IL-2、IL-15对NK细胞亚群表型和功能的调节作用

目的 探讨IL-2和IL-15对NK细胞亚群表型和功能的调节作用。方法 采用双色免疫荧光染色和流式细胞仪分析，比较IL-2和IL-15对新鲜分离外周血单个核细胞（PBMC)中NK细胞及经混合淋巴细胞培养(MLC)活化的NK细胞表型的调节作用；用4h ^51Cr释放实验检测IL-2和IL-15对NK细胞杀伤水平的影响。结果 在PBMC培养中，当IL-2或IL-15最终为50U／mL时，能明显上调PBMC中CD56^ NK细胞的比例，且IL-15对CD56^bright亚群增殖的促进作用强于IL-2；在MLC中，当IL-2或IL-15为50U／mL时，能够促进MLC活化的NK细胞比率的增加，NK细胞亚群由CD56^dim为主转变成CD56^bright为主，此诱导作用IL-2强于IL-15。IL-2和IL-15都能上调PBMC和MLC中NK细胞杀伤水平，并呈剂量依赖关系，在PBMC中，当IL-15为50U／mL时，IL-15对NK细胞杀伤水平的促进作用强于IL-2；而在MLC中当IL-2为50U／mL时，对NK细胞杀伤水平的促进作用高于IL-15。在PBMC中，同时加入50U／mL的IL-15和IL-2，NK细胞杀伤率高于单独加入IL-15或IL-2。结论 IL-15促进PBMC中NK细胞向CD56^bright亚群分化和杀伤水平强于IL-2，而IL-2促进同种异体抗原诱导的活化NK细胞向CD56^bright亚群分化和杀伤水平则高于IL-15。这种反应格局的差别，可能与不同条件下NK细胞IL-2或/和IL-15细胞因子受体表达不同以及同时有其他细胞因子的协同作用有关。     

猪苓多糖、硒及红细胞对LAK细胞杀伤活性的影响

目的　探讨猪苓多糖 (PUPS)、微量元素硒 (Se)及红细胞 (RBC)对LAK细胞杀伤活性的影响。方法　分离正常人外周血单个核细胞 (PBMC)在体外分别与不同浓度的PUPS、亚硒酸钠及红细胞单独或与IL 2协同诱导 ,以MTT法检测LAK细胞活性 ,用ELISA检测培养上清中TNF α和IFN γ含量。结果　不同浓度的PUPS和Se能单独诱导LAK细胞 ,与IL 2协同诱导的LAK细胞活性明显提高 ,细胞分泌的TNF和IFN浓度增高。RBC对LAK细胞的杀伤活性有明显地增强作用 ,并呈浓度依赖关系。结论　PUPS、Se及RBC对LAK细胞杀伤性有明显增强作用 ,细胞杀伤活性的提高可能与内源性TNF和IFN的产生增加有关。本研究为PUPS、Se及RBC与IL 2协同诱导细胞的研究和临床应用提供了理论依据。     

IL-2 receptor gene expression and IL-2 production by human preterm newborns' cells.

IL-2 receptor (IL-2R) gene expression in human umbilical cord blood mononuclear cells (CBMC) of preterm and term newborns was examined following stimulation for 18 h with phytohaemagglutinin (PHA) and compared with that of adult peripheral blood mononuclear cells (PBMC; mothers and control group). mRNA for IL-2R could not be detected in CBMC of preterm infants, whereas the mRNA levels for IL-2R found in full term neonates were similar to those observed in PBMC of adults. IL-2 activity in conditioned medium (CM) of mononuclear cells stimulated with either optimal or suboptimal PHA concentrations for 24 h and 48 h was also determined. At 24 h of stimulation, IL-2 activity found in CM obtained from CBMC of preterm and term newborns was significantly higher than that found in CM of adults' PBMC. A further enhancement of IL-2 activity (six to eight times) was observed in CM of preterm and term cells stimulated for 48 h, whereas no significant difference was found in IL-2 activity in CM from adult cells tested at the two incubation periods. The present findings may provide an additional explanation for the impaired function of the immune system, and the high susceptibility to infections observed in preterm newborns.     

硒对LAK细胞活性的影响及其机理研究

研究了硒在体外对LAK细胞活性的影响及作用机理,结果证明在LAK细胞的诱导阶段加入10~5～10mol/L亚硒酸纳能增强LAK细胞的杀伤和增殖活性。采用流式细胞仪测Tac(IL—2Rα)表达,Slot-Blot检测硒对LAK细胞的IL—2RαmRNA水平的影响,结果表明硒能增强LAK细胞的Tac抗原表达和IL-2RαmRNA的水平,提示硒可能通过促进Tac的表达增强了LAK细胞对IL—2的敏感性,从而提高了LAK细胞的增殖及杀伤活性。因此硒可望作为一种新型的免疫调节剂用于抗肿瘤治疗。     

IL-1 beta and IL-3-like activity in preterm infants.

