must be weighed against suppression of IFN-gamma production and ultimately, perhaps the long-term utility of penicillin prophylaxis in patients with SCD.     

In vitro effects of gold sodium thiomalate on IL-1, IL-2 production, IL-2 receptor expression and IL-2 responsiveness in thymocytes and peripheral blood mononuclear cells.

The aim of this study was to examine the effect in vitro of gold sodium thiomalate (GST) on interleukin 1 (IL-1), and interleukin 2 (IL-2) production and IL-2 receptor expression in thymocytes of mice and in human peripheral blood mononuclear cells (PBMC). GST increased the proliferation of thymocytes and PBMC to suboptimal doses of T cell mitogens Con A and PHA. It induced IL-1 production of the accessory adherent cells within thymocytes and PBMC, IL-1 production in the murine macrophage line P388D1 and induced the PHA reactive thymocytes to produce IL-2 and to express IL-2 receptors. The significance of these findings is discussed.     

BCG-induced suppressor T cells optimal conditions for in vitro induction and mode of action.

In vitro activation with BCG of T cells from healthy individuals vaccinated with BCG lead to the induction of suppressor cells that suppressed the proliferation of fresh T cells in response to specific antigen. Kinetics of their induction revealed that they became radioresistant by day 8 and persisted up to 18 days of the culture period. Optimal antigen and monocyte concentrations as assessed by proliferation during the induction phase also resulted in maximum suppression. The strongest suppressor activity was observed when suppressor cells were added at an early time of fresh cell activation. IL-1 production from adherent cells in response to BCG was not affected, but, IL-2 production by T-cells was considerably reduced in the presence of suppressor cells. IL-1 containing supernatants and affinity purified IL-1 exogenously added to the culture system did not affect suppression. Whereas, recombinant IL-2 partially abrogated suppression in a dose-dependent manner. Further experiments suggested that suppressor cells might have inhibited BCG induced IL-2 receptor expression on fresh T cells.     

Role of endogenous prostaglandin E2 in interleukin 1 production by peripheral blood monocytes from patients with rheumatoid arthritis

We studied the effect of endogenous prostaglandin E2 (PGE2) on interleukin 1 (IL-1) production by peripheral blood monocytes from patients with rheumatoid arthritis (RA). IL-1 production by RA monocytes was not different from that of monocytes from normal controls, when the cells were either unstimulated or stimulated with lipopolysaccharide (LPS, 20 micrograms/ml), as measured by two different bioassays (thymocyte or fibroblast proliferation assay) and enzyme-linked immunosorbent assay. However, IL-1 production by LPS-stimulated monocytes from RA patients cultured in medium containing indomethacin, an inhibitor of PGE2 synthesis, was significantly greater than that of monocytes from normal controls. In addition, the levels of PGE2 in culture supernatants of unstimulated or LPS-stimulated monocytes from RA patients were higher than in culture supernatants of monocytes from normal controls. Moreover, the increase of in vitro IL-2 production by RA T cells stimulated by phytohemagglutinin (PHA) was observed when monocytes were removed from peripheral blood mononuclear cells. These results indicated that peripheral blood monocytes from RA patients could produce IL-1 in excess in vitro, but that in vivo IL-1 production by RA monocytes and IL-2 induction by RA T cells might be negatively regulated by endogenous PGE2.     

IL-1-induced production of IL-2 and IFN-gamma in subclones of human T-cell derived leukaemia HSB.2 cells: regulation by phytohaemagglutinin-mediated (poly)phosphoinositide breakdown and cyclic AMP.

A human T-leukaemic cell line, HSB.2-C5B2, which produces high levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) when stimulated with phytohaemagglutinin (PHA) plus IL-1, was recloned to obtain spontaneous variants in IL-2 production in response to the stimuli. In these subclones, the ability of one clone to produce IL-2 correlated well with that to produce IFN-gamma. Three C5B2 subclones: clone no. 28, a high IL-2 producer, clone no. 61, an intermediate IL-2 producer, and clone no. 40, a non-producer, were selected and examined for differences in signal transduction mechanisms. Since the three subclones were shown to express about the same number of IL-1 binding sites with similar affinities, the loss of ability to produce IL-2 was not due to decreased cell-surface receptor or changes in receptor property. In support of this, IL-1 induced expression of the IL-2 receptor (Tac/p55 antigen) to the same extent on the three subclones. The levels of conventional intracellular second messengers were compared and it was revealed that loss of responsiveness was closely related to the subclones' degree of (poly)phosphoinositide (PI) turnover, protein kinase C (PKC) activation and cyclic AMP formation in response to PHA. Moreover, resting intracellular cyclic AMP concentrations were found to be increased in subclones with attenuated IL-2 production. These results indicate that the variation of IL-1-induced production of IL-2 and IFN-gamma in this T-cell line is attributed to the difference in the PHA-mediated signal transduction pathway and, presumably, to the different regulation of intracellular cyclic AMP.