Several cytoplasmic proteins are required to inhibit cell division of T cells (13
). We focused on p19 because it inhibits cyclin-dependent kinases (CDKs), especially CDK6, the earliest inducible member of the CDK family involved in cell cycle progression of T lymphocytes (13
). Increasing amounts of intracellular p19 have also been shown to inhibit the kinase activity of CDK4 (13
). Therefore, p19 may restrict the number of T cells that can be produced following antigenic stimulation or infection (13
). This characteristic of p19 may also prevent tumor development (9
) and T-cell-driven or -associated diseases (18
). Little information, however, is available as to whether inhibitors of p19 will augment immune responses associated with Borrelia
vaccination and challenge. Selective enhancement or inhibition of p19 could point the way to prevention of Lyme arthritis or enhancement of the quality of the protective borreliacidal response in future Lyme vaccines.
We showed previously that B. burgdorferi
-vaccinated mice challenged with B. bissettii
develop arthritis at the tibiotarsal joint (4
). This finding is confirmed in the present report. The synovial space, synovium, and subsynovial tissue of the tibiotarsal joint of Borrelia
-vaccinated and -challenged mice showed a severe inflammatory response involving a profuse infiltration of neutrophils. Subsequently, we hypothesized that the inflammatory arthritic response could be exacerbated by neutralizing p19 in Borrelia
-vaccinated and -challenged mice. We speculated that in the absence of the p19 inhibitor, the responding immune cells in the periarticular tissues would continue to proliferate and promote the inflammatory response in the tibiotarsal joint.
Here, we show that anti-p19 antibody treatment did augment the inflammatory response normally detected at the tibiotarsal joints of Borrelia-vaccinated and -challenged mice. Although the synovial space, synovium, and subsynovial tissues contained a profuse infiltration of neutrophils, the most distinctive feature detected in anti-p19 antibody-treated Borrelia-vaccinated and -challenged mice was erosion of cartilage and bone at the tibiotarsal joint. In fact, the joints were destroyed in most of the anti-p19-treated Borrelia-vaccinated and -challenged mice compared to the non-anti-p19-treated Borrelia-vaccinated and -challenged control mice. These results show that treatment with anti-p19 antibody exacerbates the arthritic response.
Treatment with anti-p19 antibody also induced several other effects in Borrelia-vaccinated and -challenged mice. Anti-p19 antibody treatment enhanced the borreliacidal antibody response of Borrelia-vaccinated and -challenged mice. Borreliacidal antibody increased two- to fourfold against both the vaccine and challenge agent compared to the borreliacidal activity detected in non-p19 antibody-treated Borrelia-vaccinated and -challenged mice. In addition, anti-p19 treatment was effective in enhancing borreliacidal antibody even when administered several days after Borrelia-vaccinated mice were challenged, especially against the vaccine agent. These results support our claim that anti-p19 treatment can affect the immune response to Borrelia vaccination and challenge.
In an attempt to elucidate the mechanism by which treatment with anti-p19 antibody exacerbated arthritis, we determined the number of lymphocytes, CD4+
T cells, and CD4+
T cells in the lymph nodes (popliteal and inguinal) draining the site of the arthritic response. In general, the number of these cells was increased in anti-p19 antibody-treated Borrelia
-vaccinated and -challenged mice compared to the controls. This suggests that treatment with anti-p19 antibody prolongs or amplifies the immune response. In support of this hypothesis, it is known that increases in the number of lymph node cells and CD4+
T cells are associated with antigenic stimulation, cell proliferation, and activation (39
). Of special interest is the number of CD4+
T cells in the anti-p19-treated Borrelia
-vaccinated and -challenged mice. Although CD4+
T cells may play a major role in suppressing immune reactivity (8
), initially CD4+
T cells expressing CD25, the IL-2α receptor, are active participants in developing the immune response (19
). The increase (13%) in CD4+
T cells found in the lymph nodes of anti-p19 antibody-treated Borrelia
-vaccinated and -challenged mice may have aided the arthritis-inducing effector CD4+
T cells by binding the T-cell growth-promoting factor, IL-2, to the CD25 receptor. This would further stimulate proliferation of arthritic reactive CD4+
T cells (19
) and account for the enhanced histopathology of the tibiotarsal joint found in anti-p19 antibody-treated Borrelia
-vaccinated and -challenged mice.
The ability of anti-p19 antibody treatment to augment the borreliacidal antibody response of Borrelia-vaccinated and Borrelia-vaccinated and -challenged mice, especially against the vaccine isolate, was a surprise. Sustained high levels of borreliacidal antibody is an important feature for establishing the efficacy of the next generation of Lyme vaccines. A major concern, however, for vaccine manufacturers is how to stimulate production of a strong and long-lived borreliacidal antibody response that has minimal side effects. Our results show that considerable enhancement of borreliacidal antibody can be achieved with anti-p19 antibody treatment; however, the mechanism of enhancement needs to be determined. By contrast, Borrelia-vaccinated and -challenged mice treated with anti-p19 antibody exhibited enhanced lymphocyte responses and borreliacidal activity to the vaccine isolate. Anti-p19 antibody treatment of Borrelia-vaccinated mice and Borrelia-vaccinated and -challenged mice may have caused a restricted clonal expansion of T cells specifically dedicated to stimulate production of anti-297 borreliacidal antibody (vaccine isolate). This restriction may have limited the borreliacidal antibody response to the challenge agent, B. bissettii. Our results show that anti-19 antibody treatment of vaccinated mice can enhance borreliacidal antibody which kills the homologous challenge agent, even when anti-p19 antibody treatment is delayed for 7 or 15 days. Additional studies with anti-p19 antibody are needed to determine the mechanism of antibody enhancement.
The novel activities of anti-p19 antibody are difficult to explain. How was the intracellular protein, p19, neutralized by treatment with anti-p19 antibody? It is generally accepted that antibodies cannot cross the cell membrane of eukaryotic cells (33
). However, there are antibodies called intrabodies that can function within a cell (24
), and these can inhibit protein function (24
). Intrabodies contain the antigen-binding regions and are generally small fragments (light chain) of the antibody molecule (24
). Presumably, anti-p19 antibody was altered in Borrelia
-vaccinated and -challenged mice to form intrabodies that crossed the cell membrane and neutralized p19. Another explanation may be that p19 is expressed transiently on the surface of T cells and that interaction with anti-p19 antibody inactivates or down-regulates production of intracellular p19. A similar mechanism has been described for down-regulation or prevention of the production of cytokines (19
). Finally, anti-p19 antibody may be cross-reactive and bind to the cell receptors of alpha interferon or IL-10. These cytokines have been shown to increase the CDK inhibitor p19 (34
) and inhibit proliferation of immune cells (34
). Blockage of the receptors would prevent production of p19. Presently, we are investigating this latter hypothesis.
In conclusion, we show that treatment with anti-p19 antibody can alter the immune response, especially the severity of the arthritis induced in Borrelia-vaccinated and -challenged mice.