Conjunctival Goblet Cell Secretion Research Articles

Immunoresolvent Resolvin D1 Maintains the Health of the Ocular Surface.

The present review focuses on the role of one of the D-series resolvins (Rv) RvD1 in the regulation of conjunctival goblet cell secretion and its role in ocular surface health. RvD1 is the most thoroughly studied of the specialized proresolution mediators in the goblet cells. The anterior surface of the eye consists of the cornea (the transparent central area) and the conjunctiva (opaque tissue that surrounds the cornea and lines the eyelids). The secretory mucin MUC5AC produced by the conjunctival goblet cells is protective of the ocular surface and especially helps to maintain clear vision through the cornea. In health, a complex neural reflex stimulates goblet cell secretion to maintain an optimum amount of mucin in the tear film. The specialized pro-resolution mediator, D-series resolvin (RvD1) is present in human tears and induces goblet cell mucin secretion. RvD1 interacts with its receptors ALX/FPR2 and GPR32, activates phospholipases C, D, and A2, as well as the EGFR. This stimulation increases the intracellular [Ca2+] and activates extracellular regulated kinase (ERK) 1/2 to cause mucin secretion into the tear film. This mucin secretion protects the ocular surface from the challenges in the external milieu thus maintaining a healthy interface between the eye and the environment. RvD1 forms a second important mechanism along with activation of a neural reflex pathway to regulate goblet cell mucin secretion and protect the ocular surface in health.

Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion.

Resolution of inflammation is an active process mediated by pro-resolution lipid mediators. Since resolvin (Rv) D1 is produced in the cornea, pro-resolution mediators could be effective in regulating inflammatory responses to histamine in allergic conjunctivitis. Two key mediators of resolution are the D-series resolvins RvD1 or aspirin-triggered RvD1 (AT-RvD1). We used cultured conjunctival goblet cells to determine whether histamine actions can be terminated during allergic responses. We found cross-talk between two types of G protein-coupled receptors, as RvD1 interacts with its receptor GPR32 to block histamine-stimulated H1 receptor increases in intracellular [Ca2+] ([Ca2+]i) preventing H1 receptor-mediated responses. In human and rat conjunctival goblet cells RvD1 and AT-RvD1 each block histamine-stimulated secretion by preventing its increase in [Ca2+]i and activation of extracellular regulated protein kinase (ERK)1/2. We suggest that D-series resolvins regulate histamine responses in the eye and offer new treatment approaches for allergic conjunctivitis or other histamine-dependent pathologies.

Effect of Histamine on Ca2+-Dependent Signaling Pathways in Rat Conjunctival Goblet Cells

The purpose of this study was to determine the Ca(2+)-dependent cellular signaling pathways used by histamine to stimulate conjunctival goblet cell secretion. Cultured rat goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured by loading cultured cells with the Ca(2+) sensitive dye fura-2. The level of [Ca(2+)](i) was measured using fluorescence microscopy. Extracellular regulated kinase (ERK) 2 was depleted using small interfering RNA (siRNA). Histamine-stimulated conjunctival goblet cell secretion of high molecular weight glycoproteins was blocked by removal of extracellular Ca(2+) and depletion of ERK2 by siRNA. Histamine increase in [Ca(2+)](i) was desensitized by repeated addition of agonist and blocked by a phospholipase C antagonist. Histamine at higher doses increased [Ca(2+)](i) by stimulating influx of extracellular Ca(2+), but at a lower dose released Ca(2+) from intracellular stores. Activation of each histamine receptor subtype (H(1)-H(4)) increased [Ca(2+)](i) and histamine stimulation was blocked by antagonists of each receptor subtype. The H(2) receptor subtype increase in [Ca(2+)](i) was cAMP dependent. We conclude that histamine activates phospholipase C to release intracellular Ca(2+) that induces the influx of extracellular Ca(2+) and activates ERK1/2 to stimulate conjunctival goblet cell mucous secretion, and that activation of all four histamine receptor subtypes can increase [Ca(2+)](i).

Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion

The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M1AchR, M2AchR, and M3AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca2+] ([Ca2+]i) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca2+]i in a concentration-dependent manner. EGF-stimulated increase in [Ca2+]i was blocked by inhibitors of the EGF receptor and removal of extracellular Ca2+. Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M1AchR, M2AchR, and M3AchRs. Cch-stimulated increase in [Ca2+]i was blocked by inhibitors for the M1AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca2+]i and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca2+. This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca2+]i, and activate ERK 1/2 leading to an increase in mucin secretion.

Role of Histamine and Its Receptor Subtypes in Stimulation of Conjunctival Goblet Cell Secretion

The purpose of this study was to determine the effect of histamine and its receptors on goblet cell secretion. Cultured rat and human goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugate was measured by an enzyme-linked lectin assay. Cultured rat goblet cells were homogenized and either RNA was isolated for RT-PCR or proteins were isolated for Western blot analysis for presence of histamine receptors subtypes H₁ through H₄. The localization of these receptors was determined in rat and human goblet cells by immunofluorescence microscopy. Histamine stimulated goblet cell secretion in a concentration- and time-dependent manner. All four histamine receptors were present in cultured rat and human goblet cells. Use of agonists specific to individual histamine receptor subtypes indicated that the rank order of agonist stimulation was H₁ = H₃ > H₄ > H₂. Using antagonists specific to individual histamine receptor subtypes determined that H₂ and H₃, but not the H₁ and H₄, antagonists, inhibited histamine-stimulated conjunctival goblet cell secretion. Rat and human conjunctival goblet cells are a direct target of histamine, which induces secretion. All four histamine receptors are present in rat and human conjunctiva and are active in rat conjunctival goblet cells. These findings suggest that all four histamine receptor subtypes are important for conjunctival goblet cell secretion. Blockage of histamine receptor subtypes could prevent the excess mucus production associated with ocular allergy.

Advances in upper airway diseases and allergen immunotherapy in 2011

The purpose of this review is to highlight recently published important articles on upper airway diseases and allergen immunotherapy. We review articles on rhinitis, sinusitis, conjunctivitis, and immunotherapy. New insights into epidemiology, pathophysiology, diagnosis, and therapy are described for each of the above diseases.

Conjunctival Goblet Cell Secretion Stimulated by Leukotrienes Is Reduced by Resolvins D1 and E1 To Promote Resolution of Inflammation

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.

Role of Neurotrophins and Neurotrophin Receptors in Rat Conjunctival Goblet Cell Secretion and Proliferation

To determine which neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4)-and their receptors (NTrs) TrkA, TrkB, TrkC, and p75 are present in rat conjunctiva and cultured rat goblet cells (CGCs) and whether NTs stimulate glycoconjugate secretion or cell proliferation. Western blot analysis and immunofluorescence microscopy determined presence and location of NTs and NTrs. CGCs were incubated with NTs (10(-12)-10(-8) M) for 2 or 24 hours to measure secretion or proliferation, respectively. An enzyme-linked lectin assay analyzed glycoconjugate secretion. WST-8 determined cell proliferation. Western blot analysis showed all NTs and NTrs in both conjunctiva and CGCs. The cytoplasm of conjunctival stratified squamous cells and goblet cell lateral membranes contained NGF, BDNF, and NT4. Stratified squamous cell membranes contained NT3. In CGCs, NGF and BDNF had punctuate perinuclear staining. The nucleus contained NT3 and cytosol contained NT4. TrkA, TrkB, and p75 immunoreactivity was on conjunctival goblet cell lateral membranes. Plasma membranes of the basal layer of stratified squamous cells contained TrkA. Stratified squamous cell and goblet cell nuclei contained TrkC. In CGCs, all NTrs were present in the nucleus. NGF and BDNF, but not NT3 and NT4, induced a concentration-dependent stimulation of secretion from CGCs with a maximum increase of 10(-9) M each. No effect on cell proliferation was detected with any NTs. Rat conjunctival goblet cells and CGCs contain all NTs and NTrs. Only NGF and BDNF stimulated goblet cell glycoconjugate secretion, and none induced CGC proliferation.

Cholinergic agonists transactivate EGFR and stimulate MAPK to induce goblet cell secretion.

