Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G(2) arrest of the cell cycle

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is hom ologous to a family of cytolethal distending toxins (Cdt) expressed by seve ral Gram-negative bacteria. Moreover, we have shown that CdtB impairs lymph ocyte function by inducing G(2) arrest of the cell cycle. We now report tha t both CdtB as well as an extract prepared from an Escherichia coli strain that expresses all three of the A. actinomycetemcomitans cdt genes (rCdtABC ) induce apoptosis. Pretreatment of lymphocytes with either CdtB or rCdtABC leads to DNA fragmentation in activated lymphocytes at 72 and 96 h. No DNA fragmentation was induced in nonactivated cells. Flow cytometric analysis of the Cdt-treated lymphocytes demonstrates a reduction in cell size and an increase in nuclear condensation. Mitochondrial function was also perturbe d in cells pretreated with either CdtB or rCdtABC. An increase in the expre ssion of the mitochondria Ag, Apo 2.7, was observed along with evidence of the development of a mitochondrial permeability transition state; this incl udes a decrease in the transmembrane potential and elevated generation of r eactive oxygen species. Activation of the caspase cascade, which is an impo rtant biochemical feature of the apoptotic process, was also observed in Cd t-treated lymphocytes. Overexpression of the bcl-2 gene in the human B lymp hoblastoid cell line, JY, led to a decrease in Cdt-induced apoptosis. Inter estingly, Bcl-2 overexpression did not block Cdt-induced G2 arrest. The imp lications of our results with respect to the immunosuppressive functions of Cdt proteins are discussed. The Journal of Immunology, 2001.