Neutrophils Die in the Gingival Crevice, Periodontal Pocket, and Oral Cavity by Necrosis and Not Apoptosis

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Abstract
Neutrophils play important roles in the homeostasis of periodontal tissues. However, remarkably little is known about how these cells live and die in the gingival crevice and periodontal pocket. We have examined whether subgingival neutrophils die by necrosis or apoptosis and have begun to study the mechanisms controlling the functional lifespan of these cells. Neutrophils collected from clinically healthy gingival crevices, periodontal pockets, and the oral cavity (saliva) were examined with Hoechst dye 33342, propidium iodide/eithidium bromide, and FITC-annexin V to identify live, dead, and apoptotic cells. Blood neutrophils were cultured for 17 to 20 hours in the presence or absence of gingival crevice washings (GC-w) to study the effect of GC-w on neutrophil apoptosis. In addition, endotoxin was removed from GC-w by affinity resin to investigate the contribution of LPS to the inhibitory effect of GC-w on blood neutrophils. The percentage of dead neutrophils in all subgingival sites and in all oral samples far exceeded the percentage of apoptotic neutrophils. In all 3 locations, approximately 30% of neutrophils were dead, whereas less than 1% of neutrophils were apoptotic. We conclude that the majority of neutrophils which lose their viability within gingival crevices, periodontal pockets and the oral cavity die by necrosis and not by apoptosis. Washings obtained from clinically healthy gingival crevices (GC-w) variably, but significantly, delayed apoptosis of peripheral blood neutrophils (mean suppression 45.7% +/- SD 22.3). Removal of endotoxin from GC-w significantly reduced this inhibitory effect. Our findings provide insights into the mechanisms of neutrophil death and the control of the functional lifespan of neutrophils in gingival crevices and periodontal pockets and therefore into the pathogenesis of periodontal diseases.