钙网蛋白与肿瘤免疫

钙网蛋白是内质网分子伴侣,负责糖蛋白的折叠及维持细胞内Ca2＋平衡。新近研究发现,某些凋亡刺激能诱导肿瘤细胞内的钙网蛋白快速转位到肿瘤细胞膜上,这种表面包被有大量钙网蛋白的肿瘤细胞,能被抗原呈递细胞有效识别和吞噬,由此激发抗同种肿瘤的特异性免疫杀伤效应,提示钙网蛋白在肿瘤免疫治疗中的潜在应用价值。对钙网蛋白在肿瘤免疫中的研究进展作一综述。     

细胞器与细胞凋亡

细胞凋亡是由基因控制的有序生理过程,细胞内各组分在这一过程中相互协调,组成了精细的调控系统。除细胞核外,线粒体是近年发现与凋亡密切相关的细胞器,它经多种因子诱发可以释放细胞色素c等因子参与到凋亡途径中。进一步的研究发现,在一定条件下,内质网、溶酶体等也与凋亡活动有关。这些细胞器在细胞凋亡中的作用及其机制是目前的研究热点。     

胞外ATP在男性生殖道中的作用

胞外ATP除了能广泛作为神经递质外,还被认为是一种旁分泌或自分泌因子。ATP从男性生殖道中的精子或上皮细胞中释放,在调节各种生殖生理功能中起多种作用。本文综述了ATP调节附睾上皮细胞阴离子分泌的信号通路,阐述了ATP对依赖上皮细胞的输精管平滑肌收缩的调节机制,讨论了ATP在男性生殖道中的功能和作用。     

线粒体与细胞凋亡调控

细胞凋亡是一个受到一系列相关基因严格调控的细胞死亡过程。线粒体是细胞凋亡调控的活动中心。在凋亡因子的刺激下,线粒体释放出不同促凋亡因子如细胞色素C、Smac／Diablo等,激活细胞内凋亡蛋白酶Caspase。我们发现,活化后的Caspase可以反过来作用于线粒体,引发更大量线粒体细胞色素c的释放,构成细胞色素c释放的正反馈调节机制,从而导致电子传递链的中断、膜电势的丧失、胞内ROS的升高以及线粒体产生ATP功能的完全丧失。Bcl-2家族蛋白在细胞色素C释放和细胞凋亡调控中起关键作用。     

与CD95相关的细胞凋亡和免疫调节

细胞凋亡是一个十分复杂的过程,就目前研究结果来看,CD95系统的调控起到了很重要的作用。它与免疫杀伤、肿瘤免疫和自身免疫疾病密切相关。CD95系统不仅能够维持免疫系统的自身稳定,同时也能发挥免疫反应的作用。在特异性的细胞毒效应作用中,CD95通路是细胞毒性T淋巴细胞（CTL）杀伤靶细胞的一种方式;CD95通路在肿瘤中的失效使之逃避免疫监视和削弱免疫反应;活化的T细胞和B细胞增殖后所出现的细胞凋亡,主要是通过CD95／CD95L诱导的凋亡途径产生。一旦CD95－CD95L体系功能紊乱,就会造成严重的自身免疫疾病。     

DEK蛋白与细胞凋亡的研究进展

DEK蛋白是一种广泛存在于细胞核内的可磷酸化的核蛋白.研究认为DEK蛋白与DNA的特异性结合能够改变与其结合的DNA的拓扑结构,进而影响基因的活性.细胞应激反应与DEK蛋白有潜在的关系,在死亡受体介导的细胞凋亡过程中,DEK蛋白的磷酸化状态发生了变化.DEK蛋白的磷酸化、多聚腺苷化均能促使DEK蛋白从染色质上释放下来并最终到达细胞外成为导致某些自身免疫性疾病的抗原.近来研究显示,DEK蛋白与细胞凋亡密切相关,DEK蛋白的过表达对细胞凋亡的作用具有双重性,即促进或抑制细胞凋亡.     

胞外ATP在呼吸道中的作用

ATP不但是各种细胞的能量来源,而且更是一种自分泌或旁分泌的胞外信使,参与细胞一系列的生物学效应。ATP从呼吸道上皮细胞中释放,在调节呼吸道表面液体量的平衡、黏膜纤毛清除能力和呼吸道防御功能方面起重要作用,并参与呼吸道疾病及炎症的发生。本文对ATP从呼吸道上皮释放的途径,ATP调节呼吸道上皮离子转运的机制,ATP对呼吸道平滑肌的双重调节作用,以及ATP参与呼吸道疾病和炎症的发生机制等方面予以综述。     