The capacity of peripheral blood mononuclear cells (PBMC) of preterm neonates to release IL-1 beta and IL-3-like activity (IL-3-LA) has been investigated. In the present study it was found that this capacity is significantly lower than that of their mothers and of control adults. In addition, the results showed that preterm serum has a lower stimulatory effect on IL-1 beta production and an inhibitory effect on IL-3-LA secretion by PBMC of adult controls, in comparison with maternal and adult sera. These findings suggest an additional feedback mechanism for control of haematopoiesis in premature neonates. It is possible that the lower production of IL-1 beta and IL-3-LA may be involved in the increased susceptibility to infections of preterm newborns.     

Lymphokine-activated killer (LAK) cells modulate the effects of IL-2 on a T cell-mediated immune response.

The ability of LAK cells and/or IL-2 to affect the course of an established T cell response was examined in a delayed-type hypersensitivity (DTH) model. IL-2 greatly increased the magnitude of the response at 24 h, while LAK cells alone had no effect. The administration of LAK cells and IL-2 together also had no effect on the magnitude of the DTH response, demonstrating that LAK cells were able to remove the enhancement seen with IL-2 alone. The presence of LAK cells reduced the serum half-life of IL-2 significantly, but not to an extent able to account for the observed loss of IL-2 induced DTH enhancement. IL-2 administration influenced cell phenotypes in the spleen and draining lymph nodes (DLN), as well as increasing splenic weight; the additional presence of LAK cells markedly altered these effects of IL-2 in the spleen (but not the DLN). Taken together, these results suggest that LAK cells interact with activated T-cells within the immune system and modulate their function.     

IL-12对慢性乙型肝炎患者T_(H1)/T_(H2)分化的影响

目的观察 IL- 12对慢性乙肝患者 TH1 / TH2 类细胞分化的影响。方法分离 5 0例慢性乙型肝炎患者 PBMC,分别与植物血凝素 (PHA ,10 0μg/ m L )、HBc Ag(1μg/ m L )、HBe Ag(1μg/ m L )单独或联合 IL - 12 (10 ng/ m L )体外培养 48h,EL ISA法检测培养上清液中 IL - 2、IFN -γ、IL - 4、IL - 10水平。 2 0例健康人群做对照。结果 IL - 12对健康人群 PBMC产生 TH1 /TH2 类细胞因子无显著影响 ,但对慢性乙型肝炎患者则显著增强 PBMC产生 IFN-γ,且慢性中度患者最为突出。 IL - 12与HBe Ag联合诱导 ,不但显著增强慢性乙型肝炎患者 PBMC产生 IL- 2和 IFN- γ,还抑制 IL- 4和 IL- 10的产生。结论 IL- 12可增强慢性乙型肝炎患者 IFN-γ优势表达 ,可促进 HBe Ag诱导的 TH2 型优势表达向 TH1 型优势表达转换     

Natural killer cells in cross-regulation of IL-12 by IL-10 in Leishmania antigen-stimulated blood donor cells.

We have previously shown that natural killer (NK) cells play a role in protection against leishmaniasis. Furthermore, we have shown that NK cells in mononuclear cells derived from unexposed donors are induced to proliferate in vitro in response to leishmanial antigens. Since interleukin (IL)-12, a strong inducer of NK cells, acts on the early events in NK cells and T-cells, and is considered as an adjuvant for use in a potential antileishmaniasis antigen, we wished to investigate how this cytokine influences the in vitro Leishmania induced proliferative and cytokine response in healthy donors. We demonstrate that in an innate response to Leishmania antigen involving NK cells, a critical level of IL-12 is required to induce interferon (IFN)-gamma secretion below which, IL-10 is released in amounts which apparently inhibit IFN-gamma secretion and cellular proliferation. However, at higher IL-12 levels, there is simultaneous secretion of IFN-gamma and IL-10 as well as proliferation of cells. In a similar vein, exogenous IL-10 in turn inhibited IFN-gamma secretion as well as proliferation when used at low/medium concentrations, but at high concentrations this effect was abolished and replaced by the simultaneous detection of IFN-gamma, IL-10 and proliferation. The contribution of NK cells in cross regulation of these two very important immuneregulatory cytokines and the effect of exogenous IL-12 in a Leishmania driven response are discussed.     

