Conjunctival Goblet Cell Mucin Secretion in Inflammation and Its Resolution

描述（由申请人提供）：充盈（含黏液）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免受外界环境的影响，充满杯状细胞的损失对眼表有害。这种损失表明杯状细胞已经分泌了粘蛋白，但不能再合成粘蛋白，这可能是由于长期的刺激。慢性刺激可能来自结膜炎症的介质，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。炎症终止（消退）是一个活跃的过程，产生诸如消退素之类的分子，减少炎症介质刺激的杯状细胞分泌，使杯状细胞重新充盈。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、高分辨率化合物减弱杯状细胞分泌以恢复眼表正常、关键的粘蛋白层。我们假设在发炎的结膜：1。被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LTB4通过激活特异性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。通过阻断信号通路中的特定步骤，分解蛋白的产生抑制组胺、白三烯和胆碱能激动剂刺激的杯状细胞分泌，从而终止杯状细胞的分泌。我们计划解决以下问题：特异性目标1：组胺是否刺激培养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. 特异性目标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，哪些特异性受体和信号通路被诱导？和3。特异性目的3：溶解蛋白RvE1和RvD1是否抑制组胺、白三烯和胆碱能激动剂刺激的培养结膜杯状细胞分泌，以及抑制的细胞机制。将使用从大鼠和人类结膜中培养的杯状细胞。粘蛋白分泌将通过改良的ELISA法测定。细胞内[Ca2+]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通过western blotting分析来测量。受体特异性激动剂和拮抗剂将用于揭示信号通路。分解蛋白和其他促分解化合物的产生将通过脂质组学分析来测量。公共卫生相关性：由过敏原、损伤、微生物或不稳定泪膜引起的结膜炎症可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以及参与其对杯状细胞分泌的解决的化合物。这将为开发新的眼表炎症治疗方法提供重要的见解。

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.