| Periodontal disease causes the destruction of the periodontium. A. actinomycetemcomitan is a Gram-negative capnophilic bacterial pathogen that has been associated with Localized Juvenile Periodontitis (LJP). Neutrophils are initially the predominant cells in the host defense of periodontal disease due to their ability to phagocytose and remove bacteria. To understand the pathogenesis of LJP, a better characterization of the role of natural host defense mechanisms, including the maximum phagocytic and bactericidal capabilities of normal human neutrophils is essential. Neutrophil activation by various A. actinomycetemcomitans strains and a close relative, Haemophilus aphrophilus, was studied by using a chemiluminescence technique, which measures oxygen radical production. To assess the A. actinomycetemcomitans/neutrophil interaction directly, all A. actinomycetemcomitans strains were labeled genetically with green fluorescence protein (GFP). The fate of the GFP-labeled bacteria was then determined by flow cytometry and fluorescence microscopy. Fluorescence staining was also used to assess the viability of bacterial cells. Data obtained confirmed some earlier observations that, in contrast to H. aphrophilus, phagocytosis of A. actinomycetemcomitans by human neutrophils is generally inefficient in the presence of complement alone due to lack of bacterial attachment. Phenotypic variation and leukotoxin production by A. actinomycetemcomitans played only a minor role in serum resistance. In addition, the ability of this organism to evade neutrophils did not appear to be associated with a specific bacterial serotype. In conclusion, we have developed methods that provide a rapid, reliable and reproducible approach to monitor and quantitate the different steps involved in A. actinomycetemcomitans /neutrophil interaction such as adherence, ingestion and killing. |
