Mitochondrial permeability transition pore in inflammatory apoptosis of human conjunctival epithelial cells and T cells: effect of cyclosporin A.

PURPOSE: 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.