CD19(+) cells produce IFN-gamma in mice infected with Borrelia burgdorferi.

We have recently shown that production of IFN-gamma and IL-10, but not IL-4 is specifically induced in the lymph nodes of C3H/HeJ (disease susceptible) and C57BL/6J (disease resistant) mice 1 week after infection with Borrelia burgdorferi spirochetes. The present study was conducted to determine the phenotypes of ex vivo lymph node cells obtained from infected mice of both strains at this time point. The percentages of CD3(+), CD4(+), CD8(+), TCRalpha/beta (+) and TCRgamma/delta (+) cells decreased in both strains of mice compared to LN from naive mice. In contrast, there was a threefold increase in the proportion of CD19(+) cells. In view of this expansion of the B cell proportion, we examined the ability of purified CD19(+) cells and CD43(+) cells to produce both IL-10 and IFN-gamma when the cells were restimulated in vitro with B. burgdorferi freeze-thawed spirochetes. As expected, CD43(+) cells were able to produce both cytokines, but not IL-4. Surprisingly, CD19(+) (B) cells also were able to produce IFN-gamma in comparable amounts, in addition to IL-10. Intracellular staining of CD19(+) cells with anti-IFN-gamma antibody confirmed this finding. We discuss this novel phenomenon in terms of its possible underlying mechanisms and its relevance, both in the context of the immunology of Lyme disease and that of other infectious diseases.

Borrelia burgdorferi stimulates in vitro a selective expansion of CD3-CD4-CD8- (triple negative) autoreactive cells in naive rhesus monkey lymphocytes.

Autoreactive cell lines were generated from cell suspensions of freshly isolated naive monkey lymph node (LN) cells and peripheral blood mononuclear cells by cocultivation with freeze-thawed Borrelia burgdorferi spirochetes (Bb/FT). These cells produced interleukin (IL)-6 when cocultured with autologous antigen-presenting cells (APCs) alone without Bb/FT. IL-6 production was not observed when control cell lines were stimulated in the same fashion. CD4+-enriched T cell populations obtained from the LN autoreactive cell line also produced IL-6 when cultivated with APCs alone. When these cells were cultivated further in the presence of APCs, a population of cells whose phenotype was CD56+/-CD4-CD8-CD3- was predominantly selected. These cells both proliferated and produced IL-6 when cocultured with APCs alone. The possible relevance of these cells to Lyme disease pathogenesis remains to be determined.

Early induction of gamma interferon and interleukin-10 production in draining lymph nodes from mice infected with Borrelia burgdorferi.

Lymph node (LN) cells from C3H/HeJ mice (Lyme disease susceptible) infected for 1 week with Borrelia burgdorferi strain JD1 produced higher levels of gamma interferon (IFN-gamma) when stimulated in vitro with B. burgdorferi spirochetes than equivalent cells from B. burgdorferi-infected C57BL/6J mice (disease resistant). The interleukin-10 (IL-10) levels were comparable in the two strains, whereas the IL-4 levels were below detection limits. B. burgdorferi-stimulated LN cells from C57BL/6J mice produced significantly higher levels of IFN-gamma in the presence of neutralizing anti-IL-10 antibody than cells cultured with B. burgdorferi alone. No effect of IL-10 neutralization on IFN-gamma production by LN cells from C3H/HeJ mice was observed. Neutralizing antibody to IFN-gamma had no effect on the production of IL-10 by LN cells from C57BL/6J mice. A slight decrease in IL-10 production was detected in culture supernatants of equivalent cells from C3H/HeJ mice. The differential effect of IL-10 on IFN-gamma production in C57BL/6J and C3H/HeJ mice suggests that IL-10 is probably involved in the regulation of IFN-gamma production by LN cells during infection and may be at the root of the differential susceptibility to Lyme arthritis in these two strains of mice.

Identification of an Ixodes ricinus salivary gland fraction through its ability to stimulate CD4 T cells present in BALB/c mice lymph nodes draining the tick fixation site.

