Conjunctival Goblet Cell Mucin Secretion in Inflammation and Its Resolution

描述(申请人提供)：充满(含粘蛋白)杯状细胞枯竭是眼表炎症和过敏性炎症疾病的标志，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，杯状细胞的缺失对眼表是有害的。这种丢失表明杯状细胞已经分泌，不能重新合成粘蛋白，这可能是由于慢性刺激。慢性刺激可能源于结膜炎症介质，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以激活神经反射弧，导致分泌物。炎症的终止(消退)是一个活跃的过程，产生诸如分解素之类的分子，减少炎症介质刺激的杯状细胞分泌，允许杯状细胞重新充盈。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，以及抗炎、缓化解化合物减弱杯状细胞分泌以恢复正常的、关键的粘蛋白层到眼表面。我们假设在炎症的结膜中：1.激活的肥大细胞产生的组胺和白三烯LTC4和LTD4以及入侵的中性粒细胞释放的白三烯LTC4和LTD4通过激活特定的受体和信号通路引起杯状细胞粘蛋白分泌；2：反射弧激活感觉神经刺激副交感神经释放神经递质刺激杯状细胞分泌；3.杯状细胞分泌通过阻断信号通路中的特定步骤抑制组胺、白三烯和胆碱能激动剂刺激的杯状细胞分泌而终止。我们计划解决以下问题：1.特异性目标1：组胺是否刺激培养的结膜杯细胞分泌粘蛋白？哪些组胺受体和细胞信号通路被激活？2.特异性目标2：白三烯LTC4、LTD4和LTB4是否刺激培养的结膜杯细胞分泌粘蛋白？哪些特异性受体和信号通路被诱导？3.特异性目标3：RvE1和RvD1是否抑制组胺、白三烯和胆碱能激动剂刺激培养的结膜杯状细胞分泌，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。粘蛋白的分泌量将用一种改良的酶联免疫吸附试验测定。用荧光显微镜观察单个细胞内的[Ca~(2+)]。磷酸化的细胞信号蛋白将通过蛋白质印迹分析进行检测。受体特异性激动剂和拮抗剂将用于解开信号通路。将通过脂肪组学分析来测量分解素和其他有利于拆分的化合物的产量。与公共卫生相关：由过敏原、创伤、微生物或不稳定的泪膜引起的结膜炎症会导致结膜杯状细胞分泌和眼睛有刺激感。我们的建议将使我们能够研究眼部炎症的机制，以及参与其分解杯状细胞分泌的化合物。这将为开发治疗眼表炎症的新疗法提供重要的见解。

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.