Assessment of longitudinal serum neutrophil extracellular trap-inducing activity in anti-neutrophil cytoplasmic antibody-associated vasculitis and glomerulonephritis in a prospective cohort using a novel bio-impedance technique.

Neutrophil extracellular traps (NET) have been implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Here, we developed a novel, label-free, high-throughput bio-impedance technique to effectively measure serum NET-inducing activity. Using this technique, NET-inducing activity of serum derived from patients with AAV was assessed in a prospective cohort of 62 patients presenting with active AAV with major organ involvement. Thirty-five patients presented with new and 27 patients presented with relapsing AAV, of whom 38 had kidney and/or 31 had lung involvement. NET-inducing activity was assessed at diagnosis of active AAV (time zero), during the first 6 weeks of treatment, and after 6 months of treatment. Forty-seven patients revealed elevated NET-inducing activity at time zero. After initiation of immunosuppressive treatment, NET-inducing activity was reduced at six weeks. A subsequent increase at six months could potentially identify patients with relapsing disease (hazard ratio, 11.45 [interquartile range 1.36-96.74]). NET-inducing activity at time zero correlated with kidney function and proteinuria. Importantly, in kidney tissue, NETs co-localized with lesions typical of ANCA-associated glomerulonephritis and even correlated with systemic serum NET-inducing activity. Thus, our prospective data corroborate the importance of NET formation in AAV and ANCA-associated glomerulonephritis and the potential of longitudinal evaluation, as monitored by our novel bio-impedance assay and detailed histological evaluation.

CD163 and CD206 expression define distinct macrophage subsets involved in active ANCA-associated glomerulonephritis.

INTRODUCTION: Macrophages are key players in the immunopathology of anti-neutrophil cytoplasmic antibody (ANCA) mediated-vasculitis (AAV) with glomerulonephritis (ANCA GN). Different macrophage phenotypes are expected to play distinct roles in ANCA GN. Macrophages expressing CD163 and CD206 are found in lesions associated with ANCA GN. Hence, we aimed to investigate the clinicopathological significance of CD206 and CD163 in ANCA GN in a multicenter retrospective cohort study. MATERIAL AND METHODS: Patients with ANCA-associated vasculitis, with clinical data, serum and urine samples were included from three cohorts. Serum soluble CD206 (ssCD206) and urinary soluble CD163 (usCD163) levels were measured. Human kidney tissue samples (n = 53) were stained for CD206 and CD163 using immunohistochemistry and immunofluorescence, and findings were correlated with clinical and pathological data. RESULTS: In total, 210 patients were included (i.e., ANCA GN, n = 134; AAV without GN, n = 24; AAV in remission n = 52). Increased levels of both ssCD206 and usCD163 were seen in ANCA GN. High levels of ssCD206 declined after reaching remission, however, ssCD206 did not improve the accuracy of usCD163 to detect ANCA GN. Soluble markers correlated with histopathological findings. CD163+CD206- macrophages were found in the glomerulus and may play pivotal roles in glomerulonephritis, whereas CD206+CD163- and CD206+CD163+ macrophages were located tubulointerstitially and likely play a more prominent role in ANCA-associated tubulointerstitial inflammation. In ANCA GN patients increasing levels of ssCD206 increased the risk for end-stage renal disease and mortality. CONCLUSIONS: Our results confirm and extend the notion that CD206+ and CD163+ macrophages are prominent components of the cellular infiltrate in ANCA GN. We found distinct macrophage phenotypes that may play distinct roles in the immunopathology of ANCA GN and elaborate on a potential mechanism underlying the findings of this study. usCD163 remains an excellent marker to detect active ANCA GN, whereas ssCD206 seems a more prominent marker for risk prediction.

Complement in ANCA-associated glomerulonephritis.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) are found in pauci-immune necrotizing crescentic glomerulonephritis. In the past, the role of complement in ANCA-associated vasculitis (AAV) was assumed to be minimal. More recently, however, it was found that blocking the complement cascade in a mouse model of AAV reduces glomerular damage. Immune complex deposits have been found in biopsies from AAV patients. In this study, we questioned whether immune complex formation or deposition may result in complement activation in ANCA-associated glomerulonephritis. METHODS: ANCA-positive patients from the Limburg Renal Registry were included between 1979 and 2011. Renal histology was documented together with immunoglobulin and complement immunofluorescence. In addition, C3d, properdin, C4d and mannose-binding lectin (MBL) were stained. Electron microscopy was performed. Circulating immune complexes were determined in a subset of patients, as well as C3 allotypes. RESULTS: C3c was found in 78 of 187 renal biopsies (41.7%) divided over 32.3% of proteinase-3 (PR3)-AAV patients and 52.3% of myeloperoxidase (MPO)-AAV patients (P = 0.006), whereas C3d was found positive in 51.1% of PR3-AAV patients and 70.4% of MPO-AAV patients (P = 0.105). C4d was found positive in 70.8%, properdin in 38.7% and MBL in 30.4% of patients. Whereas C4d and MBL positivity was similar between the AGN groups, properdin was more common in biopsies classified as crescentic compared with biopsies classified as focal or mixed. Renal biopsies positive for C3d and/or properdin showed more cellular crescents and less normal glomeruli compared with biopsies negative for C3d and/or properdin (P < 0.05). In 3 out of 43 renal biopsies analysed by electron microscopy, small electron dense deposits were found. In 14 of 46 patients analysed, circulating immune complexes were detectable. No association between histological findings and C3 allotypes was found. CONCLUSIONS: In the majority of AAV patients, no immune complex deposits were found in their renal biopsies. C3d, C4d and C5b-9 staining, however, was found to be positive in a majority of analysed renal biopsies. Importantly, C3d and properdin staining was associated with cellular crescents. We hypothesize that local immune complexes are quickly degraded in AAV and therefore not visible by electron microscopy. Our findings are compatible with the hypothesis that complement activation in AAV occurs predominantly via alternative pathway activation.