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Jianping Gao, Reuben Sana, Margarita Calonge, Larry A. Wheeler, Michael E. Stern; Cyclosporin A (CsA) Induces T Cell Apoptosis and Protects Human Conjunctival Epithelial Cells (IOBA-NHC) from Stress-Induced Apoptosis via Modulation of the Mitochondria Permeability Transition Pore (MPTP). Invest. Ophthalmol. Vis. Sci. 2012;53(14):3978.
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CsA selectively inhibits T cell activation and attenuates ocular inflammation in dry eye. Previously we showed that topical CsA facilitated lymphocytic apoptosis and suppressed epithelial cell apoptosis in canine KCS. MPTP plays an important role in initiation of apoptosis. We hypothesized that CsA induces T cell apoptosis and preserves ocular resident cells from stress-induced apoptosis via modulation of MPTP.
IOBA-NHC and Jurkat T cells were stimulated with ionomycin, αFas, PMA/αCD3, or IFN-γ in the presence or absence of CsA. The opening status of MPTP was assessed by tracking calcein AM, combined with CoCl2, a fluorescence-quencher does not cross mitochondrial membrane, using flow cytometry and confocal microscopy. Mitochondrial membrane potential (ΔΨm) was quantified via JC-1. Apoptosis was determined by Annexin V assay. IL-2 was measured using ELISA.
MPTP opening was induced by ionomycin in a dose-dependent fashion. In Jurkat T cells: (1) calcium overflow-induced MPTP activation was not inhibited by CsA at below 10 μM; (2) at ≥ 2.5 μM, not only was MPTP opening was not prevented, T cell apoptosis was induced dose-dependently after 24 hours incubation with CsA (13, 34 and 70% cell apoptosis induced by CsA at 2.5, 10 and 50 μM, respectively, p<0.01); (3) this was concomitant with a decrease in IL-2 in PMA/αCD3-activated T cells treated with CsA. In contrast, in IOBA-NHC: (1) MPTP opening induced by ionomycin or αFas was both prevented by CsA (10 μM); (2) the loss in ΔΨm was reduced (↓43%, p<0.01) by CsA (10 μM); (3) cells remained viable in 500μM CsA for 24 hours; (4) IFN-γ-induced IOBA apoptosis was inhibited by CsA (10 μM, p<0.03).
CsA induced T cell apoptosis via inhibition of TCR/CD3-mediated T cell activation without apparent impact on MPTP or ΔΨm alterations in Jurkat T cells. CsA protected conjunctival epithelial cells from inflammation-induced apoptosis in IOBA-NHC by blocking MPTP opening and ΔΨm loss. The findings suggest the differential effects of CsA on induction of T cell apoptosis and protection of ocular resident cells in treating ocular inflammation.
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