抵抗LAK细胞杀伤的胃癌细胞株的建立及生物学特性的研究

人胃癌细胞系ＭＧＣ－８０３经反复与ＬＡＫ细胞共培养十个周期，获得对ＬＡＫ细胞杀伤抵抗的特性，命名该细胞变株为ＭＧＣ－８０３Ｒ。当效靶比为４０∶１时，对该细胞变株的杀伤率仅为４０％，而对亲代细胞的杀伤率为９０％。用“冷”靶细胞抑制试验、电镜、ＦＡＣＳ及荧光标记技术分析，证实ＭＧＣ－８０３Ｒ细胞膜结合识别位点有改变，ＭＨＣ－Ⅰ表达升高，ＩＣＡＭ－１表达降低，相互粘附的细胞数减少，胞内Ｆ肌动蛋白小体消失，微丝恢复有序排列，细胞生长速率减慢。上述结果提示经免疫筛选对杀伤有抵抗的ＭＧＣ－８０３Ｒ细胞在生物学特性上有所改变。     

IL-15 promotes regulatory T cell function and protects against diabetes development in NK-depleted NOD mice

IL-15, an anti-apoptotic cytokine, has been reported to promote the survival and function of NK cells and T cells, including regulatory T cells (Tregs). Here we examined the effect of repeated injections of IL-15 on the development of diabetes in NOD mice. Injection of recombinant murine IL-15, once a day for 2 weeks, neither inhibited nor accelerated diabetes development in untreated NOD mice. However, treatment with IL-15 significantly reduced the incidence and delayed the onset of diabetes in NOD mice that were depleted of NK cells, while NK cell depletion alone had no protection against the disease development. The protective effect in IL-15-treated, NK cell-depleted NOD mice was associated with an increase in immunosuppressive activity of CD4⁺CD25⁺ Tregs. IL-15 also enhanced Foxp3 expression in CD4⁺CD25⁺ cells in an in vitro culture system, and such an effect of IL-15 was abrogated by IL-15-activated NK cells. Inhibition of IL-15-induced Foxp3 expression by IL-15-activated NK cells likely resulted from their IFN-γ production, as recombinant IFN-γ, or the culture supernatant of IL-15-activated wild-type mouse NK cells but not of IL-15-activated IFN-γ-deficient NK cells, mediated a similar inhibition. IFN-γ also diminished the stimulatory effect of IL-15 on Treg function in vitro. These results indicate that IL-15 has the potential to promote Treg function and protect against diabetes development in NOD mice, but such an activity can be eliminated by simultaneous activation of NK cells in IL-15-treated mice.     

IL-23与IL-12诱导NK细胞功能的异同及机制初探

目的探讨细胞因子IL-23与IL-12对NK细胞功能的影响及可能的机制。方法密度梯度离心法分离人外周血单个核细胞(PBMCs)或磁珠纯化NK细胞,不刺激或用IL-23或IL-12刺激,用流式细胞术和ELISA法检测NK细胞产生IFN-γ的情况;以K562或Jurkat细胞作为靶细胞,用流式细胞术检测NK细胞的杀伤功能并分析NK细胞在不同的刺激条件下杀伤相关分子的表达情况及pSTAT的表达情况。结果与未刺激组相比,IL-23和IL-12均可以诱导NK细胞呈剂量和时间依赖方式产生IFN-γ;但IL-12而非IL-23可以增强NK细胞对靶细胞K562或Jurkat细胞的杀伤功能。进一步研究表明,IL-12而非IL-23可以诱导杀伤相关分子TRAIL及CD107a/b的表达。此外,IL-12诱导NK细胞表达更高水平的pSTAT4,而IL-23诱导NK细胞表达更高水平的pSTAT3。结论与IL-12相比,IL-23亦可以诱导NK细胞产生细胞因子但不能增强NK细胞的杀伤功能,IL-23不能诱导杀伤相关分子TRAIL及CD107a/b的表达,IL-23可以诱导低水平的pSTAT4但高水平的pSTAT3的表达。     

IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J × BALB/c) F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-γ in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

重组人IL-12和SCF促进新生儿脐带血中CD34~+细胞的分裂增殖

目的分析IL-12及SCF对CD34+细胞分裂增殖及IL-12Rβ的表达。方法分离正常足月产新生儿脐带血与健康成年人外周血中的单个核细胞,采用流式细胞术(FACS)比较2组中CD34+细胞的频率,分析IL-12及SCF对CD34+细胞的作用。结果健康成年人外周血中CD34+细胞平均频率为0.13%,显著低于新生儿脐带血中CD34+细胞的平均频率1.53%(P<0.001)。进一步研究表明,SCF可以促进CD34+细胞表达CD25;IL-12及SCF可以显著促进CD34+细胞的分裂增殖以及IL-12Rβ1的表达。结论新生儿脐带血中CD34+细胞的频率明显高于成年人外周血中CD34+细胞的频率。IL-12及SCF可以促进CD34+细胞的分裂增殖以及IL-12Rβ1的表达。     

The power of combinatorial immunology: IL-12 and IL-12-related dimeric cytokines in infectious diseases

Appropriate induction of a Th1 immune response is required for effective antimicrobial immunity. However, dysregulated Th1 immune responses after infection may also lead to immunopathology. Thus, cell-mediated immune responses have to be tightly regulated. Upon infection, the production of interleukin (IL)-12, a heterodimeric cytokine composed of a p35 and a p40 subunit, is the dominant factor in Th1 cell development. The recent discovery of novel dimeric cytokines closely related to IL-12 add now to our understanding of cellular immunity and the fine tuning of T cell responses. At the onset of infection, IL-27, a heterodimer composed of the IL-12p40-related protein EBI-3 (Epstein-Barr virus-induced gene 3) and the IL-12p35-related protein p28 induces the expression of a functional IL-12 receptor in naive CD4⁺ T cells, making these cells sensitive to IL-12-mediated Th cell development. Later during infection, IL-23, a heterodimer composed of the IL-12p40 subunit and the IL-12p35-related molecule p19, preferentially acts on Th1 effector/memory CD4⁺ T cells. The IL-12p40 subunit can also form a homodimer, IL-12p80, which act as an IL-12 and IL-23 antagonist by competing at their receptors. This review focuses on these IL-12-related cytokines contributing to fine tuning of T cell responses after infection with intracellular pathogens.     

Modulation of 2B4 (CD244) activity and regulated SAP expression in human NK cells

The adapter protein SAP is important for the signal transduction of the family of SLAM-related receptors (SRR), which have important immune-modulating functions. The importance of SAP and SRR for a functional immune reaction becomes obvious in patients suffering from X-linked lymphoproliferative disease, which is characterized by non-functional SAP. Here we investigate the regulation of SAP expression in human NK cells. We demonstrate that SAP mRNA expression and protein levels are low in freshly isolated resting NK cells. IL-2 stimulation leads to an up-regulation of SAP expression, which can be enhanced by IL-12, the stimulation of TLR3 by polyinosinic-polycytidylic acid (poly(I:C))and to a lesser extent by IFN-alpha. EAT-2, a SAP-related adapter protein, is already detectable in resting NK cells and does not change its expression after IL-2 stimulation. The regulation of SAP has functional consequences for the stimulation of NK cell cytotoxicity by 2B4. In resting NK cells, 2B4 stimulation can only enhance NK cell lysis when co-triggered with other activating NK cell receptors. In IL-2-activated NK cells with high SAP expression the triggering of 2B4 alone is sufficient to induce NK cell cytotoxicity, demonstrating a correlation between the regulated SAP expression and the function of 2B4.     

巨细胞病毒感染时免疫功能的变化及其意义

目的　探讨巨细胞病毒感染时免疫变化及其意义。方法　以 2 0名CMV IgM(Cytomegalovirus immunoglobulinM)阳性的病儿为观察对象 ,另设对照组 30例。ELISA法检测柯萨奇B组病毒抗原 (CVB Ag)、IgM抗体 (CVB IgM)、巨细胞病毒IgM抗体 (CMV IgM)、EB病毒IgM抗体 (EBV IgM) ,单克隆抗体标记间接ABC免疫法检测外周血T细胞亚群。结果　CMV感染组的LAK细胞、NK细胞活性均明显降低 (P <0 .0 1)。合并其它感染原的CMV混合感染组CD3含量减少 ,CD8含量增加 (P <0 .0 5 ) ,CD4 CD8数值明显降低 (P <0 .0 1)。病毒混合感染并合并支原体感染组CD3含量减少 (P <0 .0 5 )、CD4 CD8数值、IgA含量明显降低 (P <0 .0 1)。结论　巨细胞病毒感染影响了T细胞亚群的平衡 ,在合并其他感染时天然免疫细胞功能下降和T细胞亚群紊乱更明显     

