Tumor Necrosis Factor-α-Induced Apoptosis in Olfactory Epithelium in Vitro: Possible Roles of Caspase 1 (ICE), Caspase 2 (ICH-1), and Caspase 3 (CPP32)

We investigated the potential roles of three members of the interleukin-1beta-converting enzyme (ICE) protease family (caspases) in apoptosis in olfactory epithelium. By RT-PCR analysis, the mRNAs of caspase 1 (ICE), caspase 2 (ICH-1), and caspase 3 (CPP32) were detected in olfactory mucosa obtained from normal adults, E19 fetuses, and unilaterally bulbectomized rats. The transcript of caspase 2 disappeared in bulbectomized animals 3 and 5 days postoperatively, but reappeared 21 days postoperatively. This suggests that most of the caspase 2 transcript was in olfactory sensory neurons. We used TNF-alpha to induce cell death in organotypic cultures of E19 olfactory epithelium and assayed the ability of three caspase inhibitors to reverse the TNF-alpha effect. After 6 h of treatment with medium containing TNF-alpha, a 2.5-fold increase in apoptotic body number was observed throughout the olfactory epithelium. Pretreatment of the cultures with either of two irreversible caspase inhibitors (Z-VAD-fmk, Ac-YVAD-cmk) for 4 h, followed by a 6-h treatment with TNF-alpha plus an inhibitor, blocked TNF-alpha-induced cell death completely. Pretreatment with a third caspase inhibitor (Z-DEVD-fmk) in the same treatment schedule reduced the numbers of apoptotic cells significantly but not to the same extent as Z-VAD-fmk or Ac-YVAD-cmk. Increasing the dose of any of the inhibitors reduced the numbers of apoptotic figures below those of control cultures, indicating that the inhibitory response is dose dependent. Taken together, the results suggest that caspases 1, 2, and 3, and perhaps others that are blocked by the inhibitors we used, participate in TNF-alpha-induced cell death in vitro.