Innate Immunity

在没有抗体或T细胞的情况下，天然免疫的细胞介体具有识别和反应靶细胞的能力。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，Toll样受体(Toll Like Receptor，TLRs)，其中几个被证明可以触发对病原体衍生物质的炎症反应。我们和其他人已经证明，未成熟的树突状细胞(IDC)表达TLRs，IDC上的TLRs在对病原体的反应中驱动成熟，这是获得性免疫的重要组成部分。TLR驱动的成熟是由G蛋白偶联受体调控的，我们还证明了组胺H2受体特别是H2组胺受体可以深刻影响成熟过程。组胺是由肥大细胞产生的，肥大细胞与DC处于相同的微环境中，我们证明了人类单核细胞来源的IDC表达两种活性组胺受体H1和H2。尽管组胺不能影响内毒素诱导的IDC的成熟，但它显著改变了成熟DC分泌的细胞因子和趋化因子。特别是，组胺在短时间内作用于H2受体，增加了IL-10的产生，减少了IL-12的分泌。结果，与在没有组胺的情况下成熟的DC相比，组胺成熟的DC将幼稚的CD4+T细胞极化为Th2表型。我们还证明了血浆细胞样树突状细胞通过减少I型干扰素的产生而对组胺产生反应，这可以解释在一些特应性患者中观察到的病毒感染增加的原因。我们目前正在小鼠身上测试一种假设，即在富含组胺的微环境中成熟的DC将促进引流淋巴结中Th2细胞的生成，从而有利于IgE的产生，并导致肥大细胞组胺分泌增加。 TLR4对内毒素的反应需要MD-2与其胞外区结合。MD-2含有一个前导序列，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MD-2(SMD-2)的来源，我们使用稳定转导了表位标记的人MD-2的细胞的培养上清。我们发现SMD-2以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性集合存在，并且低至50 pm的SMD-2浓度增强了TLR4报告细胞对内毒素的反应性。正常供者的单核细胞来源的IDC也释放了MD-2样活性。当TLR4与MD-2共表达时，TLR4与不同大小的MD-2寡聚体在内质网/顺式高尔基体中不分青红皂白地结合在一起，过量的MD-2被分泌到培养液中。我们得出的结论是，正常和转基因细胞分泌一种与TLR4高亲和力结合的可溶性MD-2。因此，SMD-2可能在体内调节对内毒素和其他病原体衍生物质的反应中发挥作用。MD-2含有7个半胱氨酸残基，为了研究巯基在寡聚和内毒素应答中的作用，我们创建了22个Cys to Ser突变体。所有的MD-2突变体，包括一个完全缺乏半胱氨酸残基的突变体，都以成熟、可溶性和稳定的蛋白形式分泌。当作为可溶性蛋白加入TLR4表达细胞中时，所有突变体都没有活性，但当与TLR4共转染时，几个突变体显示出活性。齐聚不需要单个半胱氨酸残基，几个半胱氨酸残基能够形成链内或链间二硫键。我们得出结论，MD-2是一种非典型蛋白质，其中大部分(如果不是全部)巯基位于分子的表面。此外，MD-2在没有巯基的情况下是稳定的，这表明链内二硫键在三级结构的形成中不起关键作用，但可能稳定MD-2功能所需的局部结构特征。

Cellular mediators of innate immunity have the capacity to recognize and respond to target cells in the absence of antibody or T cells. 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 and others have shown that immature dendritic cells (iDC) express TLRs, and that TLRs on iDC drive maturation in response to pathogens, an essential component of acquired immunity. The TLR-driven maturation is modulated by G-protein coupled receptors, and we have also shown that the H2 histamine receptor, in particular, can profoundly affect the maturation process. Histamine is produced by mast cells, which are located in the same microenvironment as DC, and we demonstrated that human monocyte-derived 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 have also demonstrated the plasmacytoid DC respond to histamine by decreasing type-I interferon production, which could explain increased viral infections observed in some atopic patients. We are currently testing in mice the hypothesis that maturation of DC in a microenvironment that is rich in histamine will promote the generation of Th2 cells in the draining lymph nodes, thus favoring IgE production and leading to increased histamine secretion by mast cells. 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. MD-2 contains seven Cys residues and to investigate the role of sulfhydryls in oligomerization and LPS responsiveness we created 22 single and multiple Cys to Ser mutants. All of the MD-2 mutants, including one totally lacking Cys residues, were secreted as mature, soluble and stable proteins. All were inactive when added as soluble protein to TLR4-expressing cells, but several mutants showed activity when co-transfected with TLR4. No single Cys residue was necessary for oligomerization and several were capable of forming either intra- or inter-chain disulfide bonds. We conclude that MD-2 is an atypical protein in which most if not all sulfhydryls are located on the surface of the molecule. Moreover, MD-2 is stable in the absence of sulfhydryls, suggesting that intrachain disulfide bonds do not play an essential role in tertiary structure formation, but may stabilize local structural features that are required for MD-2 function.