The induction of a sustained high level of borreliacidal antibody is an important component for establishing the efficacy of Lyme borreliosis vaccines. Previously, we developed an in vitro assay to determine means by which borreliacidal antibody production can be augmented (20
). Cultivation of lymph node cells obtained from vaccinated (for 17 days) C3H/HeJ mice with macrophages and B. burgdorferi
resulted in borreliacidal antibody production of the isotypes IgG1, IgG2a, and IgG2b (20
). IL-4, a known stimulator of B lymphocytes, drives the production of antibodies of the IgG1 isotype (24
). Attempts to increase borreliacidal activity by treatment with exogenous rIL-4 were not successful (20
). The IgG2a component of the borreliacidal antibody response suggested that IFN-γ may act upon IL-4 in an antagonistic fashion (24
) and consequently induce borreliacidal antibody production. However, treatment of borreliacidal antibody-producing cells with exogenous IFN-γ resulted in a marked suppression of borreliacidal activity (19
). Surprisingly, the neutralization of IFN-γ augmented the borreliacidal antibody response (21
). Taken together, these data suggest that IL-4 and IFN-γ play only a minor role in the generation of borreliacidal antibodies.
These data also imply that borreliacidal antibody production may not have a large dependency on T-helper lymphocyte influence but may be driven by an immunological mediator, such as IL-6, that is liberated from other immune cells (9
). The role of IL-6 in the elicitation of humoral immunity has been characterized in a variety of systems (9
). Weis and colleagues (17
) also demonstrated that B. burgdorferi
functions as both a B-lymphocyte mitogen and a stimulus for the release of IL-6 in both in vitro and in vivo models of B. burgdorferi
infection. Anguita et al. (1
) demonstrated the necessity of IL-6 for the development of a complete humoral response upon stimulation by B. burgdorferi.
However, the effects of IL-6 on the borreliacidal antibody component of the total humoral response was not investigated.
Here we show that borreliacidal antibody-producing cells obtained from 17-day vaccinates and treated with a neutralizing antibody to murine IL-6 caused a significant reduction of borreliacidal activity compared to immune cells treated with an isotype control (Fig. ). In addition, the incorporation of anti-murine IL-6 into an established cell culture model of augmented borreliacidal activity (20
) resulted in a reduction of borreliacidal antibody production (Fig. ). Other studies performed by EIA showed that IL-6 was effectively neutralized by treatment with anti-IL-6. More importantly, treatment of borreliacidal antibody-producing cells with rIL-6 enhanced anti-OspA antibody production fourfold. Remington et al. (27
) also reported that rIL-6 increased the production of another borreliacidal antibody, OspC. These results suggest that IL-6 may play a major role in the regulation of antibodies that kill B. burgdorferi
Surprisingly, borreliacidal activity was greatly depressed after treatment of borreliacidal antibody-producing cells with rIFN-γ, despite the presence of high concentrations of IL-6 in the cultures. This suggests that other immune mechanisms or cytokines may participate in IL-6-mediated production of anti-OspA borreliacidal activity. It is also likely that IFN-γ can directly suppress borreliacidal activity, even in the presence of IL-6. In support of this statement, we showed previously that treatment of borreliacidal antibody-producing cells with rIFN-γ resulted in the preferential death of B lymphocytes (19
). Taken together, our results suggest that IL-6 induces borreliacidal antibodies while IFN-γ down-regulates the response. This may explain why borreliacidal antibodies (5
) are not sustained in naturally infected or vaccinated subjects (11
In this study, exogenous macrophages were required for IL-6-enhanced production of anti-OspA borreliacidal antibody. In contrast, Remington et al. (27
) showed that enhanced in vitro production of anti-OspC borreliacidal activity occurred in the absence of supplementation with macrophages. Since macrophages produce IL-6 (9
), these results imply that the level of IL-6 necessary to augment the anti-OspA borreliacidal antibody level is considerably higher than that for anti-OspC borreliacidal activity. In support of this suggestion, the neutralization of IL-6 had its greatest reduction effect on the production of anti-OspC borreliacidal antibodies (27
). This suggests that the levels of IL-6 present in the host after infection with B. burgdorferi
may contribute to the development of anti-OspC and the subsequent production of anti-OspA borreliacidal antibodies. We hypothesize that low levels of IL-6 present during early infection may stimulate anti-OspC production. Once processing and elimination of spirochetes occur by macrophages, IL-6 levels increase along with the production of anti-OspA borreliacidal antibodies.
An interesting observation was made by Western immunoblot analysis of lymph node supernatants. As expected, treatment of borreliacidal antibody-producing cells with rIL-6 resulted in an eightfold increase in anti-OspA titer that coincided with the observed increase in borreliacidal activity. However, when borreliacidal antibody-producing cells were treated with anti-murine IL-6, anti-OspA titers were not significantly decreased, even though a fourfold reduction in anti-OspA borreliacidal antibody titer was recorded. Further studies by flow cytometric analysis revealed an increase in B-lymphocyte populations in cultures of borreliacidal antibody-producing cells treated with rIL-6 compared to control cultures. Moreover, no decrease in the number of B lymphocytes was detected following treatment with anti-murine IL-6, despite a significant decrease in borreliacidal antibody production in these cell cultures.
These results mimic the previously reported bipartite humoral response that is induced by rOspA preparations (22
). The vaccination of LSH hamsters with rOspA resulted in borreliacidal antibody titers that peaked 6 weeks after vaccination, with significant waning thereafter. Immunization of human volunteers with rOspA also generated a significant borreliacidal antibody response that peaked at 60 days of immunization and waned rapidly thereafter. Only one individual had detectable borreliacidal activity half a year after immunization (22
). In contrast, anti-OspA EIA titers remained elevated in humans and hamsters (23
), despite the decline in borreliacidal activity.
These findings are important because they may facilitate the design of future Lyme borreliosis vaccines. Ma et al. (16
) reported that OspA possesses epitopes that do not generate borreliacidal antibodies. The data described in this investigation also suggest that macrophages process OspA borreliacidal epitopes and elicit IL-6 production. This results in borreliacidal antibody production without a strong influence from T-lymphocyte-specific cytokines. In contrast, nonborreliacidal OspA epitopes are processed in a fashion that results in a sustained high level of nonborreliacidal anti-OspA antibody through the enlistment of a T-lymphocyte interaction. Therefore, the waning of borreliacidal antibodies may follow classical T-cell-independent pathways (25
). The enhancement of borreliacidal activity may require the conjugation of borreliacidal epitopes with nonborreliacidal amino groups that induce IL-6 but not IFN-γ.
In conclusion, we demonstrate here the importance of IL-6 in the production of borreliacidal antibodies. Understanding the cytokine mechanisms responsible for the production of anti-OspA borreliacidal antibodies will facilitate the development of a more effective Lyme borreliosis vaccine.