title: Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions.
abstract: Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-β2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-β2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-β2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.
authors.affiliation: Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Soriano-Romaní
authors.affiliation: Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: García-Posadas
authors.affiliation: Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: López-García
authors.affiliation: Department of Eye and Vision Science, University of Liverpool, Liverpool, UK.
authors.forename: Luminita
authors.initials: L
authors.lastname: Paraoan
authors.affiliation: Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain. Electronic address: yol@ioba.med.uva.es.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Diebold
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism; D051928:CD47 Antigen / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002460:Cell Line; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology; D006706:Homeostasis; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D011804:Quinolines / Q000633:toxicity; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D019700:Thrombospondin 1 / Q000235:genetics / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2 / Q000378:metabolism
chemical_list: D018955:CD36 Antigens; D051928:CD47 Antigen; D015850:Interleukin-6; D011804:Quinolines; D012333:RNA, Messenger; D019700:Thrombospondin 1; D053781:Transforming Growth Factor beta2; C047117:bicinchoninic acid; D053148:Caspase 3; D053179:Caspase 7
keywords: Apoptosis; Conjunctiva; Cornea; Epithelial cell; Inflammation; Thrombospondin-1