外泌体分泌在1,4-苯醌诱导的HL-60细胞凋亡中起保护作用

慢性苯暴露损害造血系统,可引起再生障碍性贫血,甚至白血病。外泌体(exosomes)是细胞分泌的纳米级膜泡,在许多生理和病理过程中发挥重要作用。然而,苯及其代谢产物对外泌体分泌的影响仍不清楚。本研究旨在观察苯的活性代谢产物1,4-苯醌(1,4-benzoquinone,1,4-BQ)能否引起人早幼粒白血病细胞HL-60外泌体分泌量的变化以及外泌体释放在1,4-BQ诱导的细胞凋亡中的作用。应用不同浓度1,4-BQ处理细胞24 h,超高速离心法提取细胞培养基中的外泌体,结果发现1,4-BQ能促进外泌体分泌,呈剂量反应关系。进一步应用外泌体抑制剂GW4869抑制外泌体分泌,流式细胞仪检测细胞凋亡率、蛋白质免疫印迹法检测抑凋亡蛋白质Bcl-2、凋亡通路关键蛋白质cleaved caspase-9和cleaved caspase-3的表达,探讨外泌体分泌对1,4-BQ所致细胞凋亡的影响。结果显示:与对照组相比,1,4-BQ单独处理组的凋亡率、Bcl-2、cleaved caspase-9和cleaved caspase-3的表达均显著增高(P<0.05),而1,4-BQ+GW4869组的凋亡率及凋亡相关蛋白质的表达均显著高于1,4-BQ单独处理组(P<0.05),表明抑制外泌体分泌可增加1,4-BQ诱导的细胞凋亡。综上表明,1,4-BQ能促进外泌体分泌,并在1,4-BQ诱导的细胞凋亡中起保护作用。本研究为了解苯的毒性效应和毒性机制提供了新的实验证据。     

ATP 调控大鼠胰腺β细胞胰岛素分泌的双重作用

实验以大鼠胰腺β细胞为研究对象,采用荧光测钙和全细胞膜片钳膜电容测量技术,研究 ATP 对胞内钙离子信号和细胞分泌的影响,并初步探讨了其作用机制 . 实验表明：胞外 ATP 刺激通过动员细胞内 thapsigargin 敏感的钙库 Ca2+ 释放,使大鼠胰腺β细胞内的游离钙离子浓度显著升高,细胞外的 ATP 信号对β细胞胰岛素分泌有双向调节作用,其一,主要通过降低去极化引起的钙电流而对β细胞胰岛素分泌产生较弱的抑制作用,其二,细胞在静息状态下, ATP 通过动员胞内钙库的 Ca2+ 释放使胞浆中的钙离子浓度显著增加,触发β细胞强烈分泌胰岛素 . ATP 的这种双向调节可能对胰岛素分泌的精确调控具有重要的生理意义 .     

微生物细胞ATP释放条件研究

在ATP生物发光法微生物细胞ATP释放剂的筛选研究中,发现采用环糊精等环状化合物可以解除表面活性剂类细胞ATP释放剂对发光反应系统的抑制作用,其中,7．5g／L环糊精CD（a）能中和1．5g／L的表面活性剂Ec和Es、Et,可以完全排除Ec、Es、Et对发光反应的抑制作用。通过比较各种细胞ATP释放剂释放ATP效果,筛选、组合出以表面活性剂Ec为代表的微生物细胞ATP释放剂;通过优化实验,发现用0．25～0．50g／L的Ec处理菌液1～2min时,细胞ATP的释放效果最好,从而建立了室温条件下简便、快速的微生物细胞ATP释放方法和ATP释放剂对发光反应系统抑制的消除方法。     

Chemotherapy induces ATP release from tumor cells

Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies.     

Na+/H+ exchange in Ehrlich ascites tumor cells. Regulation by extracellular ATP and 12-O-tetradecanoylphorbol 13-acetate

The effects of extracellular ATP and/or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the intracellular pH of Ehrlich ascites tumor cells were measured using both distribution of [14C]5,5-dimethyloxazolidine-2,4-dione, and the fluorescent indicator 5(6)-carboxyfluorescein. Micromolar concentrations of extracellular ATP induce a biphasic change in the intracellular pH characterized by a rapid acidification of 0.04 pH units followed by an alkalinization of 0.11 pH units. Concurrently with the alkalinization, an increase in the total cellular [Na+] from 37.5 to 45.0 mM is observed. The pH change is half-maximally activated by 0.5-2.5 microM extracellular ATP. The intracellular alkalinization, but not the initial acidification, phase requires extracellular Na+, with half-maximal alkalinization in the presence of 24-32 mM Na+, and is inhibited by amiloride. Exposure of Ehrlich ascites tumor cells to TPA alone produces a slight alkalinization of approximately 0.04 pH units. Conversely, preincubation of the cells with TPA partially inhibits the ATP-induced changes in intracellular pH. Under identical conditions TPA also inhibits the ATP-induced increase in the cytosolic [Ca2+]. The half-maximal dose for both effects is produced by 3-10 nM TPA. These data indicate that extracellular ATP triggers the activation of Na+/H+ exchange. Furthermore, activation of protein kinase C mediates at least part of the Na+/H+ exchange, although a second mechanism may also exist.     

