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Exploring the mechanisms of membrane blebbing and their relationship to interleukin-1beta release in murine macrophages.

机译：探索膜起泡的机制及其与鼠巨噬细胞中白介素-1β释放的关系。

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摘要

The proinflammatory cytokine interleukin-1beta (IL-1beta) plays an important role in the innate immune system, inducing fever and activation of the vascular endothelium. However, the mechanism by which IL-1beta is secreted remains a mystery given that it is expressed in the cytosol and therefore not targeted through the endoplasmic reticulum/Golgi secretory pathways. Furthermore, IL-1beta exists as an inactive, 33 kDa procytokine which must be cleaved to its secreted, biologically active 17 kDa form by caspase-1, itself a complexly regulated zymogen. Given that ATP activation of the P2X7R on LPS-primed macrophages can facilitate secretion of IL-1beta, while simultaneously mediating membrane bleb formation, we hypothesized that membrane blebbing plays an important role in IL-1beta secretion. To test this hypothesis, we used time-lapse videomicroscopy to assess the response of macrophages to various, bleb-inducing stimuli coupled with biochemical analyses of IL-1beta release. We found that P2X7R stimulation induces different forms of membrane blebbing, depending on cell type and extracellular ionic milieu. In HEK-P2X7 cells in BSS, this blebbing was characteristically zeiotic, while in macrophages in BSS, this blebbing was morphologically distinct, prompting us to name it "tethered." We further demonstrated that P2X7R stimulation led to activation of the small GTPase Rho, which in turn signaled the formation ROCK-dependent blebs. Finally, we showed inhibition of tethered membrane blebbing had no effect on ATP-induced IL-1beta release, nor did blockade of IL-1beta release by caspase-inhibition impact tethered membrane blebbing. In a separate study, we demonstrated that removal of halide anions from the extracellular BSS media led to ATP-induced formation of ROCK-independent dilated blebs and biphasic release of IL-1beta whereby one phase was blocked by the tyrphostin tyrosine kinase inhibitor AG126 while the second phase was lytic and blocked by glycine. Finally, we demonstrated that maitotoxin, which recruits the same pore-forming moiety as the P2X7R, can also facilitate massive dilated membrane blebbing and IL-1beta release. This release can be blocked by AG126, but is primarily lytic in nature, since it can be blocked by glycine. In summary, we have shown that membrane blebbing and IL-1beta secretion are regulated by many common signaling pathways but can be pharmacologically dissociated.
机译：促炎性细胞因子白介素1β（IL-1beta）在先天免疫系统中起重要作用，诱导发烧和激活血管内皮。然而，IL-1β分泌的机制仍然是个谜，因为它在细胞质中表达，因此不通过内质网/高尔基体分泌途径作为靶标。此外，IL-1β作为无活性的33 kDa前细胞因子存在，必须通过caspase-1（本身是复杂调节的酶原）将其裂解为其分泌的，具有生物活性的17 kDa形式。鉴于LPS引发的巨噬细胞上P2X7R的ATP激活可以促进IL-1beta的分泌，同时介导膜起泡的形成，我们假设膜起泡在IL-1beta的分泌中起重要作用。为了验证这一假设，我们使用延时摄影显微镜来评估巨噬细胞对各种诱导起泡的刺激的反应以及对IL-1beta释放的生化分析。我们发现，P2X7R刺激会诱导不同形式的膜起泡，具体取决于细胞类型和细胞外离子环境。在BSS的HEK-P2X7细胞中，这种起泡是典型的沸石，而在BSS的巨噬细胞中，这种起泡在形态上是截然不同的，这促使我们将其命名为“束缚”。我们进一步证明，P2X7R刺激导致小GTPase Rho的激活，进而标志着ROCK依赖性气泡的形成。最后，我们显示抑制拴系膜起泡对ATP诱导的IL-1beta释放没有影响，胱天蛋白酶抑制对IL-1beta释放的阻断也不会影响拴系膜起泡。在另一项研究中，我们证明了从细胞外BSS介质中除去卤化物阴离子会导致ATP诱导的ROCK依赖性扩张小球形成和IL-1β的双相释放，从而一相被酪氨酸酪氨酸激酶抑制剂AG126阻断，而第二相裂解并被甘氨酸封闭。最后，我们证明了招募与P2X7R相同的成孔部分的毒素，也可以促进大量的扩张膜起泡和IL-1beta释放。该释放可以被AG126阻断，但本质上是裂解性的，因为它可以被甘氨酸阻断。总而言之，我们已经表明膜起泡和IL-1β分泌受许多常见的信号传导途径调节，但可以药理学上分离。

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  • 作者

    Verhoef, Philip Alan.;

  • 作者单位

    Case Western Reserve University (Health Sciences).;

  • 授予单位 Case Western Reserve University (Health Sciences).;
  • 学科 Biology Cell.; Health Sciences Immunology.; Health Sciences Pharmacology.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 160 p.
  • 总页数 160
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;预防医学、卫生学;药理学;
  • 关键词

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