NEUTROPHIL ACTIVATION BY VIRUSES AND MAMMALIAN LECTINS

在呼吸道病毒中，甲型流感病毒(IAV)特别容易 会引起细菌的双重感染。这些感染是导致 IAV流行期间的发病率和死亡率。有令人信服的 获得性IAV引起的吞噬细胞功能缺陷是一种 细菌重叠感染的重要原因。IAV的约束力 中性粒细胞唾液酸受体部位的血凝素 调节细胞功能的抑制。初步研究表明， 白唾液酸苷、CD45、唾液酸脂/x和神经节苷脂都在这些部位中 被绑住了。我们建议确认IAV与这些站点绑定，建立 结合的特定sialyl-Le/x蛋白决定了 各种结合蛋白被IAV干扰(即内化、封顶)， 确定哪些地点在数量上是最重要的， 在功能上。我们的初步数据与假设是一致的 一种单一的糖蛋白受体调节失活。最终的 对这一理论的检验将涉及消除特定的表达 利用重组(即反义)技术在HL60细胞中表达中性粒细胞糖蛋白 RNA或基因打靶)技术。 IAV IS功能重要糖蛋白受体的鉴定 不太可能为停用提供完整的解释。我们还将 需要确定IAV颗粒是如何扰乱这些受体的 停用就会发生。IAV颗粒与肺组织预孵化的研究 表面活性蛋白D(SP-D)降低IAV产生中性粒细胞的能力 功能障碍，同时显著增强IAV刺激 呼吸爆发反应。然而，与SP-D复合的IAV结合到 中性粒细胞通过与未调理的IAV相同的机制(即通过附着 含唾液酸的中性粒细胞膜成分)。vbl.使用 重组、野生型和突变型SP-D制剂我们将确定 SP-D对IAV颗粒的“调理”作用机制 IAV与中性粒细胞相互作用的功能结果。我们会 表征IAV与SP-D孵育后形成的聚集体(通过 光散射、荧光和EM技术)，并确定这些 聚集体与中性粒细胞结合[通过识别特定结合部位和 展示了这些结合位点是如何被干扰的(例如， 内化)在完整的细胞中]。我们的工作假设是SP-D的 保护作用不是通过改变中性粒细胞的哪种受体来实现的 绑定IAV，而不是来自SP-D改变IAV性质的能力 粒子。

Among respiratory viruses influenza A virus (IAV) is particularly prone to cause bacterial superinfections. These infections are the major cause of morbidity and mortality during IAV epidemics. There is compelling evidence that an acquired IAV-induced defect in phagocyte function is an important cause of bacterial superinfection. Binding of the IAV hemagglutinin to sialic acid-bearing receptor sites on the neutrophil mediates depression of cell function. Preliminary studies indicate that leukosialin, CD45, sialyl-Le/x, and gangliosides are among the sites bound. We propose to confirm that IAV binds to these sites, establish specific sialyl-Le/x bearing proteins which are bound, determine how the various binding proteins are perturbed (i.e. internalized, capped) by IAV, and establish which sites are most important quantitatively and functionally. Our preliminary data are compatible with the hypothesis that a single glycoprotein receptor mediates deactivation. The definitive test of this theory will involve eliminating expression of specific neutrophil glycoproteins in HL60 cells using recombinant (i.e. antisense RNA or gene targeting) techniques. Identification of functionally important glycoprotein receptors for IAV is unlikely to provide a full explanation for deactivation. We will also need to establish how IAV particles perturb these receptors such that deactivation occurs. Pre-incubation of IAV particles with pulmonary surfactant protein D (SP-D) reduces the ability of IAV to cause neutrophil dysfunction, while markedly enhancing the ability of IAV to stimulate a respiratory burst response. However, IAV complexed with SP-D binds to neutrophils via the same mechanism as unopsonized IAV (i.e. via attachment to sialic-acid bearing neutrophil membrane components). Using recombinant, wild type and mutant, SP-D preparations we will determine the mechanisms through which "opsonization" of IAV particles with SP-D alters the functional outcome of IAVs interaction with neutrophils. We will characterize the aggregates formed after incubation of IAV with SP-D (by light scattering, fluorescent and EM techniques) and determine how these aggregates bind to neutrophils [by identifying specific binding sites and demonstrating how these binding sites are perturbed (e.g. capped, internalized) in intact cells]. Our working hypothesis is that SP-D's protective effect results not from changing which neutrophil receptors bind IAV but rather from SP-D's ability to change the nature of the IAV particle.