Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P [41][42][43][44][45][46][47][48][49] ) with 100% homology to the bacterial adhesin FimH, with which they Correspondence should be addressed to R.K. (renate.kain@meduniwien.ac.at). 7 Present addresses: Interne, Hämato-Onkologie, Krankenhaus der Elisabethinen, Fadingerstrasse 1, 4010 Linz, Austria (R.Z.) and Vela Laboratories. Entwicklung und Laboranalytik Gesellschaft mit beschränkter Haftung, Brunnerstrasse 69/3, 1230 Wien, Austria (R.J.). 8 These authors contributed equally to this work. Here we establish that autoantibodies to human LAMP-2 are highly prevalent in pauci-immune FNGN and provide evidence of their pathogenicity by showing that they activate neutrophils and kill human blood microvascular endothelium in vitro and cause pauci-immune FNGN when administered to rodents. Unexpectedly, auto-antibodies to LAMP-2 in individuals with FNGN commonly recognize an epitope with considerable homology to the bacterial adhesin FimH and cross-react with it. We therefore determined whether exposure to FimH could induce antibodies to human LAMP-2 and initiate pauci-immune FNGN through molecular mimicry. The results lead us to propose a previously undescribed molecular mechanism both for the induction and development of injury in this human disease. RESULTS Autoantibodies to human LAMP-2 are common in FNGNWe established the prevalence of autoantibodies to hLAMP-2 in sera from 84 individuals with biopsy-proven active pauci-immune FNGN, either at presentation (n = 62) or during relapse (n = 22). ANCA were detectable by standard immunofluorescence assays in 80 of them (95%), and ELISA for the canonical ANCA were positive in 70 of them (83%); myeloperoxidasespecific ANCA were found in 38 people, and proteinase-3-specific ANCA were found in 39 people, including seven with antibodies to both antigens. Using a specific ELISA, we detected antibodies to human LAMP-2 in 78 of the 84 (93%) sera (Fig. 1a), and we validated the results by western blotting and indirect immunofluorescence on the O-glycosylation deficient CHO cell line ldlD cells stably expressing human LAMP-2 on their surface ( Supplementary Fig. 1a online). Notably, the human LAMP-2 ELISA was negative in all but six of the individuals when they were in remission after immunosuppressive therapy. Assays for human LA...
The involvement of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is controversial because of the absence of confirmatory data subsequent to the initial reports of their high prevalence in this disease. We characterized three assays for anti-hLAMP-2 antibodies: ELISA and Western blotting assays using unglycosylated recombinant hLAMP-2 expressed in Escherichia coli, and an indirect immunofluorescence assay using stably transfected ldlD cells that expressed glycosylated full-length hLAMP-2 on the plasma membrane. The assays detected autoantibodies to hLAMP-2 in human sera reproducibly and with comparable sensitivity and the assays gave the same results in 80.5% of the test panel of 40 selected positive and negative sera. In untreated patients at presentation, the frequencies of autoantibodies to LAMP-2 were 89%, 91%, and 80%, respectively, among three groups of patients with ANCA-associated vasculitis from Vienna, Austria (n=19); Groningen, the Netherlands (n=50) and Cambridge, United Kingdom (n=53). Prevalence of LAMP-2 autoantibodies was similar in both those with myeloperoxidase-ANCA and proteinase 3-ANCA. Furthermore, we detected LAMP-2 autoantibodies in two ANCA-negative patients. LAMP-2 autoantibodies rapidly became undetectable after the initiation of immunosuppressive treatment and frequently became detectable again during clinical relapse. We conclude that when robust assays are used, circulating autoantibodies to hLAMP-2 can be detected in most European patients with ANCA-associated vasculitis. Large-scale prospective studies are now needed to determine whether they are pathogenic or merely an epiphenomenon.
Pauci-immune focal necrotizing GN (piFNGN) is usually associated with ANCAs that are thought to be pathogenic. However, 10%-15% of patients are ANCA negative and the cause of their injury is unknown. We previously reported a high frequency of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in ANCA-associated piFNGN, and have now investigated whether the same is true in ANCA-negative patients. Of 11 patients, 8 (73%) had anti-hLAMP-2 antibodies detected by ELISA and confirmed by immunoblotting and indirect immunofluorescence. The autoantibodies from all 8 patients bound to native LAMP-2 purified from human glomeruli and recombinant hLAMP-2 expressed in ldlD cells, both with molecular masses of 110 kD. However, in contrast to anti-LAMP-2 antibodies from ANCA-positive patients, these antibodies from ANCA-negative patients failed to bind the more complexly glycosylated native neutrophil hLAMP-2 (190 kD). Treatment with the deglycosylating enzyme, endo-b-galactosidase, reduced the mass of neutrophil hLAMP-2 to 110 kD and enabled autoantibody binding. Similarly, pretreating neutrophils with endo-b-galactosidase or neuraminidase converted ANCA assay results from negative to positive. Finally, IgG from LAMP-2-positive ANCAnegative patients bound specifically to normal human kidney sections and to human glomerular endothelial cells in culture. In conclusion, in patients with ANCA-negative piFNGN, we have identified autoantibodies to hLAMP-2 that bind native glomerular but not neutrophil hLAMP-2, suggesting a role in pathogenesis.
FimH-mediated adhesion of Escherichia coli to bladder epithelium is a prerequisite for urinary tract infections. FimH is also essential for blood-borne bacterial dissemination, but the mechanisms are poorly understood. The purpose of this study was to assess the influence of different FimH mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study bacterial adhesion to mammalian cells under flow conditions. We showed that E. coli MSC95-FimH adheres more efficiently to microvascular endothelium than to bladder epithelium, and that only endothelium supports adhesion at physiological shear stress. The results confirmed that mannose binding pocket mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel insights into screening methods to determine the effect of FimH mutants and potentially FimH antagonists.Electronic supplementary materialThe online version of this article (doi:10.1007/s10096-016-2820-8) contains supplementary material, which is available to authorized users.
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