RESUMEN
PURPOSE: Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function. METHODS: Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed. RESULTS: Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin ß1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups. CONCLUSION: For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.
Asunto(s)
Proliferación Celular , Células Epiteliales , Epitelio Corneal , Limbo de la Córnea , Células Madre , Antígenos de Diferenciación/biosíntesis , Técnicas de Cultivo de Célula , Células Cultivadas , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Epitelio Corneal/metabolismo , Epitelio Corneal/ultraestructura , Femenino , Humanos , Limbo de la Córnea/metabolismo , Limbo de la Córnea/ultraestructura , Masculino , Células Madre/metabolismo , Células Madre/ultraestructuraRESUMEN
Limbal stem cell deficiency can be treated with transplantation of cultured human limbal epithelial cells (LEC). It can be advantageous to produce LEC in centralized labs and thereafter ship them to eye clinics. The present study used transport simulations of LEC to determine if vigorous shaking during transport altered the viability, morphology and phenotype during a 4 day-long storage of LEC with a previously described serum-free storage method. Inserts with LEC cultured on amniotic membranes were sutured to caps inside air-tight containers with generous amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered minimal essential medium (MEM). The containers were distributed among the following testing conditions: 6 hours with full containers, 36 hours with full containers, 36 hours with container three quarters full of medium, and 36 hours with container full of medium containing a shear-protecting agent (Pluronic-F68). Compared to stored, but non-transported controls, no statistically significant changes in viability and immunohistochemical staining were observed. The epithelial sheets remained intact. However, an air-liquid interface in the containers reduced the number of desmosomes and hemi-desmosomes compared to the controls. In conclusion, cultured LEC sheets appear to endure vigorous shaking for at least 36 hours if the container is full.
Asunto(s)
Enfermedades de la Córnea/cirugía , Epitelio Corneal/trasplante , Limbo de la Córnea/patología , Trasplante de Células Madre/métodos , Transportes , Anciano , Anciano de 80 o más Años , Adhesión Celular , Supervivencia Celular , Células Cultivadas/trasplante , Células Cultivadas/ultraestructura , Enfermedades de la Córnea/patología , Epitelio Corneal/citología , Humanos , Limbo de la Córnea/citología , Masculino , Microscopía Electrónica de Transmisión , Células Madre/patología , Células Madre/ultraestructuraRESUMEN
Conjunctival goblet cells play a major role in maintaining the mucus 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 proresolution 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/FPR2 (formyl peptide receptor2) was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA4 significantly increased mucin secretion, [Ca2+]i, and extracellular regulated kinase 1/2 (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 with its initial response. LXA4 activated phospholipases 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.
Asunto(s)
Conjuntiva/patología , Conjuntivitis Alérgica/inmunología , Síndromes de Ojo Seco/inmunología , Células Caliciformes/fisiología , Inflamación/inmunología , Lipoxinas/metabolismo , Receptores de Lipoxina/metabolismo , Animales , Secreciones Corporales , Señalización del Calcio , Células Cultivadas , Humanos , Masculino , Mucinas/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Lágrimas/fisiologíaRESUMEN
Goblet cells populate wet-surfaced mucosa including the conjunctiva of the eye, intestine, and nose, among others. These cells function as part of the innate immune system by secreting high molecular weight mucins that interact with environmental constituents including pathogens, allergens, and particulate pollutants. Herein, we determined whether interferon gamma (IFN-γ), a Th1 cytokine increased in dry eye, alters goblet cell function. Goblet cells from rat and human conjunctiva were cultured. Changes in intracellular [Ca(2+)] ([Ca(2+)](i)), high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with IFN-γ with or without the cholinergic agonist carbachol. IFN-γ itself increased [Ca(2+)](i) in rat and human goblet cells and prevented the increase in [Ca(2+)](i) caused by carbachol. Carbachol prevented IFN-γ-mediated increase in [Ca(2+)](i). This cross-talk between IFN-γ and muscarinic receptors may be partially due to use of the same Ca(2+)(i) reservoirs, but also from interaction of signaling pathways proximal to the increase in [Ca(2+)](i). IFN-γ blocked carbachol-induced high molecular weight glycoconjugate secretion and reduced goblet cell proliferation. We conclude that increased levels of IFN-γ in dry eye disease could explain the lack of goblet cells and mucin deficiency typically found in this pathology. IFN-γ could also function similarly in respiratory and gastrointestinal tracts.
