We determined previously that lipoproteins of

Brain invasion by Borrelia burgdorferi, the agent of Lyme disease, results in an inflammatory and neurodegenerative disorder called neuroborreliosis. In humans, neuroborreliosis has been correlated with enhanced concentration of glial fibrillary acidic protein in the cerebrospinal fluid, a sign of astrogliosis. Rhesus monkeys infected by us with B. burgdorferi showed evidence of astrogliosis, namely astrocyte proliferation and apoptosis. We formulated the hypothesis that astrogliosis could be caused by spirochetal lipoproteins. We established primary cultures of rhesus monkey astrocytes and stimulated the cells with recombinant lipidated outer surface protein A (L-OspA), a model B. burgdorferi lipoprotein, and tripalmitoyl-S-glyceryl-Cys-Ser-Lys 4 -OH (Pam 3 Cys), a synthetic lipopeptide that mimics the structure of the lipoprotein lipid moiety. L-OspA elicited not only astrocyte proliferation but also apoptosis, two features observed during astrogliosis. Astrocytes produced both IL-6 and TNF- § in response to L-OspA and Pam 3 Cys. Proliferation induced by L-OspA was diminished in the presence of an excess of anti-IL-6 antibody, and apoptosis induced by this lipoprotein was completely suppressed with anti-TNF- § antibody. Hence, IL-6 contributes to, and TNF- § determines, astrocyte proliferation and apoptosis, respectively, as elicited by lipoproteins. Our results provide proof of the principle that spirochetal lipoproteins could be key virulence factors in Lyme neuroborreliosis, and that astrogliosis might contribute to neuroborreliosis pathogenesis.

The mechanism of lymphedema development in individuals with lymphatic filariasis is presently poorly understood. To investigate whether Wolbachia, symbiotic bacteria living within filarial nematodes, may be involved in disease progression, Wolbachia-specific immune responses were assayed in a group of Brugia malayi-infected rhesus monkeys. Serum IgG antibodies specific for a major Wolbachia surface protein (WSP) were detected in 2 of 12 infected monkeys. It is interesting that both of these monkeys developed lymphedema after becoming amicrofilaremic. WSP-specific antibody responses were temporally associated with increases in antifilarial IgG1 antibodies as well as lymphedema development. These findings suggest that Wolbachia may be important in understanding disease caused by filarial worms.

We have recently demonstrated that interleukin-10 (IL-10), produced by THP-1 monocytes in response to Borrelia burgdorferi lipoproteins, dampens the production of concomitantly elicited inflammatory cytokines. Thus, IL-10 could potentially down-regulate inflammatory and microbicidal effector mechanisms of the innate immune response to a B. burgdorferi infection, facilitating the establishment of the spirochete. To understand the mechanism(s) implicated in the regulation of the synthesis and release of IL-10 during early infection, we investigated the autocrine effects of IL-6, IL-12, tumor necrosis factor alpha (TNF-␣), and IL-10 itself, as well as the exocrine effect of IFN-␥ on the production of macrophage-derived IL-10 with lipoprotein as a stimulant. In addition, in view of the differences in the receptor and signal transduction pathways of lipopolysaccharide (LPS) and bacterial lipoproteins, we also investigated the effects described above with LPS as a stimulant. Borrelia burgdorferi, the spirochete that causes Lyme disease, is spread to humans and other mammals through the bite of infected Ixodes ticks (7). Infection may involve multiple organs (3, 42) and may persist in certain tissues for prolonged periods of time (35,48). Spirochetal persistence in the tissues has been associated with severe pathology (9, 16, 48) and both acute and chronic inflammatory conditions (37, 42). B. burgdorferi lacks lipopolysaccharide (LPS) (43), but its genome contains no fewer than 150 genes coding for putative lipoproteins (18); this amounts roughly to 11% of its genome coding capacity. Lipoproteins can directly elicit inflammatory responses both in vitro and in vivo (20,36,38,39,47). Recent findings that lipoproteins can elicit not only inflammatory but also antiinflammatory mediators (e.g., interleukin-10 [IL-10]) from mononuclear cells present in peripheral blood (21, 22) and synovial fluid (49) have added support to the contention that lipoproteins play a crucial role in Lyme disease pathogenesis.One of the hallmarks of Lyme disease is arthritis. This form of the disease has been studied extensively in the mouse model, with an emphasis on the role of cytokines in its etiology. Infection of different inbred mouse strains with B. burgdorferi results in distinct disease outcomes (2,32,48). B. burgdorferi infection elicits severe arthritis in C3H/HeN mice, but mild arthritis in C57BL/6N mice, although both strains harbor similar numbers of spirochetes in their ankles (32). These findings indicate that C57BL/6N mice either produce less proinflammatory cytokines or are better able to regulate inflammation in response to B. burgdorferi lipoproteins than C3H/HeN mice, resulting in a less intense inflammatory response and decreased arthritis severity. Recently, Brown and colleagues (6) corroborated this hypothesis by showing that macrophages from C57BL/6N mice, when stimulated with the lipoprotein outer surface protein A (OspA) or LPS, secreted significantly less proinflammatory cytokines (IL-6 and tumor necrosis factor alpha ...

We previously showed that heat-killed Borrelia burgdorferi spirochetes and lipidated outer surface protein A (L-OspA) stimulated the in vitro production of interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC) from uninfected humans and rhesus monkeys (G. Giambartolomei et al., Infect. Immun. 66:2691–2697, 1998). Here we demonstrate that uninfected human peripheral blood monocytes, but not B or T cells, are the cells that transcribe the IL-10 cytokine gene in response to heat-killed B. burgdorferi. B. burgdorferi similarly induced an upregulation of the IL-1β and IL-6 cytokine genes in monocytes and the production of IL-10 and IL-6 in culture supernatants of the human monocytic cell line THP-1. Purified L-OspA (but not unlipidated OspA [U-OspA] or U-OspC) also stimulated the production of both cytokines in THP-1 cells in a dose-dependent fashion, suggesting that acylation of the OspA protein molecule is required for the production of both anti- and pro-inflammatory cytokines in naive monocytes. A lipohexapeptide that contained the tripalmitoyl-modified cysteine motif (Pam3Cys-Hex) of B. burgdorferi lipoproteins but with an arbitrary peptide sequence had the same effect. Monoclonal antibodies (MAbs) MY4 and 60bca, both of which bind to CD14 and are known to block lipopolysaccharide (LPS)-mediated cytokine production, were able to block L-OspA-mediated IL-10 and IL-6 cytokine production. In contrast, MAb 26ic, which also binds to CD14 but does not block LPS function, failed to inhibit L-OspA-mediated cytokine production. These data suggest that activation of monocytes and production of both anti- and pro-inflammatory cytokines induced by lipoproteins proceeds via the CD14 receptor. LPS binding protein was not required for OspA-induced cytokine production. Our results demonstrate that pro- and anti-inflammatory cytokines induced by B. burgdorferilipoproteins in PBMC are produced by monocytes and that lipoprotein and LPS signaling pathways share at least the initial signaling event that involves the CD14 receptor.

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