Conjunctival goblet cells are the primary source of mucins in the mucous layer, the innermost layer of the tear film. Conjunctival goblet cell mucin secretion is under neural control because exogenous addition of parasympathetic agonists stimulates goblet cell secretion. To elucidate the intracellular signal pathways used by cholinergic agonists to stimulate goblet cell mucin secretion, we determined whether p42/p44 mitogen-activated protein kinase (MAPK) is activated during cholinergic agonist-stimulated mucin secretion. Rat conjunctiva was removed, preincubated with or without antagonists, and stimulated with the cholinergic agonist carbachol (10(-4) M). Carbachol statistically significantly stimulated the phosphorylation of MAPK in a time- and concentration-dependent manner. U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The M(1) muscarinic antagonist pirenzepine, the M(2) muscarinic antagonist gallamine, and the M(1)/M(3) muscarinic receptor antagonist N-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP) also inhibited carbachol-stimulated MAPK activation. Increasing the intracellular Ca(2+) concentration with a Ca(2+) ionophore increased MAPK activation, and chelation of extracellular Ca(2+) inhibited carbachol-stimulated activation. Carbachol also increased tyrosine phosphorylation of Pyk2, p60Src, and the epidermal growth factor receptor (EGFR). The Src inhibitor PP1 and the EGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPK activation. AG-1478 also inhibited goblet cell secretion. We conclude that carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR.

Role of mitogen-activated protein kinase in cholinergic stimulation of conjunctival goblet cell secretion.

Conjunctival goblet cells are a primary source for the mucous layer of the tear film. We showed previously that conjunctival goblet cell mucin secretion is under neural control as activation of afferent sensory nerves in the cornea stimulates mucin secretion.1 This neural reflex is mediated either by the parasympathetic and sympathetic nerves that surround the goblet cells, or by antidromic stimulation of the sensory nerves in the conjunctiva and collateral sensory nerves from the cornea. Exogenous addition of the parasympathetic agonist carbachol or the parasympathetic neuropeptide VIP stimulated conjunctival goblet cell secretion.2,3 Cholinergic agonists released from parasympathetic nerves bind to and activate G protein linked- M2 and M3 muscarinic receptors. In most tissues, activation of these receptors stimulates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 binds to specific receptors on the endoplasmic reticulum to increase intracellular Ca2+. The increased intracellular Ca2+ either directly, or in combination with Ca2+/calmodulin-dependent protein kinases, activates mucin secretion. DAG in turn stimulates protein kinase C (PKC). In many tissues, cholinergic agonists also activate the mitogen-activated protein kinase (MAPK) signaling pathway used by growth factor receptors, which usually regulate cell growth and proliferation. Activation of the MAPK pathway by cholinergic agonists can occur through several mechanisms. One such mechanism is through transactivation of the EGF receptor in an EGF-independent manner.4 The activated recepter then serves as a scaffold for recruitment of Grb2-SoS complex via tyrosine phosphorylation of Shc. KeywordsHormone Replacement TherapyLacrimal GlandMeibomian GlandCorneal DystrophyKeratoconjunctivitis SiccaThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Regulation of Conjunctival Goblet Cell Secretion by Ca2+and Protein Kinase C

Conjunctival goblet cells secrete mucus in response to cholinergic (muscarinic) agonists, but the underlying signaling pathways activated in this tissue are not well understood. Cholinergic agonists usually activate phospholipase C to produce inositol 1,4,5 trisphosphate and diacylglycerol. Inositol 1,4,5 trisphosphate increases the intracellular Ca2+concentration ([Ca2+]i) while diacylglycerol activates protein kinase C (PKC). PKC and Ca2+, either by itself or with calmodulin, activate cellular functions. Goblet cell glycoprotein secretion, our index of mucin secretion, was measured from pieces of rat conjunctiva with an enzyme-linked lectin assay using the lectin Ulex europaeus agglutinin I (UEA-I). UEA-I selectively recognizes high molecular weight glycoproteins secreted by the goblet cells. Increasing the [Ca+]iwith the Ca2+ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca2+and by the Ca2+/calmodulin-dependent protein kinase inhibitors KN93 and W7 as well as their inactive analogs KN92 and W5. Activation of classical and novel PKC isozymes by phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate stimulated goblet cell glycoprotein secretion. When ionomycin and PMA were added simultaneously, secretion was additive. PKC isozymes were identified by Western blotting analyses with antibodies specific to nine of the 11 PKC isozymes (PKCγ and ζ were not tested). All nine PKC isozymes were identified in the conjunctival epithelium. The cellular location of the PKC isozymes was determined by immunofluorescence microscopy. Goblet cells contained the classical PKC isozymes PKCα, -βI and -βII, the novel PKC isozymes PKCϵ, -θ, and -μ , and the atypical PKC isozyme PKCζ. We were unable to determine if PKC activation is required for cholinergic-agonist induced secretion because the PKC inhibitors chelerythrine and staurosporine alone greatly increased secretion. We conclude that Ca2+plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca2+/calmodulin-dependent protein kinases. We also conclude that activated PKC can stimulate goblet cell secretion and that seven different PKC isoforms are present in the goblet cells.

ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE IN CHOLINERGIC STIMULATION OF CONJUNCTIVAL GOBLET CELL SECRETION.

Conjunctival goblet cells are a primary source for the mucous layer of the tear film. We showed previously that conjunctival goblet cell mucin secretion is under neural control as activation of afferent sensory nerves in the cornea stimulates mucin secretion.1 This neural reflex is mediated either by the parasympathetic and sympathetic nerves that surround the goblet cells, or by antidromic stimulation of the sensory nerves in the conjunctiva and collateral sensory nerves from the cornea. Exogenous addition of the parasympathetic agonist carbachol or the parasympathetic neuropeptide VIP stimulated conjunctival goblet cell secretion.2,3 Cholinergic agonists released from parasympathetic nerves bind to and activate G protein linked- M2 and M3 muscarinic receptors. In most tissues, activation of these receptors stimulates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 binds to specific receptors on the endoplasmic reticulum to increase intracellular Ca2+. The increased intracellular Ca2+ either directly, or in combination with Ca2+/calmodulin-dependent protein kinases, activates mucin secretion. DAG in turn stimulates protein kinase C (PKC). In many tissues, cholinergic agonists also activate the mitogen-activated protein kinase (MAPK) signaling pathway used by growth factor receptors, which usually regulate cell growth and proliferation. Activation of the MAPK pathway by cholinergic agonists can occur through several mechanisms. One such mechanism is through transactivation of the EGF receptor in an EGF-independent manner.4 The activated recepter then serves as a scaffold for recruitment of Grb2-SoS complex via tyrosine phosphorylation of Shc.

Vasoactive Intestinal Peptide-Stimulated Glycoconjugate Secretion from Conjunctival Goblet Cells

We have developed an in vitro and in vivo method to determine if VIP-stimulates conjunctival goblet cell secretion in the rat as nerves which contain vasoactive intestinal peptide (VIP) and are most likely parasympathetic are localized around these cells. For the in vitro method, pieces of rat conjunctiva were incubated for 1hr with no additions or increasing concentrations of VIP (10 -10-10 -6 m). Goblet cell secretion was measured by determining the amount of Helix pomatia agglutinin (HPA)-detectable glycoconjugates secreted into the medium. HPA-detectable glycoconjugates were assayed using an enzyme-linked lectin assay. For the in vivo method, drops of buffer containing no additions or varying concentrations of VIP (10 -10-10 -6 m) were placed on the ocular surface of anesthetized rats for 60min. The rats were killed, the ocular surface chemically fixed, and a button of conjunctiva removed. Mucin-containing goblet cells were stained by Alcian blue–periodic acid Schiff's reagent and the number of cells per 0.16mm 2was quantified. A decrease in the number of mucin-containing goblet cells indicated an increase in mucous secretion. By immunofluorescent histochemistry, we found that the lectin HPA was localized predominantly in the secretory granules of rat conjunctival goblet cells with little binding present in the remainder of the conjunctiva. Nerves containing VIP surrounded goblet cells labelled with HPA. In pieces of conjuctiva, in vitro VIP (10 -8–10 -6 m) stimulated HPA-detectable glycoconjugate secretion in a concentration dependent manner. When applied topically to the ocular surface, in vivo VIP at 10 -8 mstimulated mucous secretion from conjunctival goblet cells. We conclude that VIP is present in nerves around conjunctival goblet cells and stimulates glycoconjugate secretion from these cells.