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

Lipoxin A4 Counter-regulates Histamine-stimulated Glycoconjugate Secretion in Conjunctival Goblet Cells

Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A4 (LXA4), are produced during inflammation returning the tissue to homeostasis and are also produced in non-inflamed tissues. The purpose of this study was to determine the actions of LXA4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca2+] ([Ca2+]i) and on histamine-stimulated responses. LXA4 increased mucin secretion and [Ca2+]i, and activated ERK1/2 in human goblet cells. Addition of LXA4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA4 responses. LXA4 inhibited histamine-stimulated increases in mucin secretion, [Ca2+]i, and ERK1/2 activation through activation of β-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases

Phospholipase D1, but Not D2, Regulates Protein Secretion Via Rho/ROCK in a Ras/Raf-Independent, MEK-Dependent Manner in Rat Lacrimal Gland

The authors describe a novel signaling pathway for PLD in nontransfected, primary cells of the rat lacrimal gland, where Rho and ROCK1 activate PLD1. Formation of this signaling complex results in the downstream activation of PKC, MEK, and ERK in a novel Ras-, Raf-, Pyk2- and cSrc-independent pathway that attenuates cholinergic agonist-stimulated protein secretion

Isolation and characterization of progenitor cells in uninjured, adult rat lacrimal gland

PURPOSE. The purpose of this study was to investigate the presence of progenitor cells in the uninjured, adult rat lacrimal gland (LG). METHODS. The presence of progenitor cells was examined in LG sections from male rats using antibodies against selected stem cell markers and α-smooth muscle actin (SMA), which marks myoepithelial cells (MECs), by immunofluorescence microscopy (IF). Small, immature cells were isolated after digestion of LG with collagenase and culture in RPMI 1640 for 2 weeks. Immature cells were examined for expression of stem cell markers by IF. Immature cell were grown in neuronal, epithelial, and myoepithelial cell media, and examined by light morphology and IF using antibodies to markers of different cell lineages. RESULTS. In the intact LGs, MECs expressed the stem cell markers nestin, Musashi 1, ABCG2, Pax6, Chx 10, ΔN p63, and Sox 2. All markers colocalized with SMA. Isolated immature cells contained Ki-67, nestin, Musashi 1, Pax 6, and CHX 10. In neuronal media, immature cells differentiated and assumed a neuronal cell morphology expressing neurofilament 200. In media for human corneal endothelial cells, immature cells differentiated, assumed cobblestone morphology, and labeled with the epithelial marker AE1/AE3. In RPMI media immature cells differentiated into cells with MEC-like morphology, and expressed the MEC markers SMA, α-actinin, adenylate cyclase II, and vimentin. CONCLUSIONS. We conclude that uninjured, adult LG contains progenitor cells that may be MECs, which can be isolated and differentiated into multiple lineages

Increase of Intracellular Ca2+ by Purinergic Receptors in Cultured Rat Lacrimal Gland Myoepithelial Cells

This article describes for the first time the culturing of myoepithelial cells from rat lacrimal gland and characterization of purinergic receptors in these cells

Lipoxin A4 activates ALX/FPR2 Receptor to Regulate Conjunctival Goblet Cell Secretion

Conjunctival goblet cells play a major role in maintaining the mucous layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis.. Resolution of inflammation is mediated by pro-resolution agonists such as lipoxin A4 (LXA4) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA4 on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca2+] ([Ca2+]i) and identify signaling pathways activated by LXA4. ALX/FPR was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA4 significantly increased mucin secretion, [Ca2+]i, and ERK 1/2 activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA4 increased [Ca2+]i to the same extent as LXA4. Sequential addition of either LXA4 or resolvin D1 followed by the second compound decreased [Ca2+]i of the second compound compared to its initial response. LXA4 activated phospholipase C, -D, and A2 and downstream molecules protein kinase C, ERK 1/2, and Ca2+/calmodulin dependent kinase to increase mucin secretion and [Ca2+]i. We conclude that conjunctival goblet cells respond to LXA4 to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases

Role of cPKC␣ and nPKC in EGF-Stimulated Goblet Cell Proliferation

PURPOSE. The authors determined the role of the protein kinase C (PKC) isoforms cPKC␣ and nPKC in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells. METHODS. Rat and human conjunctivas were minced, and goblet cells were allowed to grow. Passage 1 cells were serum starved for 24 to 48 hours and were incubated with the PKC inhibitors calphostin C and Gö 6983 (10 Ϫ10 -10 Ϫ7 M) for 20 minutes before stimulation with EGF (10 Ϫ7 M) for 24 hours. The presence and localization of PKC isoforms in cultured rat goblet cells were determined by Western blot analysis and immunofluorescence microscopy, respectively. Cultured rat goblet cells were serum starved and incubated with adenoviruses containing genes for dominant-negative cPKC␣ (Ad DNPKC␣, 10 4 pfu), dominant-negative nPKC (Ad DNPKC, 10 4 pfu), and wild-type cPKC␣ (Ad WTPKC␣, 10 7 pfu), and proliferation was measured. RESULTS. In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Gö 6983 inhibited proliferation by 53% Ϯ 15%. In human goblet cells, EGFstimulated proliferation was completely inhibited by calphostin C. PKC␣, -␤I, -␤II, -␦, -, -/, -, -␥, and -were found in cultured rat goblet cells. Ad DNPKC␣ and Ad DNPKC inhibited EGF-stimulated proliferation in rat goblet cells by 78% Ϯ 6% and 92% Ϯ 8%, respectively. Incubation with Ad WTPKC␣ alone significantly increased proliferation. CONCLUSIONS. cPKC␣ and nPKC play key roles in conjunctival goblet cell proliferation. (Invest Ophthalmol Vis Sci. 2009;50: 614 -620