SUMMARYT 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.

It has been established that interleukin-10 (IL-10) inhibits inflammatory cytokines produced by macrophages in response toLyme disease, caused by the spirochete Borrelia burgdorferi, is spread to humans and other mammals through the bite of infected Ixodes ticks (9). The spirochete can invade multiple organs (4, 59) and persist in them for a long time (47,65). Spirochetal persistence in the tissues has been associated with severe pathology (14,21,65) and both acute and chronic inflammatory conditions (50,59). Numerous studies have shown that B. burgdorferi and its lipoproteins can induce in a variety of cell types the release of proinflammatory cytokines, such as interleukin

We have recently shown that production of IFN‐γ 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+, TCRα/β + and TCR γ/δ + 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‐γ 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‐γ in comparable amounts, in addition to IL‐10. Intracellular staining of CD19+ cells with anti‐IFN‐γ 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.

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We investigated the effect of Borrelia burgdorferi lipoproteins (outer surface protein A) and the synthetic lipohexapeptide tripalmitoyl-S-glyceryl-Cys-Ser-4(Lys) (Pam 3 -Cys) on isolated lymph node (LN) cells from Lyme disease-susceptible (C3H/HeJ) and -resistant (C57BL/6J) mice. Mice were either infected with B. burgdorferi for 1 week or left uninfected. Lipoproteinstimulated LN cells from infected C3H/HeJ mice produced significantly higher levels of the inflammatory cytokines IL-6 and IFN-+ than did cells from C57BL/6J mice. Cells from uninfected mice did not respond. No TNF- § or IL-1 g were produced by LN cells from infected mice of either strain in response to lipoprotein or B. burgdorferi spirochetes. Unlike with IL-6 or IFN-+ , LN cells from either strain failed to produce IL-10 in response to lipoproteins. However, the LN cells were able to produce this cytokine in response to B. burgdorferi spirochetes or after incubation with phorbol-12-myristate-13-acetate/ionomycin, anti-CD3 antibody alone or anti-CD3 combined with anti-CD28 antibodies. Addition of exogenous IL-10 to lipopeptide-stimulated cultures significantly reduced IFN-+ and IL-6 production in a dosedependent fashion. This inhibition was more effective with cells from disease-resistant C57BL/6J mice than with cells from disease-susceptible C3H/HeJ mice. The proclivity to disease of the C3H/HeJ mouse could be simultaneously based on the phenomena of enhanced inflammatory responsiveness to lipoproteins and diminished ability to respond to IL-10. An investigation of the determinants of these two phenomena could be used as a blueprint to elucidate the pathogenesis of Lyme disease in humans.

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.

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.

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