RESUMEN
To determine whether pro-inflammatory cytokines modulate intercellular adhesion molecule-1 (ICAM-1; CD54) expression on cultured primary human corneal epithelial cells (HCEs), confluent HCEs were treated with various concentrations of interferon-gamma(IFN-gamma), interleukin-1alpha(IL-1alpha), IL-1beta, IL-4, tumor necrosis factor-alpha (TNF-alpha), or combinations over time. ICAM-1 expression was measured by flow cytometry and/or a cell-based ELISA using a monoclonal mouse anti-human CD54 antibody. The apparent MW of ICAM-1 protein was determined by immunoprecipitation of biotinylated HCEs. RT-PCR was used to detect ICAM-1 RNA. The mature cell surface form of HCE ICAM-1 was approximately 110 kDa as determined by immunoprecipitation. IFN-gammaand TNF-alpha induced both dose- and time-dependent increases in ICAM-1 expression. An approximately 20-fold increase in ICAM-1 was seen at 50-100 U IFN-gamma ml-1. ICAM-1 specific mRNA accumulated approximately 4.5-fold after IFN-gammatreatment. TNF-alpha(100 U ml-1) induced a consistent approximately 6.0-fold increase in ICAM-1 expression. When IFN-gammaand TNF-alpha were mixed, at sub-optimal concentrations of each, a synergistic effect on ICAM-1 expression was not detected. Neither IL-4, IL-1alpha nor IL-1beta affected ICAM-1 expression in a consistent fashion. In summary, ICAM-1 was modulated on primary human corneal epithelial cells by the cytokines IFN-gamma and TNF-alpha in a dose- and time-dependent fashion. Cytokine modulation of corneal epithelial cell ICAM-1 during inflammation may contribute to corneal epithelial cell injury by aiding the attachment of inflammatory cells such as eosinophils which express the receptor for ICAM-1, the beta2 integrins (CD11a,b,c/CD18).
Asunto(s)
Citocinas/farmacología , Epitelio Corneal/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Técnicas de Cultivo de Célula , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/citología , Citometría de Flujo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/farmacología , Interleucinas/farmacología , Pruebas de Precipitina , ARN Mensajero/genética , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
PURPOSE: There is mounting evidence that eosinophil granule proteins may cause tissue injury during allergic inflammation of the eye. Therefore, the authors investigated the in vitro effects of human eosinophil major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN) on cultured human corneal epithelial cell viability and morphology. METHODS: Confluent primary human corneal epithelial cell cultures were exposed to each of the four human eosinophil cationic granule proteins at concentrations ranging from 0 to 100 micrograms/ml (0, 12.5, 25, 50, and 100 micrograms/ml) for up to 48 hours in serum-free media. Morphologic changes were assessed by light microscopy at 1, 6, 24, and 48 hours; cell viability was measured using the MTT cell viability assay at 24 hours. RESULTS: Cells treated with MBP and ECP induced a dose-dependent gradual increase in morphologic changes; in contrast, EPO and EDN induced minimal changes in cell morphology. At 24 hours, both MBP and ECP induced statistically significant (P < 0.05) decreases in cell viability at a concentration of 100 micrograms/ml; EPO induced a significant (P < 0.05) decrease in cell viability at all concentrations tested, and EDN showed no significant reduction of cell viability at any of the concentrations tested. CONCLUSIONS: The current study suggests that the human eosinophil granule proteins MBP and ECP affect human corneal epithelial cell viability and morphology in vitro, whereas the protein EPO affects cell viability only. EDN had no significant effect on cell viability or morphology. Hence, MBP, ECP, and EPO perturb the corneal epithelium differentially and may contribute to keratopathy associated with severe ocular allergy.
Asunto(s)
Proteínas Sanguíneas/farmacología , Córnea/citología , Mediadores de Inflamación/farmacología , Neurotoxinas/farmacología , Peroxidasas/farmacología , Ribonucleasas , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colorantes , Córnea/efectos de los fármacos , Proteínas en los Gránulos del Eosinófilo , Peroxidasa del Eosinófilo , Neurotoxina Derivada del Eosinófilo , Células Epiteliales , Epitelio/efectos de los fármacos , Humanos , Sales de Tetrazolio , TiazolesRESUMEN
Several corneal complications have been reported in patients with long standing diabetes, but their exact pathogenesis is not well understood. It has been observed that the rate of epithelial wound healing in diabetic rats is delayed compared to those in normal animals. Here we present the effect of the free radial scavenger, Trolox, a water soluble vitamin E analogue, on epithelial wound healing in diabetic rat cornea. Three groups of rats were included: 1) normal, 2) diabetic, 3) diabetic + Trolox. After 3 months, rats were sacrificed and corneas removed. Standard 3 mm diameter corneal epithelial defects were made and residual epithelial defects were measured after 18 hours at 37 degrees C in a sterile cell culture incubator. Wound healing data measured in mm2 was used for statistical analysis. There were significantly larger (p < 0.05) epithelial defects in diabetic corneas as compared to control. Treatment with Trolox antioxidant in diabetic rats produced a significantly smaller (p < 0.05) epithelial defect than that of untreated diabetic rats. These studies suggest the involvement of free radicals in the delay of corneal epithelial wound healing in diabetes.