Characteristics and responses of EBV immortalized B cells from periodontal disease patients

This study was designed to examine human B cell responses to Actinobacillus actinomycetemcomitans (Aa). The general hypothesis to be tested was that Epstein-Barr virus (EBV) immortalized B cells could be used to investigate variations in B cell responsiveness of periodontitis patients to periodontal pathogens, and that B cells derived from the peripheral blood of periodontal disease patients infected with Aa demonstrate differences in in vitro activities compared to periodontally healthy subjects.

EBV-transformed B cell lines were used to analyze immunoglobulin and Aa-specific antibody responses, as well as to determine the frequencies of cells producing immunoglobulin (Ig) of a specific isotype and detect clones secreting antibodies specific for Aa. Lymphoblastoid cells lines (LCL) were derived by clonal transformation of peripheral blood lymphocytes from 10 Aa-infected patients with adult periodontitis (Aa-AP) and seven normal subjects.

The B cells were incubated in Aa-coated polystyrene plates to separate adherent and non-adherent cells, and stimulate the cells with the whole bacteria. In addition, the B cells were stimulated with Aa LPS, E. coli LPS, or the polyclonal B cell activators (PBAs), pokeweed mitogen (PWM) and Staphylococcus aureus protein A (SpA). Both adherent and non-adherent cell populations were cultured for up to 15 days.

Total immunoglobulins (Igs) and antibody (IgG, IgA, IgM) levels to Aa in the culture supernatants were assessed using an ELISA. The distribution of IgG, IgA, IgM and Aa-specific antibody producing cells was analyzed by a double immunoenzymatic staining technique.

IgM levels produced by the LCLs were significantly increased vs IgG and IgA (P < 0.001). Three days after Aa stimulation, a marked increase in the level of total Igs and Aa-specific antibody was observed in adherent cells from Aa-AP (P < 0.05-0.03). Aa-specific antibody levels were significantly higher in the supernatants from Aa-AP vs normals throughout the culture interval (P < 0.03). There was also a significant increase in Aa-specific antibody levels after stimulation with Aa LPS or E. coli LPS (P < 0.05), whereas PWM and SpA had no significant effect on antibody to Aa. There was a predominance of IgM cells compared to IgG and IgA isotypes (P < 0.04) in LCLs from Aa-infected patients. After stimulation with Aa, a significant increase in the number of IgA (111%) and IgG (48%) secreting cells was observed, concomitant with a 74% decrease in the Ig-negative cell population. Total Aa+ cells increased significantly after stimulation (P < 0.001), predominated by Aa-specific IgG and IgM antibody producing cells.

These results showed that LCLs from Aa-infected patients were polyclonal with respect to isotype distribution. Further stimulation with Aa revealed a shift to cytoplasmic IgG and IgA expression, as well as increases in the Aa-specific B cell population. In contrast, the PBAs stimulated the LCLs to synthesize primarily IgM. Additionally, the findings indicated that: (1) without T cells, polyclonal activation of B cells may lead to elevated Aa-specific B cell populations; and (2) the presence of previously sensitized B cells is required to exert an antigen specific antibody response in the LCL. We conclude that secondary activation of primed B cells by oral bacteria or their products in advanced periodontal lesions may contribute to the local accumulation of significant numbers of Ig-producing cells. This report also suggested that EBV-mediated transformation can be used to probe B cell-bacterial interactions in studies of periodontitis.