Small-vessel vasculitis (SVV) is a chronic autoinflammatory condition linked to antineutrophil cytoplasm autoantibodies (ANCAs). Here we show that chromatin fibers, so-called neutrophil extracellular traps (NETs), are released by ANCA-stimulated neutrophils and contain the targeted autoantigens proteinase-3 (PR3) and myeloperoxidase (MPO). Deposition of NETs in inflamed kidneys and circulating MPO-DNA complexes suggest that NET formation triggers vasculitis and promotes the autoimmune response against neutrophil components in individuals with SVV.SVV is a relapsing-remitting autoinflammatory disorder leading to necrotic inflammation of small-sized blood vessels and capillaries 1 . ANCAs directed against granule proteins of neutrophils, namely against PR3 in Wegener's granulomatosis and MPO in microscopic polyangiitis, are implicated in the pathogenesis of SVV 2 . In vitro studies have demonstrated an activating effect of ANCAs on cytokine-primed neutrophils 3 , which was further corroborated by animal models of these diseases 4,5 . However, the basic mechanism that induces the life-threatening exacerbations of vasculitis and the sustained autoimmune response against neutrophil components remains elusive.A unique type of cell death of neutrophil granulocytes has recently been discovered that is characterized by the active release of chromatin fibers, so-called NETs, that trap and kill invading microbes extracellularly 6 . However, this glutinous DNA web can also stick to the endothelium and cause tissue damage during sepsis 7 , similar to neutrophil-induced As ANCA can activate the respiratory burst by binding to PR3 or MPO on the neutrophil surface 3 , we examined whether ANCA-mediated activation of neutrophils induces NET formation. We primed isolated neutrophils with tumor necrosis factor-α and incubated them with purified IgG from individuals with SVV or healthy donors as performed previously 3 . We observed robust NET formation (as determined by immunofluorescence 6,8; Supplementary Methods online) in neutrophils incubated with ANCA-IgG (Fig. 1a and Supplementary Table 1 online) but not in those incubated with control IgG, in which most nuclei retained the typical lobulated structure (Fig. 1b). After 180 min, we found that 23% of neutrophils incubated with ANCA-IgG produced NETs, compared to 11% of control IgG-treated neutrophils (Fig. 1c). Incubation with phorbol 12-myristate 13-acetate (PMA), known as a strong inducer of NETs, triggered NET production in 38% of all neutrophils (Fig. 1c). We also induced NETs with a PR3-specific mouse monoclonal antibody ( Supplementary Fig. 1 online), supporting the hypothesis that PR3-specific autoantibodies within the ANCA-IgG fraction trigger NET formation. ANCA-induced cell death of neutrophils was previously regarded as a dysregulated form of apoptosis 9 , but the link to NETs had not been noticed. The morphological changes of neutrophil nuclei clearly indicated to us that ANCA-induced NETs were of nuclear rather than of mitochondrial origin, as recently desc...

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ANCA testing is useful due to its high sensitivity and specificity, especially for cANCA/PR3-ANCA in WG. We found a divergence in the disease spectrum between PR3- and MPO-ANCA-positive patients, characterized by higher DEI and extrarenal manifestations in the PR3-ANCA group. MPO-ANCA was rarely found in WG and was associated with less organ involvement.

Steroid-resistant focal segmental glomerulosclerosis (FSGS) often recurs after renal transplantation. In this international survey, we sought to identify genotype-phenotype correlations of recurrent FSGS. We surveyed 83 patients with childhood-onset primary FSGS who received at least one renal allograft and analyzed 53 of these patients for NPHS2 mutations. The mean age at diagnosis was 6.7 years, and the mean age at first renal transplantation was 13 years. FSGS recurred in 30 patients (36%) after a median of 13 days (range, 1.5 to 152 days). Twenty-three patients received a second kidney transplant, and FSGS recurred in 11 (48%) after a median of 16 days (range, 2.7 to 66 days). None of the 11 patients with homozygous or compound heterozygous NPHS2 mutations developed recurrent FSGS compared with 45% of patients without mutations. These data suggest that genetic testing for pathogenic mutations may be important for prognosis and treatment of FSGS both before and after transplantation.

