Innate Immunity

先天免疫的细胞介质在缺乏抗体或T细胞的情况下具有识别和应答靶细胞的能力。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（TLRs），其中一些已被证明可引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tlr，并且iDC上的tlr驱动成熟以应对病原体，这是获得性免疫的重要组成部分。tlr驱动的成熟是由g蛋白偶联受体调节的，我们也发现H2组胺受体可以深刻地影响成熟过程。组胺是由肥大细胞产生的，它们与DC位于相同的微环境中，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lps驱动的iDC的表型变化或初始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。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。我们还证明浆细胞样DC通过减少i型干扰素的产生对组胺有反应，这可以解释在一些特应性患者中观察到的病毒感染增加。我们目前正在小鼠实验中验证一种假设，即DC在富含组胺的微环境中成熟会促进引流淋巴结中Th2细胞的产生，从而有利于IgE的产生，并导致肥大细胞分泌组胺增加。

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.