organisms are easily killed when they bind specific antibody that activates complement (8
). Several outer surface proteins of B. burgdorferi
, including OspA, OspB, OspC, and the 39-kDa protein (2
), induce complement-dependent borreliacidal antibody. This feature makes these outer surface proteins ideal candidates for development of a vaccine to prevent infection with B. burgdorferi
. Of these proteins, OspA has been used to vaccinate humans (34
) and animals (11
Although rOspA is immunogenic, the antibody response is dominated by production of antibody that is not protective (10
). Most importantly, vaccinated animals challenged with B. burgdorferi
during periods with concomitant high levels of nonbactericidal anti-OspA and low levels of anti-OspA borreliacidal antibodies develop arthritis (11
). Recently, we showed that hamsters vaccinated with 30, 60, or 120 μg of rOspA with or without alum developed severe destructive arthritis when challenged with B. burgdorferi
). Similarly, hamsters vaccinated with a commercially available canine rOspA vaccine developed severe destructive arthritis after challenge with the Lyme spirochete (11
). Only the presence of high levels of borreliacidal antibody prevented infection with B. burgdorferi
from inducing arthritis in vaccinated animals (19
). Maximum protection due to borreliacidal antibody, however, is restricted to 7 to 9 weeks after vaccination.
One approach to increase the duration of protection and lessen the potential for adverse effects is to determine the immune mediators responsible for the production and maintenance of borreliacidal antibody. We showed previously (23
) that IL-4 does not augment the production of anti-OspA borreliacidal antibody, even though IL-4 is known to up-regulate B-lymphocyte growth and differentiation (27
). In fact, IL-4 inhibited the production of anti-OspA borreliacidal antibody, including IgG2a, by lymph node cells obtained from vaccinated mice (23
). It is known that IgG2a is negatively regulated by IL-4 but up-regulated by IFN-γ (26
). Our data and those of others (26
) suggested that IL-4 counteracted the effects of IFN-γ on the production of borreliacidal antibody.
When lymph node cells producing borreliacidal antibody were exposed to rIFN-γ, borreliacidal antibody production was inhibited. The time of exposure (10 min to 4 days) of immune lymph node cells to rIFN-γ did not affect the inhibition of production of borreliacidal antibody. In addition, treatment with rIFN-γ inhibited the production of other anti-B. burgdorferi
antibodies. The suppression of borreliacidal and other anti-B. burgdorferi
antibodies by rIFN-γ was unexpected. Flow cytometric analysis of rIFN-γ-treated and untreated immune lymph node cells showed that treatment with rIFN-γ reduced the number of viable lymphocytes, especially B lymphocytes. In addition, macrophages cultured in the presence of exogenous rIFN-γ were rounded and exhibited rare pseudopodia. By contrast, macrophages obtained from cultures of untreated lymph node cells producing borreliacidal antibody were spindle shaped with many pseudopodia. IFN-γ has been shown to induce apoptosis in T (1
) and B (15
) lymphocytes. These events could have affected the activation of macrophages and their ability to process borrelial antigen.
It is possible that inhibition of borreliacidal antibody was due to the use of toxic concentrations of rIFN-γ. However, when more physiologically relevant concentrations (1.0 or 0.1 μg) of rIFN-γ were added to immune lymph node cells, borreliacidal activity also failed to increase. In addition, immune lymph node cells were treated with anti-CD119, which blocks the binding of murine IFN-γ to cellular receptors (9
). No significant decrease in borreliacidal-antibody production was detected in these cultures compared to immune lymph node cells treated with an isotype-nonspecific antibody (data not shown). These results suggest that IFN-γ is not a major force in driving the production of borreliacidal antibody.
Perhaps the most compelling evidence that IFN-γ is not responsible for production of borreliacidal antibody came from experiments utilizing neutralizing antibody to IFN-γ. Treatment of immune lymph node cells with various concentrations of anti-IFN-γ failed to suppress borreliacidal activity. In fact, borreliacidal antibody production was marginally enhanced, especially when treatment with anti-IFN-γ occurred early in cultivation of the immune lymph node cells. Furthermore, treatment with anti-IFN-γ resulted in polyclonal expansion of the anti-B. burgdorferi antibody response. In support of this idea, by using flow cytometric analysis, we also detected a significant increase in the number of B lymphocytes in cultures of immune lymph node cells treated with anti-IFN-γ.
IFN-γ performs numerous immunologic functions, including T helper lymphocyte differentiation and stabilization (4
), enhancement of major histocompatibility complex expression on both B lymphocytes and macrophages (4
), antiviral effects (4
), and amelioration of production of IgG2a (14
). Our results, however, show that IFN-γ suppresses antibody production, including IgG2a borreliacidal antibody (23
). An explanation may be that IgG2a antibody expression is not completely dependent upon IFN-γ (14
). In addition, IFN-γ may prevent the expression of IL-4 functions (26
). However, we showed previously that IL-4 suppressed the production of borreliacidal antibody (23
). This suggests that other cytokines, but not IL-4 or IFN-γ, may be responsible for the induction, production, and maintenance of borreliacidal antibody. In support of this idea, treatment with anti-IFN-γ augmented not only borreliacidal antibody but other antibodies directed against B. burgdorferi
. Additional studies are needed to determine which cytokines are responsible for the production of borreliacidal antibody.
In conclusion, we showed that IFN-γ plays a major role in suppression of the production of borreliacidal antibody. Effective neutralization of endogenous IFN-γ slightly augmented the production of borreliacidal antibody and expanded the anti-B. burgdorferi antibody responses. Determination of the mechanism that inhibits the production of borreliacidal antibody by rIFN-γ may lead to the development of a safe and effective Lyme borreliosis vaccine.