Role of Fc Gamma Receptors in Triggering Host Cell Activation and Cytokine Release by Borrelia burgdorferi

RESULTS

Previous studies demonstrated that low-passage-numberB. burgdorferi spirochetes are able to activate MC/9 mast cells to upregulate and/or stabilize message for the proinflammatory cytokine TNF-α at 4 h postchallenge and to secrete bioactive TNF-α at 8 h postchallenge (53). To determine whether B. burgdorferi spirochetes were inducing other genes in MC/9 cells, we employed RNase protection assays to look for upregulation of additional cytokine and chemokine mRNAs. As shown in Fig. 1 and2, we observed a 10-fold increase in IL-6 mRNA at 4 h and a 10- to 20-fold increase in mRNAs for the chemokines MIP-1α, MIP-1β, and TCA-3 at 1 h following in vitro stimulation with low-passage-number B. burgdorferispirochetes (B31-LO). In contrast, no increases in chemokine mRNA levels were observed when MC/9 mast cells were stimulated with high-passage-number B. burgdorferi spirochetes (B31-HI), despite the fact that these spirochetes retained the ability to induce comparable levels of spleen cell proliferation to low passage numbers (data not shown), thereby confirming their expression of bioactive lipoproteins. The ability of low-passage- but not high-passage-numberB. burgdorferi spirochetes to induce and/or stabilize chemokine MIP-1α, MIP-1β, and TCA-3 mRNAs suggests that induction of these genes is mediated by the same novel LIA previously shown to induce TNF-α (53).

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Fig. 1.

Induction of increased IL-6 mRNA expression in MC/9 mast cells exposed in vitro to low-passage-number B. burgdorferispirochetes. MC/9 cells (3 × 10⁶/24-well plate) were treated with medium alone (Med.), B. burgdorferi B31 clone 5.1 spirochetes (Bb) (100:1 multiplicity), or 1 μM ionomycin (Iono) for 4 h at 37°C. Total RNA was isolated from MC/9 cells by using a Qiagen RNeasy kit, and the presence of cytokine mRNA was determined by using the Riboquant RPA system. Protected cytokine mRNA bands were visualized by autoradiography (A), and bands were quantitated by using ImageQuant software (B). mRNA data presented are relative to mRNA levels for the internal-control L32 housekeeping gene.

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Fig. 2.

Low-passage-number but not high-passage-number B. burgdorferi spirochetes induce chemokine MIP-1α, MIP-1β, and TCA-3 mRNAs in MC/9 mast cells MC/9 cells (3 × 10⁶/24-well plate) were treated with medium alone (Med.), 1 μM ionomycin (Iono), low-passage-number B. burgdorferi B31 clone 5.1 spirochetes (Bb B31 5.1), or high-passage-number B31 spirochetes (B31-HI) (both at a multiplicity of 100:1) for 1 h at 37°C. RNA was isolated by using a Qiagen RNeasy kit, and the presence of message was determined by using the Riboquant RPA system. Protected mRNA bands were visualized by autoradiography (A), and chemokine mRNA was quantitated by using ImageQuant software (B). mRNA data presented are relative to mRNA levels for the internal-control L32 housekeeping gene.

Recent data indicate that B. burgdorferi lipoproteins activate host cell cytokine secretion through interaction with the CD14–Toll-like receptor 2 (TLR2) complex (19, 26). Our failure in the previous study to detect mast TNF-α secretion when cells were incubated with either purified recombinant B. burgdorferi Osp lipoproteins or lipoprotein-expressing high-passage-number B. burgdorferi spirochetes suggests that this CD14- and TLR2-dependent pathway may be missing in MC/9 mast cells, allowing for the detection of the novel activity. Thus far, only a handful of mast cell receptors have been linked to TNF-α secretion. These include the high-affinity Fc epsilon receptor for IgE (FcɛRI) (37, 46), the low-affinity Fc gamma III receptor for IgG (FcγRIII) (25, 59), CD8 (18), CD43 (4), CD48 (31), and the substance P receptor (2). To explore the possible roles of these receptors in TNF-α induction by B. burgdorferi spirochetes, we examined the ability of wortmannin, a specific irreversible inhibitor of PI 3-kinase (38), to affect TNF-α secretion. PI 3-kinase is known to be involved in both FcɛR and FcγR signaling (36,41). As shown in Fig. 3, wortmannin inhibited B. burgdorferi-mediated TNF-α secretion by MC/9 mast cells. These results indicate that PI 3-kinase is a necessary component in the B. burgdorferi-mediated signaling cascade leading to mast cell TNF-α secretion and suggest the possibility that signaling occurs through either FcɛR or one of the FcγRs.

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Fig. 3.

Specific PI 3-kinase inhibitor wortmannin inhibitsB. burgdorferi-mediated TNF-α secretion from MC/9 mast cells. Untreated MC/9 cells (−) or MC/9 cells (10⁵/well) pretreated for 15 min with 10 nM wortmannin (Wortmn. tx.) (+) were incubated with either medium alone (Med.), 1 μM ionomycin (Iono), orB. burgdorferi B31 clone 5.1 spirochetes (100:1 multiplicity) (Bb) for 8 h. Supernatants were removed, and TNF-α was measured as described previously (53). The data are the means and standard errors of the means of triplicate determinations. The data presented are from a single experiment representative of three experiments with similar results.

We next examined the possibility that B. burgdorferi was triggering mediator release by interacting with one of the FcγRs. In addition to the high-affinity IgE receptor FcɛRI, murine mast cells also express two low-affinity IgG receptors, FcγRIIb and FcγRIII (6). These two receptors, which have nearly identical binding properties due to a 95% sequence homology in their extracellular domains, are incapable of binding monomeric IgGs but can bind aggregated or antigen-bound mouse IgG1, IgG2a, and IgG2b, but not IgG3 (27). As shown in Fig.4, prior incubation of MC/9 mast cells with preparations of mouse IgG1 and IgG2a, but not mouse IgG3, antibodies blocked the ability of B. burgdorferi spirochetes to induce TNF-α secretion, presumably by blocking interactions between B. burgdorferi LIA and a low-affinity FcγR. Furthermore, the FcγRII/III-specific MAb 2.4G2 (55) also blocked B. burgdorferi-mediated TNF-α secretion. While these antibody blocking data do not indicate whether FcγRIIb or FcγRIII is the receptor, previous studies have indicated that cross-linking of FcγRIII, but not FcγRIIb, triggers TNF-α production in transfected rat basophilic leukemia (RBL) cells (25), making it more likely that B. burgdorferispirochetes signal cytokine production by interacting with FcγRIII.

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Fig. 4.

Mouse IgG1 and IgG2a antibodies and rat FcγRII/III-specific MAb 2.4G2 block the induction by low-passage-number B. burgdorferi spirochetes of TNF-α secretion in MC/9 mast cells. MC/9 cells (10⁵/well) were (+) or were not (−) treated with 25 μg of murine IgG1, IgG2, or IgG3 antibodies (Ab) or rat MAb 2.4G2/ml for 15 min and then were incubated for 8 h with B. burgdorferi B31 clone 5.1 spirochetes (100:1 multiplicity) or medium (Med.) alone. Supernatants were removed and tested for TNF-α as described previously (53). The data presented are from a single experiment representative of three experiments with similar results. Error bars indicate standard errors of the means.

Few published reports have revealed antibody-independent cellular activation via Fc receptors (49, 50). Thus, it was possible that the mast cell cytokine-inducing activity expressed byB. burgdorferi spirochetes grown in medium supplemented with rabbit serum and assayed in medium containing bovine serum was due to binding of bovine or rabbit IgGs or other serum components. To exclude this possibility, TNF-α induction was assayed under serum-free conditions, using spirochetes that had been treated to remove any contaminating serum components. To remove antibodies, B. burgdorferi spirochetes were incubated overnight at 4°C in Tris-buffered isotonic saline, pH 5 (12). Untreated controls were incubated at pH 7.5. Following washing and neutralization with pH 7.5 isotonic saline, pH 5-treated spirochetes were incubated for 15 min in phosphate-buffered saline containing 1 mM EDTA (49) to remove any bound serum-associated protein or C-reactive protein, which also bind FcγRs (49, 50). However, the ability of spirochetes treated in this way to elicit TNF-α release from MC/9 cells under serum-free conditions was comparable to that of untreated spirochetes (pH 5 treated, 262 ± 35 pg/ml; pH 7.5 treated, 256 ± 22 pg/ml [data not shown]), indicating that a bacterial protein, and not a serum contaminant, is responsible for FcγR-mediated signaling of TNF-α release from mast cells.

DISCUSSION

B. burgdorferi lipoproteins have the ability to activate a variety of host cell types. These activation events have been shown to occur through TLR2 (26) in conjunction with CD14 (19). Prior studies in our lab have shown thatB. burgdorferi spirochetes can activate mast cells to secrete TNF-α via a lipidation-independent, protease-sensitive moiety that is expressed on the cell surface (53). We considered it likely that this moiety had other mast cell-activating properties. Furthermore, due to the lipidation-independent character of thisB. burgdorferi-associated activity, it was likely thatB. burgdorferi LIA acted through a receptor distinct from CD14-TLR2.

