The results presented here lead us to propose a previously unrecognized molecular explanation for the origin and development of injury in pauci-immune FNGN. We show that (i) autoantibodies to human LAMP-2 are present in over 90% of individuals with active pauci-immune FNGN; (ii) antibodies to human LAMP-2 injure human microvascular endothelium in vitro and induce FNGN when administered to rats; (iii) autoantibodies from individuals with pauci-immune FNGN commonly bind an epitope shared by human LAMP-2 and the bacterial adhesin FimH and cross-react with it; (iv) immunization with FimH induces pauci-immune FNGN associated with antibodies that bind human and rat LAMP-2; and (v) individuals with pauci-immune FNGN commonly have infections with FimH-expressing bacteria shortly before presentation of FNGN. Thus, FimH-triggered autoimmunity to human LAMP-2 could be responsible for the initiation and development of injury in pauci-immune FNGN, either operating alone or synergistically with antibodies to myeloperoxidase and proteinase-3.
Our hypothesis depends on the high prevalence of antibodies to human LAMP-2 in individuals with active pauci-immune FNGN. Consequently, we validated the results of our standard ELISA, not only by western blotting but also in other assays using mammal-expressed human LAMP-2. Thus, we can be certain that over 90% of our subject cohort with active pauci-immune FNGN had circulating autoantibodies to human LAMP-2. This exceeds the prevalence of antibodies to myeloperoxidase and proteinase-3, which, as in other series, were each present in around half of the subjects. Thus, autoantibodies to human LAMP-2 are the first to be found in all types of pauci-immune FNGN. Furthermore, they bind fully glycosylated human LAMP-2 on the cell surface, suggesting that they could cause injury.
The frequent co-existence of autoantibodies to LAMP-2 with those to myeloperoxidase and proteinase-3 precluded the use of patients’ sera for testing whether the autoantibodies to LAMP-2 cause injury. However, we showed that pathologically plausible concentrations of monoclonal and polyclonal antibodies to human LAMP-2 cause injury both in vitro
and in vivo
without the need for additional stimuli—such as LPS or cytokines—that are commonly required in such experiments7,30,31
. Uniquely among autoantibodies found in pauci-immune FNGN, the monoclonal human LAMP-2–specific antibody H4B4 injured human blood microvascular endothelium, the primary target of damage in pauci-immune FNGN. Although the molecular mechanisms for this have yet to be defined, activation of endothelial caspase-3 is consistent with the role of LAMP-2 in apoptotic and autophagocytic cell death32,33
. Antibodies to human LAMP-2 cause pauci-immune FNGN with up to 25% crescents when injected into WKY rats. Taken together, our data provide strong, though indirect, evidence that autoantibodies to human LAMP-2 contribute to glomerular injury in individuals with pauci-immune FNGN.
The data raise the intriguing question as to why autoantibodies to human LAMP-2 so commonly occur together with antibodies to either myeloperoxidase or proteinase-3. An essential synergistic effect on injury provides an obvious explanation, but an alternative (and not mutually exclusive) possibility is that the autoantibodies to human LAMP-2 dysregulate the role of LAMP-2a in presentation of cytoplasmic antigens, including lysosomal proteins15,34,35
. This, in turn, could facilitate the generation of autoantibodies to myeloperoxidase and proteinase-3.
Characterization of the epitopes recognized by autoantibodies from subjects with pauci-immune FNGN provided our most unexpected result. It uncovered a hitherto unsuspected homology between one of these epitopes (P41–49) and the bacterial fimbrial adhesin FimH. The homology has profound functional consequences, first because auto-antibodies that recognize P41–49 cross-react with FimH, as proved by our reciprocal inhibition experiments, and second because WKY rats immunized with FimH develop antibodies to the shared epitope. The immunized rats also developed autoantibodies to rat LAMP-2 and pauci-immune FNGN with pulmonary small vessel vasculitis similar to the human disease. This clearly demonstrates that FimH can induce FNGN by operating as a molecular mimic for LAMP-2, at least when administered together with adjuvant. These results identify P41–49 as a pathogenic epitope and support our proposal that autoimmunity to LAMP-2 causes FNGN, even in the absence of antibodies to myeloperoxidase or proteinase-3.
Molecular mimicry has frequently been proposed as a mechanism for autoimmune disease, but convincing evidence has been difficult to obtain, as has been previously reviewed21
, except possibly in neurological diseases in the presence of cross-reacting antibodies36,37
. Our demonstration that human LAMP-2 cross-reacts with FimH and that immunization with FimH induces antibodies to LAMP-2 and FNGN shows the molecular mimicry between the two proteins. This conclusion is supported by two additional pieces of data: it has previously been shown that FNGN is induced in rats immunized with attenuated E. coli
but not with Staphylococcus aureus38
, and we have found that nine of thirteen subjects had microbiologically proven infections with FimH-bearing organisms shortly before the clinical appearance of FNGN. Although this provides some of the most complete evidence yet for molecular mimicry as mechanism of human autoimmune disease, it doesn’t identify the exceptional circumstances needed for FimH to induce the cross-reactive auto-antibodies. FimH is required for bacterial colonization of bladder epithelium39,40
and so has been proposed as an attractive target for immunization programs for urinary tract infections in humans41,42
. Our data clearly question the safety of this approach.
In conclusion, we propose that infection with fimbriated bacteria induces autoantibodies to human LAMP-2 through molecular mimicry and that these antibodies bind microvascular endothelium and cause injury (Supplementary Fig. 4
). This presents a new model for the pathogenesis of pauci-immune FNGN and related ANCA-associated diseases and could provide the foundation of a new therapeutic strategy for this devastating disease.