Conjunctival Goblet Cell Mucin Secretion in Inflammation and Its Resolution

项目摘要 充满的(含粘蛋白的)杯状细胞枯竭是眼表炎症和眼球疾病的一个标志。 过敏性炎症，如干眼和大多数形式的过敏性结膜炎。杯状细胞粘蛋白保护 眼表不受外界环境的影响，杯状细胞的充盈丢失对眼表是有害的。 这种缺失表明杯状细胞已经分泌了粘蛋白，不能重新合成粘蛋白，这可能是因为 是一种慢性刺激。慢性刺激可能来自结膜炎症介质，如 白三烯和组胺，刺激杯状细胞分泌。此外，感觉神经的激活通过 炎症介质可以激活神经反射弧，导致分泌物。消炎止痛 (分解)是一个产生分子的活跃过程，例如减少炎症的分解素。 中介物刺激杯状细胞分泌，使杯状细胞重新充盈。这个项目的总体目标是 确定促炎介质诱导杯状细胞分泌和抗炎的细胞机制 炎性亲和化合物减弱杯状细胞分泌以恢复正常的临界粘蛋白 层到眼球表面。我们推测在炎症的结膜中：1.组胺和白三烯 活化的肥大细胞产生的LTC4和LTD4以及入侵的中性粒细胞释放的LTB4导致杯状 激活特定受体和信号通路的细胞粘蛋白分泌，2：通过激活感觉神经 反射弧刺激副交感神经释放神经递质，刺激杯状细胞分泌， 3.杯状细胞分泌被抑制组胺、白三烯和 胆碱能激动剂通过阻断信号通路中的特定步骤刺激杯状细胞的分泌。我们 计划解决以下问题：1.具体目标1：组胺是否刺激培养的结膜杯状细胞 分泌粘蛋白和哪些组胺受体和细胞信号通路被激活？2.特定目标2： 白三烯LTC4、LTD4和LTB4是否刺激培养的结膜杯状细胞分泌粘蛋白和 诱导了哪些特定的受体和信号通路？3.具体目标3：做好RvE1 和RvD1抑制组胺、白三烯刺激的结膜杯状细胞分泌。 胆碱能激动剂及其抑制的细胞机制。大鼠和人培养的杯状细胞 将使用结膜。粘蛋白的分泌量将用一种改良的酶联免疫吸附试验测定。细胞内[Ca2+]将 在单细胞中用荧光显微镜观察。磷酸化的细胞信号蛋白将是 经免疫印迹分析检测。受体特异性激动剂和拮抗剂将被用来解开 信号通路。拆分药物和其他有利于拆分的化合物的生产将通过脂组分进行测量 分析。

Project Summary Depletion of filled (mucin-containing) goblet cells is a hallmark of diseases of ocular surface inflammation and allergic inflammation such as dry eye and most forms of allergic conjunctivitis. Goblet cell mucins protect the ocular surface from the external environment and loss of filled goblet cells is deleterious to the ocular surface. This loss indicates that goblet cells have secreted and not been able to resynthesize mucins, perhaps because of chronic stimulation. The chronic stimulation could arise from mediators of conjunctival inflammation, such as leukotrienes and histamine, stimulating goblet cell secretion. In addition, activation of sensory nerves by inflammatory mediators could activate a neural reflex arc causing secretion. Termination of inflammation (resolution) is an active process producing molecules such as the resolvins that decrease inflammatory- mediator stimulated goblet cell secretion, allowing goblet cells to refill. The overall goal of this project is to determine the cellular mechanisms by which pro-inflammatory mediators induce goblet cell secretion and anti- inflammatory, proresolution compounds attenuate goblet cell secretion to restore the normal, critical mucin layer to the ocular surface. We hypothesize that in the inflamed conjunctiva: 1. Histamine and the leukotrienes LTC4 and LTD4 produced by activated mast cells and LTB4 released from invading neutrophils cause goblet cell mucin secretion by activating specific receptors and signaling pathways, 2: Activation of sensory nerves by a reflex arc stimulates parasympathetic nerves to release neurotransmitters that stimulate goblet cell secretion, and 3. Goblet cell secretion is terminated by production of resolvins that inhibit histamine-, leukotriene- and cholinergic agonist-stimulated goblet cell secretion by blocking specific steps in the signaling pathways. We plan to address the following: 1. Specific Aim 1: Does histamine stimulate cultured conjunctival goblet cells to secrete mucin and which histamine receptors and cellular signaling pathways are activated? 2. Specific Aim 2: Do the leukotrienes LTC4, LTD4, and LTB4 stimulate cultured conjunctival goblet cells to secrete mucins and which specific receptors and signaling pathways are induced? and 3. Specific Aim 3: Do the resolvins RvE1 and RvD1 inhibit cultured conjunctival goblet cell secretion stimulated by histamine, leukotrienes, and cholinergic agonists and what are the cellular mechanisms inhibited. Goblet cells cultured from rat and human conjunctiva will be used. Mucin secretion will be measured by a modified ELISA assay. Intracellular [Ca2+] will be investigated in single cells by fluorescence microscopy. Phosphorylated cellular signaling proteins will be measured by western blotting analysis. Receptor specific agonists and antagonists will be used to unravel signaling pathways. Production of resolvins and other pro-resolution compounds will be measured by lipidomic analysis.