Abstract
Purpose:
We demonstrated that CsA inhibited stress-induced apoptosis in human conjunctival epithelial cells (IOBA-NHC); promoted T cell death via differential regulation of mitochondria permeability transition pore (MPTP). This study is to investigate molecular mechanisms of apoptotic signaling in both cell types under immune and inflammatory stimulation.
Methods:
IOBA-NHC and Jurkat T cells were stimulated with aFas, PMA/aCD3, TNFa and/or IFNγ, in the presence or absence of CsA, and/or caspase inhibitors. Cell death was quantified by Annexin V/PI assay. MPTP and mitochondrial membrane potential (ΔΨm) were determined using calcein-cobalt technique and JC-1 staining, respectively. Mediators of the intrinsic and extrinsic apoptosis pathways (Fas/FasL, Bax/Bcl-2, cytochrome c, and caspase activities) were evaluated using flow cytometry or Western blot analysis.
Results:
In IOBA-NHC: (1) MPTP activation was elicited by aFas, TNFa, and/or IFNγ, but significantly inhibited by CsA; (2) TNFa and IFNγ provoked Fas upregulation, Bax mitochondrial translocation , mitochondrial membrane depolarization, cytochrome c release, and increased cell death. Inflammation-induced IOBA-NHC cell death was caspase-dependent. CsA attenuated inflammation-induced caspase 8, 9 and 3 activation, and inhibited IOBA-NHC apoptosis. In Jurkats: (1) PMA/aCD3-induced MPTP opening was not affected by CsA (1nM-10uM); (2) at subnanomolar concentration (>1nM), CsA completely abolished T cell IL-2 production; (3) at 10uM, CsA induced FasL expression, enhanced caspase activation, and exacerbated activation-induced T cell apoptosis.
Conclusions:
The intrinsic (mitochondria-mediated) and extrinsic (death receptor-initiated) pathways are both involved in inflammatory apoptosis of IOBA-NHC. CsA preserves conjunctival epithelial cells by stabilization of MPTP, maintaining mitochondrial membrane potential, and inhibition of Fas, Bax and caspase activation. In contrast, CsA elicited resting T cell death, and exacerbated activated T cell apoptosis primarily through extrinsic apoptosis signaling with minimal effect on MPTP. The results suggest that the differential effects of CsA on the fate of ocular resident cells versus inflammatory T cells may contribute to its therapeutic efficacy in treating ocular inflammation and restoration of immune homeostasis of the ocular surface.
Keywords: 426 apoptosis/cell death •
600 mitochondria •
489 cyclosporine