Mechanisms of apoptosis in human retinal pigment epithelium induced by TNF-alpha in conditions of heavy metal ion deficiency.

PURPOSE

To investigate the mechanism underlying apoptosis in retinal pigment epithelium (RPE) induced by TNF-alpha in conditions of heavy metal ion deficiency.

METHODS

Apoptotic morphology was assessed with Hoechst 33342. FITC-VAD-fmk or antibody specific to cleaved caspase 3 was used to detect in situ activated caspases or cleaved caspase 3, respectively. JC-1 and carboxylated dichlorodihydrofluorescein diacetate were used as probes to measure mitochondrial membrane potential (Deltapsi(m)) and intracellular reactive oxygen species (rOx).

RESULTS

The apoptotic response of RPE cells was markedly enhanced when TNF-alpha plus actinomycin D (act-D) was coapplied with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a heavy metal ion chelator. The apoptosis was caspase dependent, and a blockade with cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition (MPT), but not FK506, a calcineurin inhibitor, abolished caspase activation and subsequent apoptosis, suggesting that apoptosis requires the MPT, and that caspase activation is downstream to the MPT. MPT, observed in situ as Deltapsi(m) loss, was prevented when cells were pretreated with either CsA or the pan-caspase inhibitor z-VAD-fmk. This result suggests that apoptotic signaling is initiated by the MPT and further amplified by downstream caspases, probably through a feedback loop. An increase in rOx was observed in cells exposed to TNF-alpha+act-D+TPEN, and rOx production did not require MPT or caspase activation. In addition, application of antioxidants significantly inhibited rOx production, Deltapsi(m) loss, and apoptosis. These data suggest that the rOx may play a role as a proapoptotic signal.

CONCLUSIONS

The data suggest that intracellular heavy metal ions play a role in determining the apoptosis induction threshold of the inflammatory response to TNF-alpha in RPE.