Modulation of gamma interferon-induced major histocompatibility complex class II gene expression by Porphyromonas gingivalis membrane vesicles.

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, has been shown elsewhere to be a major etiologic agent of destructive adult periodontitis (37). Recent epidemiological studies have suggested that adult periodontitis may cause conditions more serious than tooth loss. These include cardiovascular disease (1, 28) and preterm labor and low birth weight (27). Like other gram-negative bacteria, P. gingivalis produces outer membrane vesicles (OMVs), by the outgrowth of its outer membranes, which are subsequently released into the surrounding environment (12). It has been proposed elsewhere that such vesicles are liberated from the bacteria by a mechanism involving cell wall turnover (46). Consequently, vesicles normally retain a full complement of outer membrane constituents of the bacterial cell wall, including proteins, lipopolysaccharide, muramic acid, capsule, and fimbriae (8, 12, 46). Many of these components play a crucial role in the pathogenicity of P. gingivalis. In addition, vesicles exhibit adhesion properties and strong proteolytic activity, which, together with their small size (20 to 500 nm), enable them to penetrate the intact oral mucosa and enter the underlying gingival tissue (24). By this activity, vesicles are able to deliver and release the numerous virulence factors that they contain into the surrounding tissue. It is well documented that venular endothelial cells actively participate in immunological responses by expressing certain adhesion and major histocompatibility complex (MHC) molecules in response to specific cytokines. For example, endothelial cells can participate in acquired immune responses by the induction and expression of MHC class II genes in response to gamma interferon (IFN-γ) (6), which enables them to function as antigen-presenting cells (APCs) (36, 43). As such, endothelial cells have the ability to stimulate antigen-specific CD4+ T cells, resulting in their activation and proliferation. Thus, the regulation of MHC class II synthesis is of considerable biological importance. Studies have shown that the induction of MHC class II molecules by IFN-γ requires the de novo synthesis of the class II transactivator (CIITA), which interacts with ubiquitous DNA binding proteins at MHC class II promoters (2, 3, 22, 40). The activation of CIITA occurs following the binding of IFN-γ to its specific receptor, which triggers the tyrosine phosphorylation of signaling components, i.e., the receptor-associated tyrosine kinases, Jak1 and Jak2, and Stat1α (11). The phosphorylated Stat1α forms a homodimer which functions as a transcriptional factor that translocates to the nucleus and binds to the gamma activation sequence in IFN-γ-stimulated genes, including the CIITA gene (16, 34). Previous studies in this laboratory demonstrated that treatment of endothelial cells with P. gingivalis membrane vesicles can inhibit the surface expression of IFN-γ-induced HLA-DR molecules, whereas other cytokine-induced molecules, such as tumor necrosis factor alpha-induced E-selectin or interleukin-1-induced intercellular adhesion molecule 1, were not affected (39). This suggests that the inhibitory effect observed may suppress specific immune responses, enabling the bacteria to survive and colonize host tissues. Therefore, the present study was undertaken to determine the molecular target of inhibition by P. gingivalis membrane vesicles. For this purpose, the effects of vesicles on IFN-γ-induced expression of both the HLA-DR gene and the CIITA gene as well as on the IFN-γ signaling cascade involving Jak and Stat proteins were examined.