Asunto(s)
Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Conjuntiva/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Interferón gamma/farmacología , Animales , Calcio/inmunología , Calcio/metabolismo , Señalización del Calcio , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Conjuntiva/inmunología , Conjuntiva/patología , Interacciones Farmacológicas , Síndromes de Ojo Seco/genética , Síndromes de Ojo Seco/inmunología , Síndromes de Ojo Seco/patología , Regulación de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (NADP+)(Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (NADP+)(Fosforilante)/inmunología , Glicoconjugados/biosíntesis , Glicoconjugados/metabolismo , Células Caliciformes/inmunología , Células Caliciformes/patología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Masculino , Mucina 5AC/genética , Mucina 5AC/inmunología , Ratas , Ratas Sprague-DawleyRESUMEN
Resolution of inflammation is an active process mediated by pro-resolution lipid mediators. As 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 (GPRs), as RvD1 interacts with its receptor GPR32 to block histamine-stimulated H1 receptor increases in intracellular [Ca(2+)] ([Ca(2+)]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 [Ca(2+)]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.
Asunto(s)
Conjuntivitis Alérgica/inmunología , Ácidos Docosahexaenoicos/farmacología , Células Caliciformes/efectos de los fármacos , Antagonistas de los Receptores Histamínicos H1/farmacología , Histamina/metabolismo , Animales , Aspirina/metabolismo , Secreciones Corporales/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Conjuntivitis Alérgica/tratamiento farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Caliciformes/inmunología , Histamina/inmunología , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Receptor Cross-Talk/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H1/metabolismoRESUMEN
Ocular surface inflammation associated with Sjögren's syndrome is characterized by a loss of secretory function and alteration in numbers of mucin secreting goblet cells. Such changes are a prominent feature of ocular surface inflammatory diseases and are attributed to inflammation; however, the exact effect of the inflammatory cytokines on conjunctival goblet cell function remains largely unknown. In this study, we developed a primary culture of mouse goblet cells from conjunctival tissue and evaluated the effects on their function by inflammatory cytokines detected in the conjunctiva of mouse model of Sjögren's syndrome (Thrombospondin-1 deficient mice). We found that apoptosis of goblet cells was primarily induced by TNF-α and IFN-γ. These two cytokines also inhibited mucin secretion by goblet cells in response to cholinergic stimulation, whereas IL-6 enhanced such secretion. No changes in secretory response were detected in the presence of IL-13 or IL-17. Goblet cells proliferated to varying degrees in response to all the tested cytokines with the greatest response to IL-13 followed by IL-6. Our results therefore reveal that inflammatory cytokines expressed in the conjunctiva during an ocular surface disease directly disrupt conjunctival goblet cell functions, compromising the protective function of tears, thereby contributing to ocular surface damage.
Asunto(s)
Conjuntiva/citología , Citocinas/farmacología , Células Caliciformes/fisiología , Animales , Apoptosis/efectos de los fármacos , Carbacol/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Mucina 5AC/metabolismo , Receptores de Citocinas/análisis , Células Th2/inmunología , Trombospondina 1/fisiologíaRESUMEN
The purpose of the study was to investigate if the number of goblet cells expanded ex vivo from a conjunctival explant is affected by the biopsy harvesting site on the conjunctiva and the distance from the explant. Conjunctival explants from six regions: superior and inferior bulbus, fornix, and tarsus of male Sprague-Dawley rats were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for eight days. Histochemical and immunofluorescent staining of goblet (CK-7/UEA-1/MUC5AC), stratified squamous, non-goblet (CK-4), proliferating (PCNA) and progenitor (ABCG2) cells were analyzed by epifluorescence and laser confocal microscopy. Outgrowth was measured with NIH ImageJ. For statistical analysis the Mann-Whitney test and Spearman's rank-order correlation test were used. Cultures from superior and inferior fornix contained the most goblet cells as indicated by the presence of CK-7+, UEA-1+ and MUC5AC+ cells. Superior and inferior forniceal cultures displayed 60.8% ± 9.2% and 64.7% ± 6.7% CK-7+ cells, respectively, compared to the superior tarsal (26.6% ± 8.4%; P < 0.05), superior bulbar (31.0% ± 4.0%; P < 0.05), inferior bulbar (38.5% ± 9.3%; P < 0.05) and inferior tarsal cultures (27.7% ± 8.3%; P < 0.05). While 28.4% ± 6.3% of CK-7+ goblet cells co-labeled with PCNA, only 7.4% ± 1.6% of UEA-1+ goblet cells did (P < 0.01). CK-7+ goblet cells were located at a lower concentration close to the explant (39.8% ± 3.1%) compared to near the leading edge (58.2% ± 4.5%; P < 0.05). Both markers for goblet cell secretory product (UEA-1 and MUC5AC), however, displayed the opposite pattern with a higher percentage of positive cells close to the explant than near the leading edge (P < 0.05). The percentage of CK-4+ cells was higher near the explant compared to near the leading edge (P < 0.01). The percentage of CK-7+ goblet cells in the cultures did not correlate with the outgrowth size (r(s) = -0.086; P = 0.435). The percentage of UEA-1+ goblet cells correlated negatively with outgrowth size (r(s) = -0.347; P < 0.01), whereas the percentage of CK-4+ cells correlated positively with the outgrowth size (r(s) = 0.473; P < 0.05). We conclude that forniceal explants yield the highest number of goblet cells ex vivo and thereby seem to be optimal for goblet cell transplantation. We also suggest that CK-7+/UEA-1- cells represent highly proliferative immature goblet cells. These cells could be important during conjunctival migration as they are mostly located close to the leading edge and their density does not decrease with increasing outgrowth size.