The immune response in Wegener's granulomatosis (WG) has been characterized as a predominant, potentially pathogenic Th1-like reaction by blood T cells and T-cell clones from diseased tissues. To elucidate further the immunopathogenic mechanisms, this study analysed the phenotypes of inflammatory infiltrates in frozen nasal biopsies with involvement of the upper respiratory tract only (localized or 'initial phase' WG) and with multi-organ involvement, including systemic vasculitis (generalized WG). The expression and production of Th1 and Th2 cytokines were examined in tissue specimens and peripheral blood mononuclear cells (PBMCs) of localized and generalized WG. The number of CD3+ T cells in inflammatory infiltrates ranged from 50 to 70%, together with approximately 30% CD14+ monocytes/macrophages. An average of 40% of T cells expressed CD26 in nasal biopsies of localized WG, compared with about 16% in specimens of generalized WG. In parallel, a higher number of interferon-gamma (IFN-gamma)-positive cells were detected in nasal tissue of localized than in generalized WG. PBMCs from localized WG similarly exhibited higher spontaneous IFN-gamma production in contrast to generalized WG (207 vs. 3 pg/ml, p<0.05). Interleukin-4 (IL-4) mRNA was found in higher amounts in generalized than in localized WG. IL-4 production was negligible in both disease and controls. In addition, both IL-10 mRNA and IL-10 protein levels of activated PBMCs from localized WG were elevated when compared with generalized disease (574 vs. 154 pg/ml, p<0.05) or healthy controls (574 vs. 246 pg/ml, p<0.05). It is conluded that in nasal tissues, mainly CD4+/CD26+ T cells as well as IFN-gamma-positive cells may support a polarized Th1-like immune response. Furthermore, the data suggest that this in situ immune response is already initiated and established in localized WG, accompanied by increased peripheral IFN-gamma and IL-10 production.

The current paradigm regarding sodium handling in animals and humans postulates that total body sodium is regulated predominately via regulation of extracellular volume. Active sodium storage independent of volume retention is thought to be negligible. However, studies in animals, hypertensive patients, and healthy humans suggest water-free storage of sodium in skin. We hypothesized that tissue sodium concentrations ([Na]) found in humans vary and reflect regulation due to variable glycosaminoglycan content due to variable expression of XYLT-1. Twenty seven patients on dialysis and 21 living kidney transplant donors free of clinically detectable edema were studied. During surgery, abdominal skin, muscle, and arteries were biopsied. [Na] was determined by inductively coupled plasma-optical emission spectrometry, semiquantitative glycosaminoglycan content with Alcian stain, and XYLT-1 expression by real-time PCR. [Na] of arteries were ranging between 0.86 and 9.83 g/kg wet wt and were significantly higher in arteries (4.52 ± 1.82 g/kg) than in muscle (2.03 ± 1.41 g/kg; < 0.001) or skin (3.24 ± 2.26 g/kg wet wt; = 0.038). For individual patients [Na] correlated for skin and arterial tissue ( = 0.440, = 0.012). [Na] also correlated significantly with blinded semiquantitative analysis of glycosaminoglycans staining ( = 0.588, = 0.004). In arteries XYLT-1 expression was also correlated with [Na] ( = 0.392, = 0.003). Our data confirm highly variable [Na] in human skin and muscle and extend this observation to [Na] in human arteries. These data support the hypothesis of water-independent sodium storage via regulated glycosaminoglycan synthesis in human tissues, including arteries.

Anti-neutrophil cytoplasmic Abs (cANCAs) against conformational epitopes of proteinase 3 (PR3) are regarded as an important pathogenic marker in Wegener’s granulomatosis (WG). Although the three-dimensional structure of PR3 is known, binding sites of mAbs and cANCAs have not been mapped to date. Competitive binding and biosensor experiments suggested the existence of four nonoverlapping areas on the PR3 surface. In this paper, we present an approach to identify these discontinuous surface regions that cannot be mimicked by linear peptides. The very few surface substitutions found in closely related PR3 homologs from primates, which were further varied by the construction of functional human-gibbon hybrids, resulted in the differential loss of three Ab binding sites, two of which were mapped to the N-terminal β-barrel and one to the linker segment connecting the N- and C-terminal barrels of PR3. The sera from WG patients differed in their binding to gibbon PR3 and the gibbon-human PR3 hybrid, and could be divided into two groups with similar or significantly reduced binding to gibbon PR3. Binding of almost all sera to PR3–α1-protease inhibitor (α1–PI) complexes was even more reduced and often absent, indicating that major antigenic determinants overlap with the active site surface on PR3 that associates with α1-PI. Similarly, the mouse mAbs CLB12.8 and 6A6 also did not react with gibbon PR3 and PR3–α1-PI complexes. Our data strongly suggest that cANCAs from WG patients at least in part recognize similar surface structures as do mouse mAbs and compete with the binding of α1-PI to PR3.

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