IL-18 enhances ULBP2 expression through the MAPK pathway in leukemia cells

Expression of UL16-binding proteins (ULBPs) has been reported in various cancers, such as leukemia and melanoma, and also in some other cancer cell lines. However, the factors that modulate the expression of ULBPs are not well defined. In this study, we investigated the effects of IL-18 on the expression of NKG2D ligands in leukemia cells. IL-18 treatment increased ULBP2 expression in leukemia cells at the mRNA and protein levels. In addition, PD98059 (an ERK1/2 MAPK inhibitor) and SP600125 (a JNK inhibitor) attenuated IL-18-induced ULBP2 expression in a dose-dependent manner. We observed that ERK1/2 and JNK MAPK phosphorylation increased upon treatment with IL-18. IL-18 elevated CD107a expression in cancer cells and increased the cytotoxic activity of NK cells; therefore, we propose that IL-18 increases the susceptibility of target cells by inducing surface expression of ULBP2. Taken together, these findings suggest that IL-18 may play a critical role in regulating ULBP2 expression via the ERK1/2 and JNK MAPK pathways in leukemia cells.     

Human Th2-like cell clones induce IL-12 production by dendritic cells and may express several cytokine profiles

Previous studies have shown that human T_h2 cells, unlike theirmurine counterparts, retain the ability to produce IFN- uponactivation in the presence of exogenous IL-12. Here we firstextended this notion by showing that T_h2-like cell clones (T_h2C)are also capable of inducing IL-12 production by physiologicalantigen-presenting cells (APC); we next showed that these cellsmay express several distinct cytokine profiles depending uponthe activation signal and the type of APC with which they interact.We have analyzed the production of IL-4, IL-5 and IFN- by T_h2Cstimulated by either anti-CD3 mAb or exogenous IL-2, using peripheralblood monocytes or dendritic cells (DC) as accessory cells.We found that: (i) DC but not monocytes released IL-12 and promotedIL-12-dependent IFN- production upon interaction with anti-CD3-or IL-2-stimulated T_h2C and (ii) ligation of CD3 was requiredfor the production of IL-4 but not of IL-5 or IFN-. Thus, dependingupon the type of APC with which they interacted and the modeof activation, T_h2C, expressed four distinct cytokine profiles:(i) IL-4 + IL-5, in response to anti-CD3 + monocytes; (ii) IL-4,IL-5 + IFN-, in response to anti-CD3 + DC; (iii) IL-5 + IFN-,in response IL-2 + DC; and (iv) IL-5 alone, in response to IL-2+ monocytes. The ability of human T_h2-like cells to induce IL-12production and to release the proinflammatory cytokines IFN-yandIL-5 upon IL-2-driven interactions with APC may contribute toexplain how local infection exacerbates Th2-mediated diseases,like bronchial asthma and atopic dermatitis.     

rhC5a诱导PBMC产生IL-8与PKC的关系

目的:研究PKC在rhC5a诱导外周血单个核细胞(PBMC)产生IL-8中的作用。方法:将PBMC分别与rhC5a、PMA、Cheleythrine、 Calphostin C、Dequalinium孵育24 h,取上清用法测定IL-8。将PBMC分别与rhC5a、PMA孵育,按一定时间间隔收集细胞测定 PKC活性。用 Cheleythrine、Calphostin C、Dequalinium预处理 PBMC,然后再以rhC5a诱导测定其 PKC活性。结果:PBMC经rhC5a诱导后IL-8产生水平增加,同时PKC活性呈双峰型增高;PBMC经PMA诱导后不仅PKC活性增高,而且IL-8产生水平也增加;Cheleythrine、Calphostin C、Dequalinium能抑制rhC5a介导的PBMC PKC活性和IL-8产生水平增高。结论:PKC参与rhC5a诱导PBMC产生IL-8细胞内信号转导过程。     

Enhancing effect of natural killer cell stimulatory factor (NKSF/interleukin-12) on cell-mediated cytotoxicity against tumor-derived and virus-infected cells

Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL-2, NKSF/IL-12 enhances NK cell-mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)-α, IFN-β IFN-γ, IL-2 or tumor necrosis factor (TNF)-α and, unlike the induction of IFN-γ production by peripheral blood lymphocytes, it does not require HLA class II-positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor-derived target cells, NKSF/IL-12 is also a potent stimulator of cytotoxicity against virus-infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus-1. NK cell-mediated antibody-dependent cytotoxicity or anti-CD16 antibody-redirected lysis is not significantly enhanced by NKSF/IL-12. However, the ability of resting peripheral blood T cells to mediate anti-CD3 antibody-redirected lysis is enhanced by 18-h incubation with NKSF/IL-12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.