Asunto(s)
Conjuntiva/citología , Células Caliciformes/citología , Recolección de Tejidos y Órganos/métodos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Biomarcadores/metabolismo , Biopsia , Recuento de Células , Proliferación Celular , Células Cultivadas , Conjuntiva/metabolismo , Células Caliciformes/metabolismo , Queratinas/metabolismo , Masculino , Microscopía Confocal , Mucina 5AC/metabolismo , Fenotipo , Lectinas de Plantas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/µm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/µm; P=0.98 and 0.89±1.0 microvilli/µm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/µm; P=0.47 and 0.07±0.07 microvilli/µm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.
Asunto(s)
Conjuntiva/ultraestructura , Criopreservación , Preservación de Órganos , Amnios , Biomarcadores/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Sulfatos de Condroitina/farmacología , Mezclas Complejas/farmacología , Conjuntiva/metabolismo , Medio de Cultivo Libre de Suero , Dextranos/farmacología , Epitelio , Gentamicinas/farmacología , HEPES/farmacología , Humanos , Técnicas para Inmunoenzimas , Microvellosidades/ultraestructura , Fenotipo , Factores de TiempoRESUMEN
AIM: Large variations in results of diagnostic tests for mild to moderate dry eye are widely recognised. The purpose of this study was to assess if there was concordance between common dry eye diagnostic tests. METHODS: A total of 91 subjects were recruited to the study. The tear film and ocular surface were evaluated using the phenol red thread test (PRT), tear film break-up time (TBUT), biomicroscopic examination and impression cytological assessment of conjunctival goblet cells. Dry eye symptoms were assessed using McMonnies' dry eye questionnaire (MQ) and statistical correlations between all tests were assessed. RESULTS: This study cohort did not include severe aqueous deficient dry eye patients as determined by the PRT. A statistically significant difference was noted between PRT results and all other tests (pAsunto(s)
Técnicas de Diagnóstico Oftalmológico/normas
, Síndromes de Ojo Seco/diagnóstico
, Lágrimas/metabolismo
, Adulto
, Anciano
, Anciano de 80 o más Años
, Análisis de Varianza
, Conjuntiva/patología
, Diagnóstico Diferencial
, Femenino
, Humanos
, Masculino
, Glándulas Tarsales/patología
, Persona de Mediana Edad
, Reproducibilidad de los Resultados
, Adulto Joven
RESUMEN
The purpose of this study was to investigate the expression of the EGF family of growth factors and EGF receptor subtypes (ErbB1-4) present in lacrimal gland and determine the effects of these growth factors on different functions of rat lacrimal gland. RT-PCR was used to detect mRNA expression in the lacrimal gland of selected members of the EGF family of growth factors, namely EGF, transforming growth factor alpha (TGF-alpha), heparin-binding EGF (HB-EGF), and heregulin. The presence of ErbB receptors was investigated by immunofluorescence microscopy and western blot analysis. The effects of EGF, TGF-alpha, HB-EGF, and heregulin on protein secretion from lacrimal gland acini were examined using a fluorescent assay for peroxidase, a marker of protein secretion. Fura-2 tetra-acetoxymethyl ester was used to measure the effects of the growth factors on intracellular [Ca2+] ([Ca2+]i) in acini. MAPK activation in acini by these growth factors was also examined by western blot analysis using antibodies specific to phosphorylated p42/44 MAPK and total p42 MAPK. Rat lacrimal gland expressed EGF, TGF-alpha, HB-EGF, and heregulin mRNA, and all four ErbB receptors were present in the lacrimal gland as detected by western blot analyses. ErbB 1 and ErbB2 were located in basal and lateral membranes of acinar and ductal cells. The location of ErbB3 could not be determined while ErbB4 was found in ductal cells. Heregulin (10(-7) m) significantly increased protein secretion in lacrimal gland acini whereas all growth factors tested significantly increased [Ca2+]i at 10(-7) m. TGF-alpha (10(-9) m), heregulin (10(-7) m), EGF (10(-7) m), and HB-EGF (10(-7) m) significantly increased the amount of phosphorylated MAPK in lacrimal gland acini. We conclude that all members of the EGF family of growth factors studied are synthesised in rat lacrimal gland, could activate all four ErbB receptors that are present in this tissue, and differentially activate lacrimal gland functions.
