Yoshinori Komagata

Immunopathologic co-localization of MPO, IgG, and C3 in glomeruli in human MPO-ANCA-associated glomerulonephritis.

Myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA)- associated glomerulonephritis (GN) is characterized by pauci-immune necrotizing glomerulonephritis(NGN). Although it has been thought that MPO-ANCA is involved in the pathogenesis of capillary injuries in NGN via activation of neutrophils, recent studies suggest a possible role of other factors such as immunoglobulins precipitated on the glomeruli. Here we performed a pathological study investigating a relationship of deposition of MPO, IgG, complements with regard to MPO-positive cells and glomerular capillaries in human MPO-ANCA-associated GN. Renal specimen including 317 glomeruli obtained from 20 patients with MPOANCA- associated GN were analyzed. All of the specimens showed significant focal segmental deposition of IgG. There was a significant glomerular infiltration of MPO-positive cells along with deposition of extracellular MPO in the active lesions of segmental and global NCG, with CD34 staining being decreased in the adjacent areas. IgG deposits were almost colocalized with C3 and partly with MPO, which are also associated with a decrease in CD34 staining, suggesting that immune complex formation and the resultant capillary injuries. Actually occurred, the colocalization of MPO, IgG and C3 was seen only in the glomerular lesions with low severity and activity. These results suggest that not only MPO itself released from the neutrophils but also immune complexes composed of MPO and anti-MPO antibody may play some pathogenetic roles for the glomerular injuries especially in the early phase of human MPO-ANCA-associated GN.

Adult-onset Still’s disease-like manifestation accompanied by the cancer recurrence after long-term resting state

Abstract A 72-year-old woman presented 9 months ago with skin rash on her bilateral forearms, which was followed by intermittent high fever, and stiffness and swelling of her bilateral fingers. She was diagnosed with seronegative rheumatoid arthritis (RA). She had a past history of breast cancer and had undergone breast preservation surgery 13 years previously. During admission in our hospital, she developed high fever and leukocytosis with a relapsing skin rash, sore throat, polyarthralgia and increased levels of serum ALT/AST and ferritin, all of which fulfilled Yamaguchi’s criteria for adult-onset Still’s disease (AOSD). While we tried to exclude other diseases that may show AOSD-like manifestations, pancytopenia rapidly developed and bone marrow biopsy strongly suggested the diagnosis of macrophage activating syndrome (MAS). Accordingly, steroid pulse therapy was begun, followed by oral glucocorticoid therapy. Thereafter, all of her symptoms improved, but systemic rash, inflammatory signs and pancytopenia gradually progressed. The results of bone marrow pathology, which returned 2 weeks after the beginning of treatment, revealed hemophagocytosis with CK7-positive/CK20-negative atypical cells that suggested recurrence of breast cancer in the bone marrow, thus all of her AOSD-like symptoms were considered to be paraneoplastic manifestations of late-onset metastatic breast cancer. She was treated successfully with chemotherapy. When we see the patients showing AOSD-like symptoms with a history of malignancy, we should consider the possibility of paraneoplastic syndrome due to cancer recurrence.

The role of myeloperoxidase and myeloperoxidase–antineutrophil cytoplasmic antibodies (MPO-ANCAs) in the pathogenesis of human MPO-ANCA-associated glomerulonephritis

It is well known that antineutrophil cytoplasmic antibodies (ANCAs) are pathogenic and have a diagnostic value for ANCA-associated vasculitis. We demonstrated that a rise in myeloperoxidase (MPO)-ANCA titers during remission is often predictive of a future relapse in MPO-ANCA-associated vasculitis. Pathological examination of renal biopsies indicated that not only MPO-ANCAs, but also extracellular MPO, an in situ immune complex composed of MPO and MPO antibodies, may play important roles in the pathogenesis of glomerular capillary injury in MPO-ANCA-associated vasculitis.

