Innate Immunity

先天免疫

• 批准号: 6559040
• 负责人: David M. Segal
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/6559040
• 关键词: CD16 molecule; CD44 molecule; T lymphocyte; antitumor antibody; biological signal transduction; breast neoplasms; cell adhesion molecules; cell mediated cytotoxicity; cellular immunity; cytolysis; human tissue; interleukin 2; laboratory mouse; leukocyte activation /transformation; natural killer cells; neoplasm /cancer immunology; neoplastic growth; protein tyrosine kinase; receptor

Cellular mediators of innate immunity do not express rearranged V regions, yet have the capacity to recognize and respond to target cells in the absence of antibody. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in humans and mice and several have been shown to trigger inflammatory responses to pathogen derived substances. We hypothesized that pathogen recognition by immature dendritic cells (iDC) is mediated by TLRs and showed that human iDC do express and signal through TLRs. We have also shown that histamine generates signals in iDC that interact with the LPS/TLR4 signaling pathway, profoundly altering the maturation process. Histamine is produced in the peripheral microenvironment by the binding of allergens to IgE on mast. We demonstrated that iDC express two active histamine receptors, H1 and H2. Although histamine failed to affect the LPS-driven maturation of iDC with regard to phenotypic changes or capacity to prime naive T cells, it dramatically altered the repertoire of cytokines and chemokines secreted by mature DC. In particular, histamine, acting upon the H2 receptor for a short period of time, increased IL-10 production and reduced IL-12 secretion. As a result, histamine-matured DC polarized naive CD4+ T cells toward a Th2 phenotype, as compared with DC that had matured in the absence of histamine. We propose that the Th2 cells favor IgE production, leading to increased histamine secretion by mast cells. Thus a positive feedback loop would be established that could contribute to the severity of atopic diseases such as allergy and asthma and explain the observation that Th2 cells and serum IgE antibodies are increased in atopic patients. The molecular mechanisms by which TLRs recognize pathogens and initiate inflammatory responses is poorly understood, and we have examined two aspects of the TLR signaling process. The aim of one study was to determine a function for TLR1. The mammalian TLRs constitute a family of ten members, several of which initiate inflammatory responses upon recognition of pathogen derived substances. TLR4 in particular recognizes LPS, and is essential for LPS-induced septic shock. By contrast, TLR1, the most widely expressed of the TLRs, has no defined function. Human microvascular endothelial cells (HMEC) express TLR4 but not TLR1 and activate NF-kB through TLR4 in response to LPS. When HMEC cells were transfected with TLR1, they lost their capacity to respond to LPS. Inhibition was specific for TLR1 because a different paralog, TLR5, failed to block TLR4 function. Moreover, TLR1 had no effect upon the TNF-a induced activation of NF-kB, indicating that TLR1 inhibited early events in the TLR signaling pathway. Constructs encoding the transmembrane and extracellular domains of TLR1 blocked TLR4 function. In addition, TLR1 physically associated with TLR4 in co-precipitation experiments. Our results suggest that the function of TLR1 is to moderate the potentially dangerous innate response to LPS by binding to TLR4 and diminishing its capacity to activate the TLR signaling pathway. The TLR4 response to LPS requires the binding of MD-2 to its extracellular domain. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2) we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We showed that sMD-2 exists as a heterogeneous collection of large disulfide linked oligomers formed from stable dimeric subunits, and that concentrations of sMD-2 as low as 50 pM enhanced the responsiveness of TLR4 reporter cells to LPS. An MD-2 like activity was also released by monocyte-derived iDC from normal donors. When co-expressed with MD-2, TLR4 indiscriminantly associated in the ER/cis Golgi with different sized oligomers of MD-2, and excess MD-2 was secreted into the medium. We concluded that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4. Thus, sMD-2 might play a role in regulating responses to LPS and other pathogen derived substances in vivo.

先天免疫的细胞介质不表达重排的V区，但在没有抗体的情况下具有识别和响应靶细胞的能力。最近，在人类和小鼠中发现了一个古老的病原体识别受体家族，即Toll样受体（TLR），其中一些已被证明可以引发对病原体衍生物质的炎症反应。我们假设未成熟树突状细胞（iDC）的病原体识别是由TLR介导的，并表明人类iDC确实通过TLR表达和信号传导。我们还表明，组胺在iDC中产生与LPS/TLR 4信号通路相互作用的信号，从而深刻地改变成熟过程。组胺是通过过敏原与肥大细胞上的IgE结合而在外周微环境中产生的。我们证明iDC表达两种活性组胺受体H1和H2。尽管组胺未能影响LPS驱动的iDC成熟的表型变化或引发幼稚T细胞的能力，但它显著改变了成熟DC分泌的细胞因子和趋化因子的库。特别是组胺，作用于H2受体的时间很短，增加IL-10的产生和减少IL-12的分泌。因此，组胺成熟的DC极化幼稚的CD 4 + T细胞向Th 2表型，与DC已经成熟的组胺的情况下相比。我们建议，Th 2细胞有利于IgE的生产，导致肥大细胞组胺分泌增加。因此，将建立一个积极的反馈回路，可能有助于过敏性疾病，如过敏和哮喘的严重程度，并解释观察到的Th 2细胞和血清IgE抗体增加的过敏性患者。 TLR识别病原体和启动炎症反应的分子机制知之甚少，我们已经研究了TLR信号传导过程的两个方面。一项研究的目的是确定TLR 1的功能。哺乳动物TLR构成了一个十个成员的家族，其中几个在识别病原体衍生物质时启动炎症反应。TLR 4特别识别LPS，并且是LPS诱导的败血性休克所必需的。相比之下，TLR 1，最广泛表达的TLR，没有明确的功能。人微血管内皮细胞（HMEC）表达TLR 4，但不表达TLR 1，并通过TLR 4激活NF-κ B应答LPS。当用TLR 1转染HMEC细胞时，它们失去了对LPS的反应能力。抑制作用对TLR 1是特异性的，因为不同的受体TLR 5不能阻断TLR 4的功能。此外，TLR 1对TNF-α诱导的NF-κ B活化没有影响，表明TLR 1抑制TLR信号通路中的早期事件。编码TLR 1的跨膜和胞外结构域的构建体阻断TLR 4功能。此外，在共沉淀实验中，TLR 1与TLR 4物理结合。我们的研究结果表明，TLR 1的功能是通过与TLR 4结合并降低其激活TLR信号通路的能力来缓和对LPS的潜在危险的先天反应。 TLR 4对LPS的应答需要MD-2与其胞外结构域的结合。MD-2含有前导序列但缺乏跨膜结构域，我们询问它是否作为活性蛋白分泌到培养基中。作为分泌的MD-2（sMD-2）的来源，我们使用来自用表位标记的人MD-2稳定转导的细胞的培养上清液。我们发现，sMD-2作为由稳定的二聚体亚基形成的大的二硫键连接的寡聚体的异质集合而存在，并且低至50 pM的sMD-2浓度增强了TLR 4报告细胞对LPS的反应性。来自正常供体的单核细胞衍生的iDC也释放出MD-2样活性。当与MD-2共表达时，TLR 4在ER/顺式高尔基体中不加区别地与MD-2的不同大小的寡聚体相关联，并且过量的MD-2分泌到培养基中。我们的结论是，正常和转染细胞分泌的可溶形式的MD-2结合与TLR 4的高亲和力。因此，sMD-2可能在体内调节对LPS和其他病原体衍生物质的反应中起作用。