The Reduction of Caspase-3 Expression Delays Apoptosis in LAP PMN

Objectives: Apoptosis of PMN is required for the resolution of inflammation. Oxidative stress is an important factor in the pathogenesis of several inflammatory diseases, such as periodontitis, diabetes, and arthritis. PMNs generate superoxide and other reactive oxygen species, which are major oxygen-dependent components of the anti-microbial arsenal in phagocytic leukocytes. The superoxide is converted into hydrogen peroxide (H₂O₂), which is a key factor in induction of apoptosis. PMNs from Localized Aggressive Periodontitis (LAP) patients are characterized by enhanced superoxide generation. The abnormal function of PMNs plays a key role in the pathogenesis of inflammation in this disease. We hypothesized that apoptosis of PMNs is delayed in LAP, which leads to increased tissue damage. Methods: PMNs were isolated from peripheral blood by density gradient centrifugation. Rate of cell death in H₂O₂-induced apoptotic PMNs was assessed by trypan blue dye exclusion methods. Expression and activation of caspase-3 were monitored by Western blot analysis. Results: H₂O₂ induced cell death in a dose- and time-dependent manner. PMNs from LAP patients were resistant to H₂O₂-induced apoptosis, and approximately 10 times more H₂O₂ was required to induce cell death than in PMNs from healthy subjects. A significant correlation was observed between increased superoxide generation and resistance to H₂O₂ cell death in LAP PMN (r² = 0.7787). H₂O₂ also enhanced the cleavage of caspase-3 in a dose- and time-dependent manner. However, LAP PMN exhibited reduced cleavage of caspase-3 upon cell stimulation and a 25% reduction of expression of caspase-3 in resting state (P<0.05). Conclusion: These results indicate that delayed apoptosis is consistent with reduction of caspase-3 activity and that it is a part of chronic inflammation leading to tissue destruction in LAP. Supported by NIH grants DE16191 and RR00533.