Peripheral blood was collected by using disodium EDTA as anticoagulant, and plasma was collected after centrifugation. Figure S5. Co-localization of CD88 and elastase in neutrophil-coated slides. (A) Colocalization of CD88 and elastase in resting neutrophils by Abcam antibody (catalog number, ab11884). (B) Colocalization of CD88 and elastase in activated neutrophils by Abcam antibody (catalog number, ab11884). (C) Colocalization of CD88 and elastase in resting neutrophils by Santa Cruz antibody (catalog number, sc-70812). (D) Colocalization of CD88 Vibunazole and elastase in activated neutrophils by Santa Cruz antibody (catalog number, sc-70812). Magnification, 400. ar3873-S5.TIFF (1.0M) GUID:?B92A054A-3253-4C2E-A4BD-9E46361B89A1 Additional file 6 Figure S6. Immmunohistochemical staining of CD88 in spleen. (A) Immmunohistochemical staining of CD88 Vibunazole in spleen by Abcam antibody (catalog number, ab11867). (B) Immmunohistochemical staining of CD88 in spleen by Abcam antibody (catalog number, ab11884). (C) Immmunohistochemical staining of CD88 in spleen by Santa Cruz antibody (catalog number, sc-70812). ar3873-S6.TIFF (2.3M) GUID:?977CB365-F572-461B-BCC7-6012D1F30F81 Additional file 7 Figure S7. Hematoxylin/eosin (HE) and CD88 counterstaining. CD88, open arrow; neutrophils, solid arrow. Magnification, 400. ar3873-S7.TIFF (811K) GUID:?6315ACE7-A8C1-4C75-A029-02DC09832A11 Abstract Introduction The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a plays a central role. In this study, plasma and urinary levels of C5a as well as renal C5a receptors (CD88 and C5L2) expression were investigated in patients with AAV. Methods Twenty-four patients with AAV in the active phase, 19 patients with AAV in the remission phase, and 20 patients with lupus nephritis (LN) were included. Plasma and urinary levels of C5a were measured with enzyme-linked immunosorbent assay (ELISA). The staining of CD88 and C5L2 in renal specimens was detected with immunohistochemistry. Results The level of plasma C5a was significantly higher in patients with AAV in the active phase than that in patients in remission, that in patients with LN, and that in normal controls. The urinary C5a level was significantly higher in patients with AAV in the active Vibunazole phase than that in patients in remission and that in normal controls, but not significantly different between patients with active AAV and patients with LN. The mean optical density of CD88 staining in the tubulointerstitium was significantly lower in AAV patients than that in normal controls (0.0052 0.0011 versus 0.029 0.0042; em P /em = 0.005). The mean optical density of C5L2 in glomeruli was significantly higher in AAV patients than that in normal controls (0.013 0.0027 versus 0.0032 0.0006; em P /em 0.001). The mean optical density of CD88 staining closely correlated with the initial eGFR ( em r /em = 0.835; em P /em 0.001) in AAV patients. Double-labeling immunofluorescence assay suggested that CD88 did not express on neutrophils, monocytes, or macrophages, but C5L2 expressed on neutrophils (or monocytes) and macrophages. Conclusion The elevated plasma and urinary C5a levels indicated complement activation in human AAV. The level of renal CD88 expression could reflect the disease severity of ANCA-associated glomerulonephritis. CD88 expression was downregulated, and C5L2 was upregulated in ANCA-associated glomerulonephritis. Introduction Antineutrophil cytoplasmic antibodies (ANCAs)-associated vasculitis (AAV) comprises a group of autoimmune disorders, including granulomatosis with polyangiitis (GPA, previously named Wegener granulomatosis), microscopic polyangiitis (MPA), Churg-Strauss syndrome (CSS), and renal-limited vasculitis (RLV) . These diseases are characterized by necrotizing small-vessel vasculitis. ANCAs are the serologic hallmarks for the previously mentioned primary small-vessel vasculitis. ANCAs are predominantly immunoglobulin G (IgG) autoantibodies directed against neutrophil cytoplasmic constituents, in particular, proteinase 3 (PR3) and myeloperoxidase (MPO) . The histopathologic hallmark of ANCA-associated glomerulonephritis is “pauci-immune” necrotizing crescentic glomerulonephritis (NCGN), characterized by little or no glomerular staining for immunoglobulins and complements in renal histology by immunofluorescence microscopy examination. Recent studies in a mouse model of anti-MPO IgG-mediated glomerulonephritis suggested that complement activation via the alternative pathway was crucial for the disease development [2,3]. In particular, Schreiber em et al. /em  further found that recombinant C5a dose-dependently primes KLRK1 neutrophils for an ANCA-induced respiratory burst. In animal models, C5a receptor (C5aR)-deficient animals were protected from ANCA-induced NCGN. As such, Vibunazole the interaction between C5a and C5aR (CD88) may compose an amplification loop and, thus, plays a central role in ANCA-mediated neutrophil recruitment and activation . However, the role of interaction between C5a and its receptors in the pathogenesis of human AAV is less clear. C5a is a cleavage product of complement C5 with chemotactic and anaphylatoxic properties. C5a exerts its action through two different receptors: C5aR (CD88) and C5a.