In this study, using RNase protection assays, we showed that low-passage-number B. burgdorferi spirochetes have the ability to induce mast cells to upregulate and/or stabilize message for the proinflammatory cytokines TNF-α and IL-6 and the chemokines MIP-1α, MIP-1β, and TCA-3 (Fig. 1 and 2). In contrast, high-passage-number spirochetes (≫50 passages in vitro) did not show increased chemokine or cytokine mRNA expression (data not shown) or induce TNF-α secretion (53). Because high-passage-number spirochetes still possess comparable levels of bioactive outer surface lipoproteins, as evidenced by their ability to induce levels of spleen cell proliferation comparable to those induced by low-passage-number spirochetes (data not shown), these findings strengthen prior assertions that this lipidation-independent activity is biochemically distinct.

Because the probe sets we used detected mRNAs for only nine proinflammatory cytokines and nine chemokines, it is possible that additional cytokine and/or chemokine mRNAs are also upregulated by low-passage-number B. burgdorferi. Nevertheless, these findings establish that exposure to B. burgdorferi LIA results in the activation of multiple proinflammatory cytokine and chemokine genes in MC/9 mast cells. TNF-α has a multitude of effects, ranging from neutrophil chemotaxis to macrophage activation to adhesion molecule upregulation (1). IL-6 has many of the same effects and is also a potent inducer of acute-phase proteins (23). The chemokines MIP-1α and MIP-1β are chemotactic for mononuclear cells and T cells (54). MIP-1α is also chemotactic for mast cells and appears to play a role in differentiation of type 1 T cells (28). TCA3 is chemotactic for neutrophils and macrophages (24).

Since prior studies had indicated that B. burgdorferispirochetes can bind to host cell integrins, including α₅β₁ and α_vβ₃(11), we initially examined the ability of RGD-containing peptides and anti-β1 and -β3 MAbs to block B. burgdorferi-induced TNF-α production by MC/9 mast cells. However, we observed no inhibition of TNF-α production by any of these reagents (data not shown).

A number of different surface molecules have been implicated in the triggering of TNF-α production by mast cells. These include the high-affinity receptor FcɛRI (37, 46), the low-affinity FcγRIII (25, 59), CD48 (31), CD43 (4), and the substance P receptor (2). The ability of the PI 3-kinase inhibitor wortmannin to block B. burgdorferi-mediated TNF-α secretion (Fig. 3) suggested the possible involvement of Fc receptors since signaling through both FcɛRI (41) and FcγRs (36) is wortmannin sensitive.

Mouse cells express three distinct FcγRs, FcγRI, FcγRII, and FcγRIII, each having different binding affinities for the different mouse IgG subclasses (20). FcγRI is the high-affinity IgG receptor, and it binds monomeric IgG2a with high affinity and IgG1, IgG2b, and IgG3 with low affinity (21). FcγRII and FcγRIII are low-affinity receptors with similar IgG binding characteristics because of the 95% amino acid homology in their extracellular domains. They bind aggregated mouse IgG1, IgG2a, and IgG2b, but not IgG3 (27). Murine mast cells do not express FcγRI, but they do express both FcγRIIb and FcγRIII (6). Our finding that mouse IgG1, mouse IgG2a, and the FcγRII/III-specific rat MAb 2.4G2, but not mouse IgG3, are able to block B. burgdorferi-mediated TNF-α production by MC/9 mast cells (Fig. 4) implicated one of the low-affinity FcγRs in this effect. Since these receptors are believed not to bind monomeric mouse IgG (27), blocking by mouse IgG1 and IgG2a preparations may have been mediated by aggregates.

The known properties of these two FcγRs, however, suggest that it is more likely that FcγRIII has a role in B. burgdorferi-mediated signaling. In contrast to FcγRIIb, FcγRIII acts mainly as a cell-activating receptor (20). Like FcɛRI, FcγRIII is a multisubunit receptor and it shares a common γ chain with FcɛRI (40). The γ chain is thought to be the major signaling molecule since it bears an immunoreceptor tyrosine-based activation motif in its cytoplasmic tail (7, 8). Cross-linking of FcγRIII (or FcɛRI) leads to cell activation which is accompanied by tyrosine phosphorylation (8) and Ca²⁺ mobilization (10). FcγRIIb, in contrast, appears to function as an inhibitory or regulatory receptor (39). Its single α chain bears an immunoreceptor tyrosine-based inhibitory motif (13). Cross-linking of FcγRIIb does not induce tyrosine phosphorylation or Ca²⁺ mobilization (56) and is known to downregulate B-cell activation (3). Perhaps most relevant to this study, transfection of FcγRIII, but not FcγRIIb, into nonexpressing rat RBL mast cells rendered these cells capable of TNF-α secretion when cross-linked with the FcγRII/III-specific MAb 2.4G2 (25). Furthermore, the finding that cross-linking of FcγRIIb downregulates PI 3-kinase activity (41) also argues against a role for FcγRIIb in signaling since PI 3-kinase is necessary for B. burgdorferi-induced mast cell TNF-α production by mast cells (Fig. 3).

