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.
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.
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