Extracellular ATP activates polyphosphoinositide breakdown and Ca2+ mobilization in Ehrlich ascites tumor cells

The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ homeostasis were studied in Ehrlich ascites tumor cells. Cytosolic [Ca2+] was measured using either quin 2 or the recently described indicator fura 2. Addition of 0.5-25 microM extracellular ATP to intact cells results in a rapid mobilization of Ca2+ from a nonmitochondrial, intracellular Ca2+ store. Likewise, direct addition of 0.2-2 microM myo-1,4,5-inositol trisphosphate (IP3) to digitonin-permeabilized Ehrlich cells induces a rapid and reversible release of Ca2+ from a nonmitochondrial pool. Under the same conditions which facilitate intracellular Ca2+ mobilization, extracellular ATP also triggers a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and accumulation of IP3. A maximal 18% decrease of the polyphosphoinositide is observed 40-60 s after the addition of 25 microM ATP; within 5 min PtdIns(4,5)P2 returns to or exceeds the original, prestimulus level. These conditions also trigger a rapid accumulation of phosphatidic acid (1.7-fold increase within 5 min). Paralleling these ATP-induced changes in phospholipid levels is a substantial accumulation of the mono-, bis-, and trisphosphate derivatives of inositol; most significantly, a 2-fold increase in the IP3 level is observed within 30 s after ATP addition. These results suggest that in these tumor cells, extracellular ATP elicits changes in phosphoinositide metabolism similar to those produced by a wide variety of Ca2+-mobilizing hormones and growth factors.     

Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites

Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1β (IL-1β), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.     

Superoxide responses of immune complex-stimulated rat alveolar macrophages. Intracellular calcium and priming

ATP is known to induce calcium transients in rat and human neutrophils and to "prime" these cells for enhanced oxygen radical responses after stimulation with chemotactic peptide, FMLP, or immune complexes. Calcium ionophores are also well known for their ability to prime phagocytic cells. In the current studies, nonelicited rat alveolar macrophages were analyzed for the ability of ATP as well as FMLP, C5a, platelet-activating factor and calcium ionophore (A23187) to modify levels of intracellular calcium and to enhance superoxide anion (O2-) production in response to immune complexes. Although none of these agents induced a O2- response under the conditions employed, all, except FMLP and C5a (human, recombinant) increased intracellular calcium, although the temporal features of the increases varied with the agent. In contrast to the inability of FMLP and C5a to cause intracellular calcium increases in macrophages, these same peptides caused dose-dependent intracellular calcium increases in rat neutrophils, whether the cells were derived from the blood or from the peritoneal cavity. On the basis of the effects of EGTA, the calcium increases in alveolar macrophages were caused by intracellular release of calcium in addition to some influx of extracellular calcium. Although ATP caused a dose-related increase in the level of intracellular calcium in alveolar macrophages, the cells were not "primed" for enhanced O2- responses to immune complexes. In contrast, platelet-activating factor and A23187, each of which induced increased intracellular levels of calcium, were able to prime macrophages for enhanced O2- responses. C5a and FMLP neither increased intracellular calcium levels nor primed macrophages for enhanced O2- responses to immune complexes. It is not clear if the inability of ATP to prime alveolar macrophages is caused entirely by insufficient increases in intracellular calcium or if ATP is unable to bring about additional changes that are relevant to the priming phenomenon.     

Extracellular NAD and ATP: Partners in immune cell modulation

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as ‘danger signals–to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response.     

Autophagy-dependent ATP release from dying cells via lysosomal exocytosis

ATP not only represents a key molecule for the intracellular storage and utilization of energy, but also plays a central role in the extracellular milieu, as it both recruits and activates immune cells. The secretion of ATP in the course of immunogenic cell death (ICD), a peculiar type of apoptosis that elicits adaptive immune responses, critically relies on the molecular machinery for autophagy. We have recently demonstrated that autophagy allows for the ICD-associated secretion of ATP as it contributes to the maintenance of lysosomal ATP stores. In addition, we found that ATP release in this setting is mediated by a molecular mechanism that involves lysosomal-associated membrane protein 1 (LAMP1)- and pannexin 1 (PANX1)-dependent lysosomal exocytosis.     

Modulation of P2Z/P2X(7) receptor activity in macrophages infected with Chlamydia psittaci

Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.     

Bacteriocin inhibition of two glucose transport systems in Listeria monocytogenes

Listeria monocytogenes transports glucose by proton motive force-mediated and phosphoenolpyruvate-dependent phosphotransferase systems (PEP-dependent PTS). Inhibition of both systems by nisin, pediocin JD and leuconosin S is reported here for four strains of L. monocytogenes . Intracellular and extracellular adenosine triphosphate (ATP) and extracellular inorganic phosphate were measured in energized L. monocytogenes Scott A cells to determine whether inhibition of the PEP-dependent PTS might occur as a result of bacteriocin-induced leakage of intracellular components. Addition of nisin resulted in a decrease in intracellular ATP with an increase in extracellular ATP. Leuconosin S and pediocin JD induced a depletion of intracellular ATP. ATP efflux was low for the leuconosin S-treated cells and barely detectable for pediocin JD-treated cells. Addition of nisin, leuconosin S and pediocin JD induced efflux of inorganic phosphate. It appears that bacteriocin-mediated inhibition of the glucose PEP-dependent PTS occurs as a result of hydrolysis or efflux of ATP, PEP and other essential molecules from L. monocytogenes cells.