Asunto(s)
Calcio/análisis , Factor de Crecimiento Epidérmico/análisis , Proteínas del Ojo/metabolismo , Aparato Lagrimal/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Receptores ErbB/análisis , Receptores ErbB/metabolismo , Inmunohistoquímica/métodos , Aparato Lagrimal/enzimología , Masculino , Peroxidasa/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
1. In the present investigation we examined the regulation of calmodulin (CaM)- and protein kinase C (PKC)-dependent pathways by cytosolic Ca(2+) in the contraction of cat lower oesophageal sphincter (LES). 2. Force developed in response to increasing doses of acetylcholine (ACh) was directly related to the increase of the [Ca(2+)](i) measured by fura-2. Thapsigargin, which depletes Ca(2+) stores, reduced the contraction and the [Ca(2+)](i). In addition, contraction in response to maximal ACh was reduced by the CaM inhibitor CGS9343B but not by the PKC inhibitor chelerythrine. The contraction in response to submaximal ACh was reduced by chelerythrine but not by CGS9343B. 3. In permeabilized cells, the contraction in response to low Ca(2+) (0.54 microm) was also reduced by CGS9343B. 4. The response to high Ca(2+) (1.0 microm) was reduced by CGS9343B. ACh also inhibited PKC activation induced by diacylglycerol, which activation is inhibited by the N-myristoylated peptide inhibitor derived from pseudosubstrate sequences of PKCalphabetagamma (myr-PKC-alphabetagamma), but not of myr-PKC-alpha. 5. These data are consistent with the view that activated CaM-dependent pathways inhibit PKC-dependent pathways, this switch mechanism might be regulated by Ca(2+) in the LES.
Asunto(s)
Calcio/fisiología , Calmodulina/fisiología , Unión Esofagogástrica/fisiología , Proteína Quinasa C/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Acetilcolina/antagonistas & inhibidores , Acetilcolina/farmacología , Alcaloides , Animales , Bencimidazoles/farmacología , Benzofenantridinas , Calcio/antagonistas & inhibidores , Calcio/química , Calmodulina/química , Gatos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Diglicéridos/farmacología , Unión Esofagogástrica/citología , Unión Esofagogástrica/efectos de los fármacos , Inositol 1,4,5-Trifosfato/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Fenantridinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Transducción de Señal/fisiología , Tapsigargina/antagonistas & inhibidores , Tapsigargina/farmacologíaRESUMEN
PURPOSE: To determine whether neural pathways for controlling goblet cell secretion are present in mouse and human conjunctiva. METHODS: Mouse conjunctiva was homogenized and subjected to electrophoresis and Western blotting to detect PGP 9.5 (indicates nerves), muscarinic receptor subtypes (indicates parasympathetic pathway), and adrenergic receptors (indicates sympathetic pathway). Mouse eyes and human conjunctival tissue were analyzed by immunofluorescence microscopy. Antibodies to vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and muscarinic and alpha(1)- and beta-adrenergic receptor subtypes were used. RESULTS: Western blot demonstrated PGP 9.5, M(1), M(2), and M(3) muscarinic receptors and alpha(1A)-, beta(1)-, beta(2)-, and beta(3)-adrenergic receptors in mouse conjunctiva. Immunoreactivity for VIP, TH, and DBH was found adjacent to mouse and human goblet cells. M(1) and M(2) muscarinic receptors were identified throughout mouse conjunctiva, but M(3) receptor was predominantly on goblet cells. All three muscarinic receptor subtypes were detected on goblet cells in human conjunctiva. alpha(1A)-Adrenergic receptors were found on epithelial cells and on goblet cells in mouse and human conjunctiva. beta(1)- and beta(2)-Adrenergic receptors were found on both epithelial and goblet cells in mouse conjunctiva, but not on human conjunctival cells. beta(3)-Adrenergic receptors were found on both epithelial and goblet cells in human conjunctiva but not on mouse conjunctival cells. CONCLUSIONS: The following conclusions were drawn: parasympathetic nerves and M(1), M(2), and M(3) muscarinic receptors, as well as sympathetic nerves are present on mouse and human goblet cells. The adrenergic receptors beta(1) and beta(2,) but not alpha(1A) and beta(3) are present on mouse conjunctival goblet cells, whereas alpha(1A) and beta(3,) but not beta(1) and beta(2) are present on human conjunctival goblet cells, suggesting that these nerves and receptors could activate goblet cell secretion in mouse and humans.