SAT0516 Two distinct subsets of low density granulocytes in anca associated vasculitis

Background Low density granulocyte (LDG), a proinflammatory population of neutrophils, was first described in systemic lupus erythematosus (SLE) and has been shown to contribute to lupus pathogenesis. It has been suggested that LDGs have a pathogenic role in ANCA associated vasculitis (AAV) based on the data of gene expression signature in AAV and the ability of excessive neutrophil extracellular traps (NETs) formation by LDGs in AAV. However, more detailed analysis of LDG in AAV patients has not been reported. Objectives In this study we investigated the characteristics of LDG in AAV patients using flow cytometry and proteomics approach and examined the correlations with disease activity. Methods We examined the presence of LDGs in peripheral blood of 10 AAV patients before treatment and followed them for 4 months with immunosuppressive therapy. Normal-density granulocytes (NDGs) were isolated by dextran sedimentation and PBMCs were isolated by Ficoll gradient. LDGs were assessed using cell surface expression of CD14 and CD15 by flow cytometry and isolated by magnetic bead procedure from PBMCs. We performed comparative proteomic analysis among LDGs and autologous NDGs and healthy controls (HC)-NDGs. Results LDG frequencies were 9.8-fold higher in AAV patients before treatment relative to HC. There were two distinct populations of LDGs showing different cell surface expressions of CD10, CD14, and CD15 in AAV patients. One population of LDGs was mainly CD10 positive and another one was CD10 negative. Although the frequency of CD10 positive-LDG decreased along with decrease of disease activity, the frequency of CD10 negative-LDG increased in 4 months (17.9-fold higher than before treatment). Comparative proteomic analysis revealed these two populations of LDGs were distinct from each other and had common differences from autologous NDGs and HC-NDGs. LDG frequencies were 9.8-fold higher in AAV patients before treatment relative to HC. There were two distinct populations of LDGs showing different cell surface expressions of CD10, CD14, and CD15 in AAV patients. One population of LDGs was mainly CD10 positive and another one was CD10 negative. Although the frequency of CD10 positive-LDG decreased along with decrease of disease activity, the frequency of CD10 negative-LDG increased in 4 months (17.9-fold higher than before treatment). Comparative proteomic analysis revealed these two populations of LDGs were distinct from each other and had common differences from autologous NDGs and HC-NDGs. Conclusions We identified two distinct subsets of LDGs in AAV. It is possible that they play different roles in the pathogenesis of AAV. References [1] Denny MF, et al. A distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type I Interferons. J Immunol2010;184(6):3284–3297. [2] Grayson, et al. Neutrophil-Related Gene Expression And Low-Density Granulocytes Associated with Disease Activity and Response to Treatment in ANCA-Associated Vasculitis. Arthritis Rheumatol2015;67:1922–1932. Disclosure of Interest None declared

FRI0247 The Release of Nets from MPO-AAV Neutrophils was Increased by Anti-MPO Antibody and Correlated with Disease Activity

