Membrane Remodeling in Viral Infection, Parasite Invasion, Apoptosis, and Cancer

病毒感染、寄生虫入侵、细胞凋亡和癌症中的膜重塑

• 批准号: 7968586
• 负责人: JOSHUA ZIMMERBERG
• 金额: $ 127.09万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968586
• 关键词: Activated Lymphocyte; Adhesions; Adult; Antibodies; Antigens; Apoptosis; Apoptosis Regulator; Apoptotic; Area; Astronauts; Biochemical; Bioreactors; Bone Marrow; CD95 Antigens; Cell Adhesion; Cell Death; Cell Line; Cell membrane; Cell physiology; Cell-Matrix Junction; Cell-Mediated Cytolysis; Cells; Cellular Membrane; Charge; Chemicals; Chimeric Proteins; Cholesterol; Cyclodextrins; Cytolysis; Data; Density Gradient Centrifugation; Dependence; Ensure; Escherichia coli; Event; Excision; Eye; Failure; Family; Fibroblasts; Force of Gravity; Functional disorder; Glass; Goals; Hemagglutinin; Heterogeneity; Human; Ice; Immune; Immune Cell Activation; Immune System Diseases; Immune response; Immune system; Immunosuppressive Agents; In Vitro; Infection; Integral Membrane Protein; International; Jurkat Cells; Kinetics; Lipids; Lymphocyte; Lymphoid Cell; Lymphoid Tissue; Malignant Neoplasms; Mechanics; Mediating; Membrane; Metabolic; Methods; Microgravity; Microscopic; Microscopy; Modeling; Molecular; Natural regeneration; Neurons; Organ; Pathologic Processes; Pathway interactions; Physiological; Placenta; Polylysine; Preparation; Process; Property; Relative (related person); Reporting; Research; Role; Rotation; Side; Simulate; Site; Solid; Sonication; Space Flight; Stem cells; Stromal Cells; Surface; Suspension Culture; Suspension substance; Suspensions; System; T-Lymphocyte; Techniques; Temperature; Testing; Time; Tissues; Tumor Necrosis Factor Ligand Superfamily Member 6; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Up-Regulation; Virus Diseases; Water; Work; apoptosis in lymphocytes; base; cancer cell; caspase-8; cytokine; cytotoxic; cytotoxicity; cytotrophoblast; density; design; in vivo; injured; interest; killings; neoplastic cell; parasite invasion; polyacrylamide; prevent; protein function; reproductive; research study; response; stem; tumor; tumorigenesis

1. An adhesion-based method for plasma membrane isolation: Evaluating cholesterol extraction from cells and their membranes. Techniques currently used for the isolation of plasmamembranes include zonal or density gradient centrifugation, the ripflip method developed for the microscopic observation of small pieces of the cytoplasmic side of plasma membranes, and methods based on the adhesion of negatively charged cells to a positively charged surface such as polylysine-coated polyacrylamide or glass beads. A method to isolate large quantities of directly accessible cytoplasmic surface of the plasma membranes suitable for both microscopy and biochemical analysis was developed. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water. Optimal conditions were established for all preparation steps, including polylysine coating, cell adhesion, and membrane washing, ensuring both high purity and high yield of the membrane preparation. This method allows (i) the creation of isolated plasma membranes without chemical (high-salt) or mechanical (vortexing or sonication) treatments, (ii) direct lipid extraction on glass plates, and (iii) the ability to study both biochemical and structural properties of isolated plasma membranes. Methyl-b-cyclodextrin (MbCD)1 is used to alter the cholesterol content of cells and, in particular, the cholesterol content of the plasma membrane. The cholesterol content of cellular membranes and the relationships between cholesterol-enriched domains and physiological function is an active area of research. Although questions remain unanswered with regard to detailed kinetics, dependence on composition and temperature, and specific and nonspecific effects, exposure of cells to MbCD reduces cellular cholesterol. However, reduction in cellular cholesterol following MbCD treatment might not quantitatively reflect the changes in plasma membrane cholesterol content. Having developed a method to isolate plasma membranes in quantities suitable for biochemical examination, MbCD cholesterol depletion evaluated using intact cells and their plasma membranes were compared. MbCD treatment extracted cholesterol from the plasma membrane of HAB2 cells, a hemagglutinin-expressing fibroblast cell line, and intact HAB2 cells in a temperature-dependent way, and the reduction in plasma membrane cholesterol content was not proportional to the decrease observed using intact cells. Nearly complete removal of plasma membrane cholesterol can be achieved by extraction at physiological temperature (37 _C). At 4 _C, MbCD extraction of cholesterol from the plasma membrane is less. These data indicate that one cannot predict the loss of cholesterol from the plasma membrane based on the loss determined from intact cells. Treatment with 10 mM MbCD for 30 min at 37 _C did not deplete cholesterol from all membrane fractions but essentially depleted all of the cholesterol in the plasma membrane. The roles of cholesterol in membrane heterogeneity/domains (rafts vs. nonrafts) and the association and function of proteins to specialized domains are of considerable interest. MbCD is often used to selectively deplete cholesterol from low- and high-density membrane fractions. Not only are MbCD concentration and exposure time important parameters in perturbing the cholesterol content of the membrane, but also extraction temperature is critical. Having a method to isolate and evaluate biochemical quantities of pure plasma membrane will benefit those studies where cholesterol perturbation needs to be minimized. The combined effects of MbCD concentration, exposure time, and temperature extraction now can be evaluated easily. 2. Cytotoxicity Mediated by the Fas Ligand (FasL)-activated Apoptotic Pathway in Stem Cells Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. We tested the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(/) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues. We hypothesize that BMSC-mediated cytotoxicity of lymphocytes involves the FasL-activated apoptotic machinery. FasL is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) family. FasL interacts with its receptor, Fas (CD95/APO-1) and triggers a cascade of subcellular events culminating in apoptotic cell death. FasL and Fas are key regulators of apoptosis in the immune system. In addition, FasL is expressed by cells in