Asunto(s)
Conjuntiva/inervación , Células Caliciformes/metabolismo , Sistema Nervioso Parasimpático/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Muscarínicos/metabolismo , Sistema Nervioso Simpático/metabolismo , Anciano , Animales , Western Blotting , Conjuntiva/metabolismo , Dopamina beta-Hidroxilasa/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-Dawley , Tioléster Hidrolasas/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina Tiolesterasa , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Photorefractive keratectomy and laser in situ keratomileusis can induce or exacerbate dry eye after surgery. This manifests as an increase in degree and frequency of symptoms, corneal findings, such as superficial punctate keratopathy, and abnormal results of dry eye tests, such as the Schirmer test and tear break-up time. The cause mainly involves decreased corneal sensation, resulting in decreased feedback to the lacrimal gland and reduced tear production. Other causes may include increased evaporation, inflammation, or toxicity of medications. Dry eye may result infrequently in impaired wound healing and decreased optical quality of the cornea, but it is transient, lasting from a few weeks up to 1 year. Patients should be warned about this distressing complication. During a period of dry eye, artificial tears and punctal plugs are helpful in preventing or alleviating patient discomfort.
Asunto(s)
Síndromes de Ojo Seco/etiología , Queratomileusis por Láser In Situ/efectos adversos , Queratectomía Fotorrefractiva/efectos adversos , Procedimientos Quirúrgicos Refractivos , Córnea/inervación , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/terapia , Humanos , Láseres de Excímeros , Nervio Oftálmico/cirugía , Lágrimas/metabolismoRESUMEN
We previously reported an increased secretion of amyloid precursor-like protein 2 (APLP2) in the healing corneal epithelium. The present study sought to investigate signal transduction pathways involved in APLP2 shedding in vitro. APLP2 was constitutively shed and released into culture medium in SV40-immortalized human corneal epithelial cells as assessed by Western blotting, flow cytometry, and indirect immunofluorescence. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC-epsilon-specific, N-myristoylated peptide inhibitor. Epidermal growth factor (EGF) also induced APLP2 accumulation in culture medium. Basal APLP2 shedding as well as that induced by PMA and EGF was blocked by a mitogen-activated protein kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-epsilon may be involved in the induction of APLP2 shedding in corneal epithelial cells.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Epitelio Corneal/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas del Tejido Nervioso/metabolismo , Línea Celular Transformada , Factor de Crecimiento Epidérmico/farmacología , Epitelio Corneal/citología , Epitelio Corneal/efectos de los fármacos , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Fosforilación , Proteína Quinasa C/fisiología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
PURPOSE: To isolate, culture, and characterize goblet cells from the conjunctiva of rats. METHODS: Conjunctival tissue was surgically removed from Sprague-Dawley rats. Goblet cells were then isolated from the nictitating membrane and fornix using explant cultures. Cells derived from the explants were grown and propagated in RPMI medium supplemented with 10% fetal bovine serum. They were characterized using an enzyme-linked lectin assay (ELLA) with the lectin Ulex europaeus agglutinin-1 (UEA-1), Western blot analysis, PCR, light and electron microscopy, specialized histochemistry and indirect immunofluorescence microscopy. RESULTS: Goblet cells were successfully isolated from conjunctival explants by scraping nongoblet cells from the culture vessel. To date, cultures have been passaged a minimum of three times without the loss of their specific cellular markers. Cells identified as goblet cells fulfilled the following criteria: positive staining for alcian blue/periodic acid Schiff reagent, cytokeratin (CK)-7, the lectins UEA-I and Helix pomatia agglutinin (HPA), MUC5AC, and M(3) muscarinic receptor; detection of MUC5AC mRNA using RT-PCR; and negative staining for CK-4, M(1) muscarinic receptor, and Banderia simplicifolia lectin. The authors also measured, using the ELLA, substantial amounts of UEA-I-detectable high-molecular-weight glycoproteins and MUC5AC released into the medium. CONCLUSIONS: Cultured goblet cells retain many characteristics of goblet cells in vivo and thus may serve as a useful tool in delineating the pathobiology of the ocular surface.
