title: Mitochondrial permeability transition pore in inflammatory apoptosis of human conjunctival epithelial cells and T cells: effect of cyclosporin A.
abstract: OBJECTIVE
  To investigate the role of mitochondrial permeability transition pore (MPTP) and effect of cyclosporin A (CsA) on inflammatory apoptosis of human conjunctival epithelial cells (IOBA-NHC) and T cells.
  
  
  METHODS
  IOBA-NHC and Jurkat cells were stimulated with IFNγ, TNFα, αFas, or PMA/αCD3, in the presence or absence of CsA. MPTP was determined using the calcein-cobalt technique. Mitochondrial membrane potential (ΔΨm) was measured with JC-1. Apoptosis was quantified by Annexin V/PI staining. Apoptosis mediators were evaluated by flow cytometry or Western blot.
  
  
  RESULTS
  In IOBA-NHC, TNFα, and IFNγ induced MPTP opening, ΔΨm loss, and increased cell apoptosis. This was accompanied by upregulation of Fas/FasL; Bax; and caspase-3, -8, and -9 activation. Addition of CsA prevented IOBA-NHC from cell death by blocking MPTP opening, ΔΨm loss, Fas/FasL, and caspase activation. In PMA/αCD3-activated Jurkat T cells, MPTP opening and ΔΨm loss were increased along with cell apoptosis and upregulated Fas/FasL/caspase expressions. CsA further promoted T-cell apoptosis, ΔΨm loss, and upregulation of Fas/FasL/caspase.
  
  
  CONCLUSIONS
  Inflammation induces aberrant MPTP opening, resulting in an increased apoptosis in conjunctival epithelial cells. CsA protected IOBA-NHC from cell death by blocking both intrinsic and extrinsic apoptosis pathways. CsA promoted T-cell apoptosis via upregulating Fas/FasL and caspase activities with a minimal effect on MPTP. The findings suggest that the differential effect of CsA on T cells versus ocular surface resident epithelial cells may contribute to its therapeutic efficacy in treating ocular inflammation such as dry eye disease.
authors.affiliation: Biological Sciences, Inflammation Research Program, Allergan, Inc., Irvine, California 92612, USA.
authors.forename: Jianping
authors.initials: J
authors.lastname: Gao
authors.forename: Reuben
authors.initials: R
authors.lastname: Sana
authors.forename: Virginia
authors.initials: V
authors.lastname: Calder
authors.forename: Margarita
authors.initials: M
authors.lastname: Calonge
authors.forename: Wanju
authors.initials: W
authors.lastname: Lee
authors.forename: Larry A
authors.initials: LA
authors.lastname: Wheeler
authors.forename: Michael E
authors.initials: ME
authors.lastname: Stern
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology; D003229:Conjunctival Diseases / Q000188:drug therapy; D016572:Cyclosporine / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects*; D053222:Fas Ligand Protein / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007249:Inflammation / Q000188:drug therapy; D007371:Interferon-gamma / Q000494:pharmacology; D019169:Jurkat Cells / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects* / Q000502:physiology; D000083162:Mitochondrial Permeability Transition Pore; D013601:T-Lymphocytes / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism
chemical_list: D053222:Fas Ligand Protein; D007166:Immunosuppressive Agents; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D007371:Interferon-gamma; D016572:Cyclosporine; D020169:Caspases
keywords: T cells; apoptosis; conjunctival epithelial cells; cyclosporin A (CsA); dry eye; inflammation; mitochondrial permeability transition pore (MPTP)