These studies were originally intended to extend our previous studies of B cell SAg mediated immune complex inflammation [3
]. We turned our attention to PL because we believed it to be a “cleaner” B cell SAg than SpA since it possessed V-region binding activity outside the CDRs but lacked the Fcγ binding activity of SpA. Moreover, the B cell superantigenic Ig-binding properties of PL are thought to be an important virulence factor of the F. magna
strains that produce it [19
The results of the studies reported herein indicate that the PL cell wall component of F. magna
induces an inflammatory reaction characterized by the rapid accumulation of PMNs in the BALF and a peribronchial and perivascular infiltrate of inflammatory cells peaking between 18 and 24 hours. The cellular changes were associated with the appearance of elevated levels of MIP-2, KC, TNF-α, and IL6 in BALF, peaking at 4–8 hours, but not IL-1β. The temporal pattern of appearance of these mediators suggests that one or more may contribute to the PMN infiltration. Although our studies do not allow for a conclusion to be drawn about which of these mediators is responsible for the attraction of PMNs, the CXC chemokines MIP-2 and KC are likely to be involved given their robust chemoattractant properties. Of note, the same profile of BALF chemokines and cytokines we observed in the PL-induced reaction was observed in models of lung inflammation caused by the deposition of conventional immune complexes (12) Thus, our findings were consistent with the hypothesis that PL-induced inflammation was elicited by PL/IgG containing immune complexes. Such a mechanism would not be expected to require sensitization of the mice to PL, since reactive Ig Vk-binding molecules would be present in the endogenous murine Ig repertoire [8
]. Indeed, the kinetics of the PL-induced pulmonary changes in the naïve mice were consistent with this supposition. Our findings are physiologically relevant since they were associated with a significant airway hyperresponsiveness to MCh 24 hours after PL exposure of the mice. Further, the kinetics of the PL-induced airway changes in the naïve mice strongly suggest that PL activated the innate, rather than the adaptive immune response in the lung.
Our results are highlighted by the finding that the Ig-binding property of PL is not responsible for its proinflammatory action in the lung. This unexpected result was revealed in studies of the JHT mice, which lack both B cells and Ig molecules [13
]. We therefore shifted our focus to potential innate immune mechanisms that would not require binding of PL to Igs and presensitization of the recipient mice. The preservation of lung inflammation in C3 deficient mice ruled out the recruitment of two potent complement pathway activation by-products, C3a and C5a. However, the abrogation of all components of the inflammatory responses in MyD88 knockout mice suggested a requirement for one or more TLRs, IL-1β, IL-18 or IL-33 since all signal through a MyD88-dependent pathway. We think that binding of PL to a TLR is the most likely explanation for the elicitation of the observed inflammatory reactions. It is unlikely that IL-1β is an important contributor to the pulmonary inflammation since it was not detected in the BALF of PL-challenged animals. Although we did not measure IL-18 or IL-33 levels in BALF, we think these IL-1 family cytokines are not key players since they tend to elicit TH2-cytokines and typically are not associated with PMN-rich inflammatory responses when linked to either innate or adaptive immune pathway-induced airway hyperreactivity [17
To date, thirteen types of TLRs have been described in mammals and all but TLR-3 have been associated with MyD88 signaling [reviewed in 15]. We can exclude TLR-4 as a PL target. since the pulmonary reactions were unaltered in the TLR-4 defective C3H/HeJ mouse strain. In addition, the latter observation implies that the PL-induced effects cannot be caused by the negligible amount of endotoxin in the recombinant PL preparation under study since endotoxin signals via TLR-4. Studies are currently underway in mouse strains deficient in other TLRs to indentify the putative PL-binding receptor.
Having obtained data strongly suggesting that TLR mediates PL-induced-inflammation in the lungs, we turned our attention to a resident effector cell population that expresses these innate immune receptors, namely AMs. The PL-induced reactions were markedly diminished in AM-depleted mice, suggesting an important role for AMs in their pathogenesis. However, the responses were not completely abrogated. This finding suggests that the macrophages that escaped depletion or perhaps another cell type that expresses TLRs, e.g., epithelial cells, dendritic cells and/or mast cells, accounted for the residual response.
The interaction of PL with another component of the innate immune system has recently been reported [24
]. PL was demonstrated to bind to the antibacterial proteins S100A8/A9 via a site not involved with Ig binding. It was suggested that this interaction protected PL-secreting strains from the bactericidal actions of these antibacterial proteins and thereby promoted inflammation.
In summary, our results reveal a novel pro-inflammatory mechanism initiated by a microbial protein with known B cell superantigenic properties. Whereas the virulence of F. magna has been formerly associated with the superantigenic Ig binding property of PL, our findings indicate that this property does not account for PL-induced pulmonary inflammation. Rather, the experimental results strongly suggest pulmonary inflammation is mediated by a MyD88-dependent activity of this microbial protein, which we believe is most likely initiated by its binding to a TLR expressed by pulmonary macrophages. Accordingly, our results add to the growing list of mechanisms by which this bacterial protein may exploit components of the adaptive and innate immune system to elicit inflammation.