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Separación Celular/métodos , Conjuntiva/citología , Células Caliciformes/citología , Animales , Western Blotting , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Conjuntiva/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Células Caliciformes/metabolismo , Masculino , Microscopía Fluorescente , Mucina 5AC , Mucinas/genética , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
Factor de Crecimiento Epidérmico/fisiología , Aparato Lagrimal/metabolismo , Neurotransmisores/farmacología , Agonistas alfa-Adrenérgicos/farmacología , Animales , Agonistas Colinérgicos/farmacología , Receptores ErbB/fisiología , Humanos , Ligandos , Ratones , Ratas , Transducción de Señal/fisiología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
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 Ca(2+)concentration ([Ca2(+)](i)) while diacylglycerol activates protein kinase C (PKC). PKC and Ca(2+), 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(+)](i)with the Ca(2+)ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca(2+)and by the Ca(2+)/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 (PKCgamma and zeta 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 PKCalpha, -betaI and -betaII, the novel PKC isozymes PKCepsilon, -theta;, and - mu, and the atypical PKC isozyme PKCzeta. 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 Ca(2+)plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca(2+)/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.
Asunto(s)
Calcio/fisiología , Conjuntiva/citología , Células Caliciformes/metabolismo , Proteína Quinasa C/fisiología , Animales , Western Blotting , Quelantes , Glicoproteínas/metabolismo , Ionomicina/farmacología , Ionóforos/farmacología , Masculino , Microscopía Fluorescente , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
PURPOSE: The purpose of this study was to determine the role of protein kinase C (PKC) in corneal endothelial cell proliferation. METHODS: Immunocytochemistry and Western blotting were used to define the PKC isoforms expressed in primary cultures of rat corneal endothelial cells. For proliferation studies, primary cultures of rat corneal endothelial cells were serum-starved for 48 hours and incubated for 2 hours with the PKC inhibitors staurosporine (10(-9) to 10(-7) M), chelerythrine (10(-9) to 5 x 10(-8) M), or calphostin C (10(-9) to 10(-7) M). Individual PKC isoforms were inhibited using PKCalpha antisense oligonucleotide transfection or exposure for 1 hour to myristoylated, pseudosubstrate-derived peptide inhibitors against PKCalpha, -alphassgamma, -epsilon, and -delta (10(-8) to 10(-6) M). Cells were then stimulated with 2.5% serum for 24 hours. Cell proliferation was measured with bromodeoxyuridine (BrDU) and Ki67 immunocytochemistry. Protein level of cyclin E was determined by Western blotting. RESULTS: PKCalpha, -ssII, -delta, -epsilon, -iota, -eta, -gamma, and -theta were detected in corneal endothelial cells. Maximum inhibition of PKC with staurosporine, calphostin C, and chelerythrine reduced cell proliferation to 7%, 31%, and 48% of control, respectively. Myristoylated peptide inhibition of PKCalpha and -epsilon reduced cell proliferation to 57% and 59% of control, respectively. PKCalpha antisense oligonucleotide reduced cell proliferation to 35% of control. Cyclin E protein level was decreased to 70%, 38%, 57%, and 43% of control in cells treated with calphostin C, staurosporine, chelerythrine, and PKCalpha antisense, respectively. CONCLUSIONS: PKC activity, in particular PKCalpha and -epsilon activity, is important in promoting corneal endothelial cell proliferation. Inhibition of PKC activity prohibits G1/S-phase progression and reduces cyclin E protein levels.