Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCAnegative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

Objective The development of pathogenic anti-neutrophil cytoplasmic autoantibodies (ANCAs) can result in systemic small vessel vasculitis. However, the breakdown in immune tolerance that results in the induction and persistence of ANCAs is not well-understood. We hypothesized that abnormal T cell regulation is central to disease pathogenesis and demonstrate here two separate abnormalities in T cell regulation in ANCA-associated vasculitis patients. Methods Peripheral blood samples were obtained from patients with ANCA-associated vasculitis (n=63) and healthy controls (n=19) for flow cytometric analysis of CD4+ T cell populations. Functional T cell studies were performed with FACS sorted CD4+ T cell populations stimulated with anti-CD3/28. Results First, we show that the Treg frequency in the peripheral blood of active disease patients is increased, but Tregs from patients with ANCA-associated vasculitis have decreased suppressive function. Tregs from active disease patients disproportionately utilize a FOXP3 isoform lacking exon 2, which may alter Treg function. Second, we identify a CD4+ T cell population with increased frequency that is resistant to Treg suppression, produces pro-inflammatory cytokines, and is antigen-experienced. Conclusion ANCA-associated vasculitis is associated with disruption of the suppressive Treg network and increased frequency of a distinct pro-inflammatory effector T cell subset which comprises the majority of peripheral CD4+ T cells.

Lysosomal membrane protein 2 (LAMP-2) is a target of antineutrophil cytoplasmic autoantibodies (ANCA) in addition to the more commonly known targets proteinase 3 and myeloperoxidase. The prevalence of anti-LAMP-2 antibodies and their relationship to disease in ANCA glomerulonephritis are not well described. We measured anti-LAMP-2 reactivity in 680 sera samples (two academic centers) from patients with ANCA glomerulonephritis (n=329); those with ANCA-negative glomerulonephritis (n=104); those with fimbriated, gram-negative Escherichia coli urinary tract infection (n=104); disease controls (n=19); and healthy volunteers (n=124). With levels in healthy controls used to define a reference range, anti-LAMP-2 reactivity was present in 21% of ANCA sera from two of the centers; reactivity was present in 16% of the control group with urinary tract infection. Western blotting and immunofluorescence microscopy did not verify positivity. Titers of anti-myeloperoxidase and anti-proteinase 3 antibodies were 1500-fold and 10,000-fold higher than anti-LAMP-2 titers, respectively. There was no correlation between anti-LAMP-2 antibodies and disease activity. Furthermore, Wistar Kyoto rats injected with anti-LAMP-2 antibodies did not develop glomerulonephritis. In conclusion, antibodies that react with LAMP-2 may exist at very low titers in a minority of patients with ANCA disease. These data do not support a mechanistic relationship between anti-LAMP-2 antibodies and ANCA glomerulonephritis.

Anti-neutrophil cytoplasmic autoantibody (ANCA) disease rarely occurs in African Americans and risk factors for the disease in this population are unknown. Here, we genotyped MHC class II alleles and found that, among African Americans, those with proteinase 3-ANCA (PR3-ANCA) had 73.3-fold higher odds of having HLA-DRB1*15 alleles than community-based controls (OR 73.3; 95% CI 9.1 to 591). In addition, a disproportionate number of African American patients carried the DRB1*1501 allelic variant of Caucasian descent rather than the DRB1*1503 allelic variant of African descent. Among Caucasians, those with PR3-ANCA had 2.2-fold higher odds of carrying DRB1*1501 than controls (OR 2.2; 95% CI 1.2 to 4.0). A validation study supported by the Vasculitis Clinical Research Consortium confirmed the strong association between the DRB1*15 allele and PR3-ANCA disease, among African Americans. Furthermore, we found that DRB1*1501 protein binds with high affinity to amino acid sequences of sense-PR3, purportedly an antigenic epitope, and to the amino acid sequence complementary to this epitope in vitro. Peptides of sense-PR3 and complementary-PR3 also bound to TNF-␣-induced surface expression of DRB1*1501 on peripheral neutrophils. Taken together, these data suggest HLA-DRB1*15 alleles contribute to the pathogenesis of PR3-ANCA disease.

SummaryBackground and objectives B cell significance in ANCA disease pathogenesis is underscored by the finding that ANCA alone can cause disease in mouse models and by the effectiveness of rituximab as therapy in ANCA-small vessel vasculitis (ANCA-SVV). To avoid infections and adverse events from therapy, clinicians require improved markers of disease activity and impending relapse to guide immunosuppression strategies after rituximab treatment.Design, setting, participants, & measurements The B cell phenotype was investigated in patients with active ANCA-SVV and in remission. From 2003 to 2009, 54 patients were followed longitudinally for 4-99 months and compared with 68 healthy controls. In a subset of 19 patients, the B cell immunophenotype was examined in samples after rituximab therapy.Results Patients with active ANCA-SVV had lower %CD5 + B cells, whereas %CD5 + B cells from patients in remission were indistinguishable from healthy controls. After rituximab, median time to relapse was 31 months in patients maintaining normalized %CD5 + B cells, with or without maintenance immunosuppression. Among patients whose B cells repopulated with low %CD5 + B cells or had a sharply declining %CD5 + B cells, those who were on low or no maintenance immunosuppression relapsed sooner (median 17 months) than patients who were maintained on high levels of oral maintenance immunosuppression (29 months; P=0.002). Conclusions The %CD5+ B cells, as a component of the human B regulatory cell phenotype, is a useful indicator of disease activity, remission, and future relapse, and thus may guide remission maintenance therapy after rituximab treatment.