Role of CD14 molecules in internalization of Actinobacillus actinomycetemcomitans by macrophages and subsequent induction of apoptosis

We report the evidence for apoptosis in 5774.1 cells by the periodontopathic bacterium Actinobacillus actino-mycetemcomitans, suggesting that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis. In this study, we examined the role of macrophage CD14, anchored by a glycerophosphatidylinositol tail, in the induction of apoptosis by A. actinomycetemcomitans infection by using the parent 5774.1 cells and CD14-defective mutant (LR-9) cells. A small number of A. actino-mycetemcomitans Y4 cells inside the LR-9 cells compared with the number in 5774.1 cells was detected by confocal scanning microscopy, We found that LR-9 cells showed a weak cytotoxic effect after being infected with A. actinomycetemcomitans Y4. Apoptotic cell death of LR-9 cells infected with A. actinomycetemcomitans Y4, compared with that of the parent 5774.1 cells was almost undetectable, as shown by the proportion of fragmented DNA in agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP end-labeling method, Flow cytometric cell cycle analysis of 5774.1 cells infected with A, actinomycetemcomitans Y4 revealed the increased percentage of apoptotic cells,vith hypodiploid DNA, However, LR-9 cells infected with A. actinomycetemcomitans Y4 showed no increase in population of apoptotic nuclei compared with the noninfected cells. These findings suggest that the CD14 molecules may contribute to the phagocytosis of A. actinomycetemcomitans by 5774.1 cells and regulate, at least in part, apoptotic cell death of macrophages infected with A. actinomycetemcomitans.