title: Human fetal retinal pigment epithelium-induced cell cycle arrest, loss of mitochondrial membrane potential and apoptosis.
abstract: OBJECTIVE
  To investigate the mechanism of action of the soluble immune suppressive product secreted by human fetal retinal pigment epithelial (HFRPE) cells in a model system using the human T-cell line Jurkat (Jkt).
  
  
  METHODS
  Pure HFRPE cells were isolated and cultured. The supernatants of both nonactivated and IFN-gamma-activated HFRPE cells were isolated. Cells from the human T-cell line Jkt were incubated either in standard culture medium or in the supernatant isolated from HFRPE cells. In the first assay Jkt cell proliferation was measured by [(3)H]thymidine incorporation. In the second assay Jkt cell apoptosis was examined for annexin V staining by flow cytometry. In the third assay Jkt cell division was evaluated with carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye. In the last assay the mitochondrial transmembrane potential of Jkt cells was measured with the cationic lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)]. In all the assays the effect of supernatants isolated from both nonactivated and IFN-gamma-activated HFRPE cells were compared with standard culture medium. The involvement of antiapoptotic human gene bcl-x(L:)was determined by using a Jkt cell line that was stably transfected with bcl-x(L:).
  
  
  RESULTS
  The supernatant isolated from HFRPE cells significantly suppressed the cell division in Jkt cells and induced apoptosis. These effects were stronger when the supernatant was isolated from IFN-gamma-activated HFRPE cells. The apoptosis pathway induced by the secreted product of HFRPE cells involved the early disruption of mitochondrial transmembrane potential. Although the overexpression of bcl-x(L) gene rescued the Jkt cells from supernatant-induced apoptosis, it could not restore the proliferation of Jkt cells.
  
  
  CONCLUSIONS
  These data suggest that HFRPE cells secrete a product that initiates an early cell cycle arrest in the human T-cell line Jkt, which is followed by the activation of an apoptotic pathway that involves the loss of mitochondrial membrane potential. The latter could be prevented by bcl-x(L) overexpression. Also these data suggest that the HFRPE-induced T-cell apoptosis may play a significant role in maintaining the immune privilege in the subretinal space.
authors.affiliation: Department of Ophthalmology and Visual Science, Department of Pathology, University of Chicago, Illinois, USA. lfarrokh@midway.uchicago.edu
authors.forename: L
authors.initials: L
authors.lastname: Farrokh-Siar
authors.forename: K A
authors.initials: KA
authors.lastname: Rezai
authors.forename: E M
authors.initials: EM
authors.lastname: Palmer
authors.forename: S C
authors.initials: SC
authors.lastname: Patel
authors.forename: J T
authors.initials: JT
authors.lastname: Ernest
authors.forename: G A
authors.initials: GA
authors.lastname: van Seventer
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002232:Carbocyanines; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division; D002462:Cell Membrane; D002478:Cells, Cultured; D005333:Fetus / Q000166:cytology*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism / Q000473:pathology*; D008564:Membrane Potentials / Q000502:physiology*; D008928:Mitochondria / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism; D051020:bcl-X Protein
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D017304:Annexin A5; C494811:BCL2L1 protein, human; D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D019253:Proto-Oncogene Proteins c-bcl-2; D013388:Succinimides; D051020:bcl-X Protein; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine