Flow cytometric analysis of the cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human natural killer cells

The goal of this investigation was to determine if human natural killer (NK) cells were susceptible to the cytolytic effects of the Actinobacillus actinomycetemcomitans leukotoxin (LTX). Following treatment with LTX (0-200 ng/ml), NK cell activation by interleukin-2 (IL-2) was evaluated. LTX inhibited the IL-2-induced expression of both CD69 and the IL-2 receptor. Furthermore, the upregulation of CD56 was also impaired. To determine whether the observed functional deficits were the result of cell death, NK cell viability was evaluated by flow cytometry. Changes in forward and side light scatter patterns consistent with cell death were observed within 60 min. Direct analysis of cell viability by measuring propidium iodide exclusion, however, indicated little change in the viability of LTX-treated NK cells. Electron microscopic analysis of NK cells exposed to LTX revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation, and condensation of nucleoplasm. However, no change in membrane integrity was initially noted. Finally, LTX caused a rapid and sustained elevation in the intracellular levels of Ca²⁺. These morphological and biochemical changes are consistent with the notion of programmed cell death.