Conjunctival Goblet Cell Mucin Secretion in Inflammation and Its Resolution

描述（由申请人提供）：充盈（含粘蛋白）杯状细胞的耗竭是眼表炎症和过敏性炎症疾病（如干眼和大多数形式的过敏性结膜炎）的标志。杯状细胞粘蛋白保护眼表面免受外部环境的影响，并且充满的杯状细胞的损失对眼表面有害。这种损失表明杯状细胞已经分泌并且不能再合成粘蛋白，这可能是由于慢性刺激。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，炎症介质激活感觉神经可激活神经反射弧，引起分泌。炎症的终止（消退）是一个主动过程，其产生诸如消退素的分子，所述消退素减少炎性介质刺激的杯状细胞分泌，从而允许杯状细胞再填充。本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、促消退化合物减弱杯状细胞分泌以恢复眼表面的正常、关键粘蛋白层的细胞机制。我们假设，在发炎的结膜：1。组胺和由活化的肥大细胞产生的白三烯LTC4和LTD4以及从侵入的嗜中性粒细胞释放的LTB4通过活化特异性受体和信号传导途径引起杯状细胞粘蛋白分泌，2：通过反射弧激活感觉神经刺激副交感神经释放刺激杯状细胞分泌的神经递质，以及3。杯状细胞分泌通过产生消退素而终止，消退素通过阻断信号传导途径中的特定步骤来抑制组胺、白三烯和胆碱能激动剂刺激的杯状细胞分泌。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LTC4、LTD4和LTB4是否刺激培养的结膜杯状细胞分泌粘蛋白，以及诱导哪些特异性受体和信号通路？和3.具体目标3：Resolvins RvE1和RvD1是否抑制由组胺、白三烯和胆碱能激动剂刺激的培养结膜杯状细胞分泌，抑制的细胞机制是什么。将使用从大鼠和人结膜培养的杯状细胞。将通过改良的ELISA测定法测量粘蛋白分泌。将通过荧光显微镜在单细胞中研究细胞内[Ca2+]。将通过蛋白质印迹分析测量磷酸化的细胞信号传导蛋白。受体特异性激动剂和拮抗剂将用于解开信号传导途径。将通过脂质组学分析测量消退素和其他促消退化合物的产生。公共卫生相关性：由过敏原、创伤、微生物或不稳定的泪膜诱导的结膜炎症导致结膜杯状细胞分泌和眼睛刺激感。我们的建议将使我们能够研究眼部炎症的机制和参与其决议杯状细胞分泌的化合物。这将为开发眼表炎症的新型治疗方法提供重要见解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Conjunctival inflammation induced by allergens, wounding, microorganisms, or an unstable tear film results in conjunctival goblet cell secretion and feelings of irritation in the eye. Our proposal will allow us to study the mechanisms of ocular inflammation and the compounds involved in its resolution on goblet cell secretion. This will provide significant insights into developing novel treatments for ocular surface inflammation.