The nature of the physical association between B. burgdorferi LIA and FcγR resulting in host cell signaling and TNF-α production is unknown. By analogy with antibody-mediated activation (20), it probably requires receptor cross-linking. The fact that activity is destroyed by limited proteolysis of live organisms and remains associated with pelleted bacteria despite intense sonication (53) is consistent with a requirement for arrayed, surface-expressed B. burgdorferi LIA for efficient FcγR cross-linking.

Recent studies have demonstrated that B. burgdorferilipoproteins activate cells by signaling through CD14-TLR2 receptors (19, 26, 61). It is likely that our ability to efficiently detect B. burgdorferi LIA in MC/9 mast cells is a consequence of the absence of this activation pathway in this cell line. In support of this hypothesis, MC/9 cells fail to make TNF-α when stimulated with several different forms of LTA, which is known to activate cells via the TLR2 pathway (47; J. Talkington and S. P. Nickell, unpublished observations). LPS, which appears to signal through TLR4 (42), also did not induce TNF-α production in MC/9 cells (53), suggesting that the TLR4-dependent activation pathway is also absent in these cells. In vivo studies suggest that LPS may not be a significant inducer of mast cell TNF-α (16). Interestingly, despite the insensitivity of MC/9 mast cells to direct LPS stimulation, such costimulation significantly augmented B. burgdorferi-induced TNF-α production by these cells (212 ± 28 versus 118 ± 16 pg/ml [pooled data from four experiments]). While the mechanism responsible for such LPS augmentation of cytokine production is not known, it has also been observed in bone marrow-derived mast cells activated by FcɛRI cross-linking or c-kit stimulation (33). In contrast, B. burgdorferi-mediated TNF-α production in MC/9 cells was not augmented by LTA.

While this is not the first report of host-pathogen signaling through FcRs (45, 62), to our knowledge it is the first report of direct FcR signaling by a bacterial pathogen. Current views of Lyme disease pathology hypothesize that bioactive bacterial products contribute to tissue damage by provoking the release of proinflammatory cytokines or chemokines and other inflammatory mediators from host cells. Our finding that interaction between FcγRs and B. burgdorferi-associated proteins leads to host cell cytokine production raises the possibility that such signaling contributes to pathological events in vivo. However, considering the very high biological potency of lipoproteins (60), it is likely that lipoprotein-mediated effects significantly outweigh lipidation-independent effects in vivo. This is supported by preliminary studies which found that levels of B. burgdorferi spirochete-induced TNF-α produced in vitro by bone marrow-derived macrophages from either common Fc γ-chain-deficient mice [C57BL/6 (B6)-Fcer1g], which lack expression of FcɛRI, FcγRI, and FcγRIII (51, 58), or FcγRIIb-deficient mice (B6-Fcgr2) (52) were not significantly reduced compared to wild-type FcγR-expressing B6 mice (Talkington and Nickell, unpublished observations), suggesting that lipoprotein-mediated signaling via TLR2 is dominant over lipidation-independent signaling via FcγRs. Additionally, while our data suggest that positive signaling for TNF-α production by B. burgdorferi LIA occurs through FcγRIII, similar interactions between B. burgdorferi LIA and FcγRIIb may also occur, with possible regulatory consequences. It is perhaps of some relevance to the present work that recent studies have demonstrated a role for the Fc receptor common γ chain in the development of inflammation and cartilage damage in a mouse model of experimental antigen-induced arthritis (57). Also, a genetic polymorphism in human FcγRIII has recently been linked to arthritis susceptibility (35). Ultimately, the relevance of B. burgdorferi-FcγR signaling events to outcome will have to be determined empirically. In studies under way, we are addressing whether FcγR signaling can modify B. burgdorferilipoprotein-mediated cytokine production in vitro and whether the course of B. burgdorferi infection differs in FcγR-deficient and wild-type mice.