Background NETosis, a unique form of cell death of neutrophils, is characterized by the active release of chromatin fibers called NETs, that trap and kill invading microbes extracellularly. Although NETosis plays a crucial role in host defense, excessive NETs formation becomes self-defeating by promoting tissue injury and organ damage. It has been known that NETs are implicated also in the pathogenesis of autoimmune vasculitis such as SLE, RA and ANCA –associated vasculitis (AAV). Objectives We observed NETs formation of neutrophils from MPO-AAV patients which is a majority type of AAV in Asia, examined the effects of anti-MPO antibody and immunosuppressive therapy on the release of NETs in order to investigate the role of NETs and MPO-ANCA in MPO-AAV.We also observed the damage of human renal glomerular endothelial cells (HRGECs) by neutrophils in order to investigate the role of NETs in AAV. Methods Isolated peripheral blood neutrophils from healthy donors, pre/post-treatment MPO-AAV patients were incubated with phorbol myristate acetate (PMA), which is known as a strong inducer of NETs, PMA plus anti-MPO antibody or PMA plus anti-PR3 antibody. Neutrophils were stained with Hoechst 33342, SYTOX Green and the percentage of NETs producing cells were calculated. Brd-U labeled HRGECs stimulated by PMA were co-cultured with neutrophils. A cellular DNA fragmentation ELISA was used to quantitatively determine HRGECs damage. Results Neutrophils from pre-treatment MPO-AAV patients produced much more fiber-like NETs than neutrophils from controls and the shape of fiber-like NETs were different from controls. Anti-MPO antibody increased the release of fiber-like NETs both in controls and MPO-AAV patients. The release of fiber-like NETs decreased in tandem with the decrease of ANCA titer after initial treatment and increased with the rise of ANCA titer in some MPO-AAV patients. Although PMA-stimulated HRGEC were damaged in the presence of neutrophils, HRGEC without PMA stimulation were not damaged. Conclusions Neutrophils from pre-treatment MPO-AAV patients released different type of fiber-like NETs and the amount of fiber-like NETs correlated with the activity of AAV. HRGECs stimulated with PMA were damaged in the presence of neutrophils. These data suggest the release of fiber-like NETs by neutrophils at vascular endothelium is important for the pathogenesis of AAV. References Jennette JC et al: 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides, 65: 1-11, 2013. Arimura Y et al: Serum myeloperoxidase and serum cytokines in anti-myeloperoxidase antibody-associated glomerulonephritis, Clin Nephrol, 40: 256-264, 1993. Kessenbrock K et al: Netting neutrophils in autoimmune small-vessel vasculitis, Nat Med, 15: 623-625, 2009. Gupta AK et al: Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death, FEBS Lett, 584: 3193-3197, 2010. Kambas K et al: Tissue factor expression in neutrophil extracellular traps and neutrophil derived microparticles in antineutrophil cytoplasmic antibody associated vasculitis may promote thromboinflammation and the thrombophilic state associated with the disease, Ann Rheum Dis, 73: 1854-1863, 2014. Disclosure of Interest None declared

SAT0183 Autoantibodies for Several Antigens in Neutrophil Cytoplasm other than PR3 and MPO Promote Release of Nets from Neutrophils

Background Neutrophil extracellular traps (NETs) were first described as web-like structures that trap and neutralize microbes at sites of infection. NETs are comprised of chromatin components and neutrophil cytoplasmic proteins. Because these components of NETs might provide an immunogenic substrate for autoimmune responses during regular encounters with commensal and pathogenic microbes, it has been reported that NETs are involved in autoimmunity such as SLE or ANCA associated vasculitis (AAV). It has also been reported that autoantibodies themselves for important cytoplasmic auto-antigens of neutrophils such as MPO or PR3 induce NETs production. However, the role of other neutrophil cytoplasmic antigens for the production of NETs is unclear. Objectives Thus, we investigated how auto-antibodies for these antigens other than PR3 and MPO are involved in NETs productions by neutrophils. Methods Human peripheral blood neutrophils were obtained from healthy donors. Neutrophils were seed onto Polystyrene chamber slide system and incubated with PMA (control) or PMA plus several kinds of anti-neutrophil cytoplasmic antigens for 3 hours. Cells were fixed and stained with Hoechst 33342 and Sytox Green for DNA and anti-MPO antibody. The percentage NETs producing cells and the quantitation of nuclear decondensation were analyzed using Image J software. We distinguished cells that released NETs fiber from cells that just died of NETosis. Results Anti-MPO antibody strongly promoted both NETosis induction and release of NETs fiber. Although anti-PR3 antibody promoted NETosis induction, it did not increase the release of NETs fiber. Antibody for cathepsin G, found in the azunophil granule, also promoted NETosis induction only. There were some other antibodies, such as anti-lactoferrin and anti-neutrophil elastase, that promoted both NETosis and release of NETs fiber. Conclusions It has been thought that ANCAs directed against granule proteins of neutrophils are implicated in the pathogenesis of AAV, partly because ANCAs promote NETs production by neutrophils. We showed that not only anti-MPO and anti-PR3 but also antibodies for other neutrophil cytoplasmic antigens promote NETs production. The mechanism these antibodies for cytoplasmic protein induce NETosis and how important they are for the pathogenesis of AAV are to be elucidated. Disclosure of Interest None Declared