immune-privileged sites, such as cancer cells, neurons, eyes, cytotrophoblasts of the placenta, and reproductive organs. In neurons, FasL expression specifically protects against T cell-mediated cytotoxicity. The discovery that FasL is also expressed by a variety of tumor cells raises the possibility that FasL may mediate immune privilege in human tumors. Activated T cells expressing Fas are sensitive to Fas-mediated apoptosis. Thus, up-regulation of FasL expression by tumor cells may enable tumorigenesis by targeting apoptosis in infiltrating lymphocytes. In the present work, we show that BMSCs can mediate immunosuppressive activity by FasL-induced killing of activated lymphocytes. Thus, BMSCs have properties of immuneprivileged cells. 3. Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station. The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

1. 一种基于粘附的质膜分离方法：评估从细胞及其膜中提取胆固醇的情况。 目前用于分离质膜的技术包括区带或密度梯度离心、ripflip 为显微观察质膜细胞质侧小片而开发的方法，以及方法 基于带负电的细胞粘附到带正电的表面，例如聚赖氨酸涂层的聚丙烯酰胺或玻璃珠。开发了一种分离大量可直接接触的质膜细胞质表面的方法，适用于显微镜和生化分析。该方法基于细胞粘附到玻璃板上的聚赖氨酸吸附层，然后用冰冷的蒸馏水进行低渗裂解。为所有制备步骤建立了最佳条件，包括聚赖氨酸包被、细胞粘附和膜洗涤，确保膜制备的高纯度和高产率。该方法允许（i）无需化学（高盐）或机械（涡旋或超声处理）处理即可创建分离的质膜，（ii）在玻璃板上直接提取脂质，以及（iii）能够研究分离的质膜的生化和结构特性。甲基-b-环糊精 (MbCD)1 用于改变细胞的胆固醇含量，特别是质膜的胆固醇含量。细胞膜的胆固醇含量以及富含胆固醇的结构域与生理功能之间的关系是一个活跃的研究领域。尽管有关详细动力学、对成分和温度的依赖性以及特异性和非特异性效应的问题仍未得到解答，但将细胞暴露于 MbCD 会降低细胞胆固醇。然而，MbCD 治疗后细胞胆固醇的减少可能无法定量反映质膜胆固醇含量的变化。开发了一种分离质膜的方法，其数量适合生化检查，并使用完整细胞评估 MbCD 胆固醇消耗，并对其质膜进行了比较。 MbCD处理以温度依赖性方式从HAB2细胞、表达血凝素的成纤维细胞系和完整HAB2细胞的质膜中提取胆固醇，并且质膜胆固醇含量的降低与使用完整细胞观察到的降低不成比例。通过在生理温度（37℃）下提取可以实现质膜胆固醇的几乎完全去除。在 4 ℃ 时，MbCD 从质膜中提取的胆固醇较少。这些数据表明，人们无法根据完整细胞测定的胆固醇损失来预测质膜中胆固醇的损失。 在 37℃下用 10 mM MbCD 处理 30 分钟并没有耗尽所有膜组分中的胆固醇，但基本上耗尽了质膜中的所有胆固醇。胆固醇在膜异质性/结构域（筏与非筏）中的作用以及蛋白质与特殊结构域的关联和功能引起了人们的极大兴趣。 MbCD 通常用于选择性地去除低密度和高密度膜组分中的胆固醇。 不仅是 MbCD 浓度 和暴露时间是干扰膜胆固醇含量的重要参数，也是提取温度 很关键。拥有一种分离和评估纯质膜生化量的方法将有利于这些研究 需要最大限度地减少胆固醇扰动。现在可以轻松评估 MbCD 浓度、暴露时间和提取温度的综合影响。 2. Fas 配体 (FasL) 激活干细胞凋亡途径介导的细胞毒性 然而现在已经清楚，人骨髓基质细胞 (BMSC) 可以具有免疫抑制作用并逃避细胞毒性 淋巴细胞（CTL）在体外和体内的实验中，这种现象的机制仍然存在争议。我们测试了假设 BMSCs 通过 Fas 介导的活化淋巴细胞凋亡来抑制免疫反应，并发现 Fas 和 FasL 原代 BMSC 的表达。共孵育 72 小时后，Jurkat 细胞或活化的淋巴细胞均被 BMSC 杀死。在 相比之下，BMSCs对非活化淋巴细胞和caspase-8(/) Jurkat细胞的细胞毒作用非常强 低的。 Fas/Fc融合蛋白强烈抑制BMSC诱导的淋巴细胞凋亡。尽管我们检测到高水平的 Fas 尽管在 BMSC 中表达，但用抗 Fas 抗体刺激 Fas 并没有导致预期的 BMSC 凋亡，无论 浓度，表明 Fas 激活途径被破坏。因此，BMSCs 可能具有内源性机制 逃避 Fas 介导的细胞凋亡。总的来说，这些数据提供了成体干细胞/祖细胞和癌细胞之间的相似之处， 与干/祖细胞可以利用FasL通过诱导淋巴细胞凋亡来防止淋巴细胞攻击的观点一致 在受伤组织的再生过程中。我们假设 BMSC 介导的淋巴细胞细胞毒性涉及 FasL 激活的凋亡机制。 FasL 是一种 II 型跨膜蛋白，属于肿瘤坏死因子 (TNF) 家族。 FasL 与其受体 Fas (CD95/APO-1) 相互作用，引发一系列亚细胞事件，最终导致细胞凋亡 死亡。 FasL 和 Fas 是免疫系统细胞凋亡的关键调节因子。此外，FasL 在以下细胞中表达： 免疫豁免部位，如癌细胞、神经元、眼睛、胎盘的细胞滋养层和生殖器官。在神经元中，FasL 表达特异性地防止 T 细胞介导的细胞毒性。 FasL 也由多种肿瘤细胞表达的发现提出了 FasL 可能介导的可能性 人类肿瘤的免疫特权。表达 Fas 的活化 T 细胞对 Fas 介导的细胞凋亡敏感。因此， 肿瘤细胞上调 FasL 表达可能通过靶向浸润淋巴细胞的凋亡来促进肿瘤发生。 在目前的工作中，我们证明 BMSCs 可以通过 FasL 诱导杀死活化的细胞来介导免疫抑制活性。 淋巴细胞。因此，BMSC 具有免疫豁免细胞的特性。 3.旋转悬浮培养物和国际空间站上人体淋巴组织和细胞的免疫抑制。 研究了人淋巴组织外植体或从该组织分离的细胞的免疫反应 在正常重力和微重力下定量。微重力可以通过固体悬浮来模拟 在旋转的充氧培养容器中或实际上是在国际空间站（ISS）上实现的。我们的 实验表明，模型中的组织或细胞受到回忆抗原或多克隆激活剂的攻击 微重力失去了产生抗体和细胞因子以及增加代谢活动的所有能力。相比之下， 如果细胞在暴露于模拟微重力悬浮培养物之前受到挑战，它们会保持其 回应。同样，在国际空间站的微重力下，淋巴细胞对抗原或多克隆攻击没有反应， 而在太空飞行之前受到挑战的细胞在太空中仍保持其抗体和细胞因子反应。因此， 淋巴组织细胞的免疫激活在模拟微重力和真实微重力下都严重减弱。这表明 通过固体旋转的悬浮培养足以诱导真实细胞生理学的变化 微重力。这种现象可能反映了宇航员在太空飞行期间观察到的免疫功能障碍。如果 因此，上述离体系统可用于了解这种功能障碍的细胞和分子机制。