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 kille r (NK) cells were susceptible to the cytolytic effects of the Actinoba cillus actinomycetemcomitans leukotoxin (LTX). Following treatment wit h LTX (0-200 ng/ml), NK cell activation by interleukin-2 (IL-2) was ev aluated. LTX inhibited the IL-2-induced expression of both CD69 and th e IL-2 receptor. Furthermore, the up-regulation of CD56 was also impai red. To determine whether the observed functional deficits were the re sult of cell death, NK cell viability was evaluated by flow cytometry. Changes in forward and side light scatter patterns consistent with ce ll death were observed within 60 min. Direct analysis of cell viabilit y by measuring propidium iodide exclusion, however, indicated little c hange in the viability of LTX-treated NK cells. Electron microscopic a nalysis of NK cells exposed to LTX revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation, and condens ation of nucleoplasm. However, no change in membrane integrity was ini tially noted. Finally, LTX caused a rapid and sustained elevation in t he intracellular levels of Ca2+. These morphological and biochemical c hanges are consistent with the notion of programmed cell death.