We report here that an anti-TcdA MAb, A1H3, enhances TcdA-mediated cellular effects in murine macrophages and human monocytes. The observed effects included (i) inducing cell rounding and death, (ii) inactivating a small Rho GTPase via glucosylation, and (iii) eliciting TNF-α production in macrophages. Antibody-dependent enhanced cytotoxic activity of TcdA was not observed in other cell types, including human intestinal epithelial HCT8 or HT29 cells or murine colonic CT26 cells (data not shown).
Experiments demonstrated an important role for FcγRI in A1H3-dependent enhancement of TcdA toxicity. First, while blocking FcγRII and FcγRIII with anti-mouse-CD16/32 antibodies did not affect the glucosylation of Rac1 in RAW 264.7 cells, presaturation of FcγRI on THP1 cells with anti-human CD64 antibodies significantly reduced the level of Rac1 glucosylation. Anti-human CD64 blocking antibodies failed to completely eliminate the enhancement of TcdA activity on THP1 cells by mouse MAb A1H3. The exact reason for this finding is unclear, but it may have been due to the inefficient interaction between mouse-derived A1H3 and human FcγR in THP1 cells. Additionally, preincubation of the TcdA-A1H3 complex with recombinant mouse CD64 completely blocked the A1H3-mediated enhancement of glucosyltransferase activity by TcdA in RAW 264.7 cells. Finally, expression of FcγRI strikingly enhanced the sensitivity of mRG1-1 cells to TcdA when it was complexed with A1H3. Similarly, the presence of A1H3 greatly enhanced the glucosyltransferase activity of TcdA in mRG1-1 cells. Neither cytotoxicity nor glucosyltransferase activity was enhanced by A1H3 in CHO cells, the parental line of mRG1-1 cells.
The FcγR family consists of at least three members, FcγRI, FcγRII, and FcγRIII (48
). Murine FcγRI has a higher affinity for IgG2a than for other IgG subisotypes (14
). Although FcγRI is a high-affinity receptor capable of binding an IgG monomer, noticeable surface binding of A1H3 to RAW 264.7 cells occurred only when it was complexed with TcdA (Fig. ). This may be due to the relative low expression of FcγRI on these cells (data not shown). In fact, monomeric A1H3 binding was detected on mRG1-1 cells (data not shown), which were engineered to express a high level of FcγRI (4
). However, the binding of TcdA with FcγRI was significantly enhanced after its association with the A1H3 antigen (Fig. ) compared with the results for cells treated with TcdA alone (Fig. ), indicating that the presence of A1H3 facilitated recruitment of TcdA to the cell surface, which consequently might contribute to the antibody-dependent enhancement of toxin activity. Although the underlying mechanism is unclear, our data showed that A1E6, an IgG1 MAb against TcdA, neither enhanced toxin binding to macrophages nor augmented toxin activity when it was complexed with TcdA. Similar to A1E6, MAb A1B1, another anti-TcdA IgG1 MAb, had no enhancing effect (data not shown). These results are in agreement with a previous report showing that only the IgG2a isotype, but not the IgG1 isotype, of anti-protective antigen antibodies of anthrax toxin enhanced the cytotoxicity of the toxin to murine macrophages (40
Upon binding to FcγRs, the immune complexes are internalized via either phagocytosis or endocytosis. The mode of internalization is intimately linked to the size of the bound complexes (7
). Large opsonized particles are internalized by phagocytosis, while internalization of small soluble complexes most likely occurs via endocytosis. Mechanistically, the molecular processes underlying the FcγR-mediated phagocytosis and endocytosis differ dramatically. Endocytosis specifically requires assembly of clathrin at the site of receptor clustering (41
). FcγRI-mediated phagocytosis, however, requires a signal-transducing γ chain that harbors tyrosine activation motifs (8
). Cells expressing the FcγRI extracellular domain (in the absence of the γ subunit) are unable to phagocytose large particles, while their endocytic functions remain intact (8
). In our experiments, enhancement of TcdA activity mediated by A1H3 did not require the presence of the γ chain, since expression of the FcγRI α chain alone on mRG1-1 cells rendered the cells more susceptible to A1H3-dependent enhancement of the toxicity of TcdA (Fig. ), suggesting that the TcdA-A1H3 complex was taken up via FcγRI-mediated endocytosis. The involvement of an endocytic pathway in the uptake of TcdA-A1H3 by RAW 264.7 cells was further supported by the observation that TcdA-A1H3-mediated Rac1 glucosylation and TNF-α production were inhibited by chlorpromazine and ammonium chloride-chloroquine (Fig. ), chemicals that are known to target the endocytic pathway.
Our findings may have implications for understanding the antibody response in host protection and pathogenesis of C. difficile
-associated diseases. TcdA and TcdB are key virulence factors, and antibodies against these two toxins are highly protective (5
). Intravenous administration of Ig against TcdA and TcdB to patients with recurrent or severe CDAD resulted in resolution of symptoms (37
). A higher level of anti-TcdA antibodies following colonization or primary disease has been correlated with protection from CDAD or relapse (34
). Finally, vaccination of long-term relapsing humans with toxoids A and B has successfully prevented additional relapses (59
). However, different subsets of antibodies may have different roles in host protection and disease progression (26
). While patients with recurrent CDAD do not show evidence of overall humoral immune deficiency, they do have a selectively reduced IgG2 and IgG3 response against TcdA compared to patients with single CDAD (26
). Our data showed that the IgG2a subisotype of anti-TcdA actually enhanced the toxicity of TcdA on macrophages/monocytes in vitro. How these findings apply to in vivo pathogenesis of C. difficile
infection remains to be determined. Antibody-dependent enhancement of viral infection has been widely described for mammalian viruses, as well as bacteriophages (36
). Instead of neutralizing or reducing viral infectivity, the presence of virus-specific antibodies paradoxically potentiates infection of susceptible host cells, a process that is most often mediated by receptors for complement components or the Fc portion of Igs (18
). To date, no report has documented the enhancement of toxin activity in vivo by specific subsets of antibodies. Given the results of this report and a previous study (40
) using in vitro cell culture models, it is likely that some toxin-specific antibodies have detrimental effects on the host mediated by enhanced toxin activity. Such effects, which have not been illustrated in humans or in animal models yet, are being investigated in our laboratory.