BALB/c mice infested with larvae or nymphs of Ixodes ricinus develop in their lymph nodes a T cell-specific immune response triggered by salivary gland soluble antigens (SGA). SGA are apparently conserved in the 3 biological stages of I. ricinus ticks and are species specific. SGA derived from partially fed females I. ricinus stimulate lymph node T cells from mice infested with I. ricinus larvae or nymphs. In contrast, lymph node cells from mice infested with Amblyomma hebraeum nymphs do not respond. A chromatographic fraction enriched with a 65 kDa protein (IrSG65) isolated from salivary glands of I. ricinus partially fed females induces in vitro a specific T cell proliferation of lymph node cells from mice infested with I. ricinus nymphs. The depletion of CD4+ T cells drastically reduces the ability of lymphocytes from infested mice to proliferate after IrSG65 stimulation.

Cytokine production by lymph node cells from mice infested with Ixodes ricinus ticks and the effect of tick salivary gland extracts on IL-2 production.

In BALB/c mice repeatedly infested with nymphal Ixodes ricinus ticks, lymphocytes from axillary and brachial lymph nodes which drain the tick attachment site produced significant levels of IL-2, TNF-alpha and GM-CSF when stimulated in vitro with Con A or anti-CD3 antibodies. Cytokine production by cells from lymph nodes of the opposite flank was equivalent to that of cells from uninfested mice. Nine days after the first infestation and IL-2, GM-CSF were produced primarily by the CD4+ T cells, while some other cell types contributed also to the TNF-alpha production. In mice repeatedly infested, a gradual increase of lymph node cell production of IL-2 was observed. The IL-2 levels regularly increased from the first to the third infestation compared to TNF-alpha levels which gradually decreased. The in vitro production of GM-CSF was not affected by successive infestations. Spleen lymphocytes from naive mice produced higher levels of IL-2 than lymphocytes from axillary and brachial lymph nodes. Both tick salivary gland extracts and D-mannose inhibited IL-2 production by these lymphocytes.

Immunosuppression and cytokine production in mice infested with Ixodes ricinus ticks: a possible role of laminin and interleukin-10 on the in vitro responsiveness of lymphocytes to mitogens.

T cells from BALB/c mice infested 9 days before with Ixodes ricinus nymphs had a suppressed response to in vitro concanavalin A (Con A) stimulation compared to cells from uninfested mice. When laminin (the main component of the extracellular matrix) was used as a coating agent, the Con A response of naive mice was characterized by a decrease in cell proliferation, whereas there was no significant effect on the mitogen response of cells from infested mice. In contrast, an increased response to lipopolysaccharide (LPS) was observed when assaying lymph node cells of infested mice, probably reflecting an increase in B-lymphocyte number or activity. LPS cell stimulation was not modified by laminin. Supernatants of lymph node cells, taken 9 days after the first infestation of mice, stimulated with Con A in vitro, contained interleukin-10 (IL-10) but no significant levels of IL-5 as tested by enzyme-linked immunosorbent assay. At this stage of the infestation all T cells reactive with tick antigens generated in lymph nodes that drain the tick fixation site, were CD4+ cells, as determined by CD4+ depletion. With cells taken 9 days after the third infestation an increase of IL-5 and IL-10 was observed. The IL-10 levels were higher than the IL-5. According to these observations, we conclude that the reduction of T-cell proliferation in response to Con A observed in lymph node cells from infested mice, may be due to the combined effect of laminin interaction with T lymphocytes during migration and IL-10 production by these lymphocytes.

In vitro production of interleukin-4 and interferon-gamma by lymph node cells from BALB/c mice infested with nymphal Ixodes ricinus ticks.

In this study we compared the ability of lymphocytes taken from axillary and brachial lymph nodes of BALB/c mice that had been infested once three times with 15 nymphal Ixodes ricinus ticks, to produce interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) after in vitro stimulation with concanavalin A (Con A). They released high levels of IL-4 and low levels of IFN-gamma. An increase of IFN-gamma between the first and the third tick infestation was observed. Salivary gland extracts from female I. ricinus ticks induced specific in vitro proliferation of lymphocytes from infested mice. IL-4 production was correlated with the salivary gland extracts' ability to stimulate tick-specific lymphocyte proliferation. Its levels remained high from the first to the third infestation. IFN-gamma production was not necessarily associated with tick salivary gland antigen stimulation. In BALB/c mice, anti-tick immune response induction is regional and the contribution of other similar secondary lymphoid organs is negligible. Only cells from the lymph nodes which drained the tick-fixation site proliferated in vitro in the presence of tick antigens, and when stimulated with Con A produced IL-4 and IFN-gamma.