Conjunctival Goblet Cell Mucin Secretion in Inflammation and Its Resolution

项目摘要 填充的（含粘蛋白的）杯状细胞的消耗是眼表面炎症疾病的标志， 过敏性炎症如干眼症和大多数形式的过敏性结膜炎。杯状细胞粘蛋白保护 从外部环境和充满杯状细胞的损失眼表面是有害的。 这种缺失表明杯状细胞已经分泌而不能再合成粘蛋白，这可能是因为 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，通过激活感觉神经， 炎症介质可激活神经反射弧，引起分泌。炎症终止 （消退）是一个积极的过程，产生分子，如消退，减少炎症- 介质刺激杯状细胞分泌，使杯状细胞重新填充。该项目的总体目标是 确定促炎介质诱导杯状细胞分泌的细胞机制， 炎性、促消退化合物减弱杯状细胞分泌以恢复正常的关键粘蛋白 层到眼表面。我们假设，在发炎的结膜：1。组胺和白三烯 活化的肥大细胞产生的LTC 4和LTD 4以及侵入的中性粒细胞释放的LTB 4引起杯状 通过激活特定受体和信号通路分泌细胞粘蛋白，2：通过激活感觉神经 反射弧刺激副交感神经释放刺激杯状细胞分泌的神经递质， 和3.杯状细胞分泌终止的生产消退，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，哪些组胺受体和细胞信号通路被激活？2.具体目标二： 白三烯LTC 4、LTD 4和LTB 4是否刺激培养的结膜杯状细胞分泌粘蛋白， 哪些特定的受体和信号通路被诱导？和3.具体目标3：Resolvins RvE 1 和RvD 1抑制由组胺、白三烯和 胆碱能激动剂和抑制的细胞机制。大鼠和人的杯状细胞培养 将使用结膜。将通过改良的ELISA测定法测量粘蛋白分泌。细胞内[Ca 2 +]将 通过荧光显微镜在单细胞中进行研究。磷酸化的细胞信号蛋白将被 通过蛋白质印迹分析测定。受体特异性激动剂和拮抗剂将用于解开 信号通路消退素和其他促消退化合物的产生将通过脂质组学测量。 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.