Section 3 -

Pathology and Pathogenesis of ANCA Glomerulonephritis and Vasculitis J. Charles Jennette University of North Carolina Chapel Hill, USA

Introduction Antineutrophil cytoplasmic antibodies (ANCAs) are found in > 80% of patients with active untreated necrotizing and crescentic glomerulonephritis and necrotizing small vessel vasculitis that has a paucity or absence of immunoglobulin deposited in vessel walls. The primary clinicopathologic phenotypes of ANCA small vessel vasculitis are Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome and renal-limited vasculitis [1, 2, 3]. Substantial in clinical and experimental evidence indicate that ANCAs are not only a serologic marker but also are the pathogenic factors that cause ANCA disease [4]. Microscopic polyangiitis is necrotizing vasculitis with few or no immune deposits that primarily affects small vessels, such as capillaries, venules, or arterioles [5, 6]. Crescentic glomerulonephritis is common. Necrotizing arteritis of small and medium-sized arteries may or may not be present. Wegener's granulomatosis and Churg-Strauss syndrome have pauci-immune small vessel vasculitis that is indistinguishable from the vasculitis of microscopic polyangiitis. However, Wegener's granulomatosis and Churg-Strauss syndrome have their own distinguishing diagnostic features [5, 7, 8]. Wegener's granulomatosis has necrotizing granulomatous inflammation that most often involves the upper or lower respiratory tract [7]. Patients with Churg-Strauss syndrome have a history of asthma and blood eosinophilia [5, 8]. Microscopic polyangiitis, Wegener's granulomatosis and Churg-Strauss syndrome are all expressions of pauci-immune small vessel vasculitis. Pauci-immune small vessel vasculitis is almost synonymous with ANCA small vessel vasculitis; however, it is very important to recognize that a minority of patients with pauci-immune small vessel vasculitis are ANCA-negative. Pathology Acute ANCA glomerulonephritis is characterized by segmental fibrinoid necrosis with focal lysis of glomerular basement membranes [1, 2, 3, 9]. Neutrophils often are adjacent to or within areas of necrosis, but many non-necrotic segments and glomeruli look remarkably normal with no hypercellularity. Within a few days, glomerular lesions contain predominantly macrophages. Sclerosis begins to replace necrosis within a week. Severe glomerular necrosis often causes disruption of Bowman's capsule resulting in periglomerular inflammation that may have a granulomatous appearance, sometimes with multinucleated giant cells. Approximately 90% of specimens with ANCA glomerulonephritis have glomerular crescents, which usually affects around 50% of glomeruli and rarely is >75% [3]. The necrotizing and crescentic glomerular injury evolves into segmental or global glomerular sclerosis within a few weeks. Masson trichrome staining is useful for differentiating between areas of fibrinoid necrosis (red) versus sclerosis (blue or green) [2]. The waxing and waning character of ANCA disease often results in glomerular lesions of different ages in the same specimen. Extra-glomerular vasculitis, which can be arteriolitis, arteritis and/or medullary angiitis, is identified in 10% to 20% of renal biopsy specimens [2, 9]. Acute arterial and arteriolar lesions have segmental fibrinoid necrosis with varying proportions of infiltrating neutrophils, eosinophils, monocytes and macrophages. Leukocytoclasia is frequent. The vascular necrosis and inflammation progress to vascular scarring. Medullary leukocytoclastic angiitis affecting the medullary vasa recta is a distinctive but not completely specific feature of ANCA renal disease [2, 10]. By definition, ANCA-associated, pauci-immune crescentic glomerulonephritis has a paucity (2+ or less} or absence of glomerular staining for immunoglobulin and complement [2, 11]. Crescents and foci of glomerular fibrinoid necrosis stain irregularly for fibrin. Although most ANCA-associated glomerulonephritis has less than 2+ staining for immunoglobulin, approximately 30% of patients with anti-GBM crescentic glomerulonephritis and approximately 25% of patients with immune complex crescentic glomerulonephritis are ANCA-positive [2, 11]. Pathogenesis Clinical Evidence The high frequency of ANCAs in patients with pauci-immune glomerulonephritis and small vessel vasculitis suggests but does not prove that ANCAs cause the disease [4]. ANCA titers correlate to a degree with disease activity but this could be either a cause or an effect of the disease. The induction of circulating ANCA by drugs, such as propylthiouracil and hydralazine, and the subsequent development of pauci-immune necrotizing and crescentic glomerulonephritis and vasculitis supports a pathogenic role for ANCA [12]. However, the most convincing clinical evidence that ANCA are pathogenic is the report of a neonate who developed glomerulonephritis and pulmonary hemorrhage after delivery from a mother with MPO-ANCA microscopic polyangiitis [13, 14]. The transplacental transfer of MPO-ANCA IgG into the baby's circulation apparently caused glomerulonephritis and vasculitis. In Vitro Evidence Numerous in vitro experimental studies have demonstrated that PR3-ANCA and MPO-ANCA IgG can activate neutrophils to release mediators of inflammation such as granule enzymes, cytokines and oxygen metabolites [4, 15, 16]. Some circulating neutrophils have MPO and PR3 on the surface to interact with ANCA IgG [17, 18]. Other neutrophils are induced to express surface MPO and PR3 by priming, for example by exposure to proinflammatory cytokines (e.g. TNF alpha) [19, 20]. In vitro, ANCA IgG causes TNF-primed neutrophils to release toxic oxygen species, inflammatory cytokines, lytic enzymes and toxic enzymes, e.g. PR3 and MPO [23, 24]. ANCA-induced neutrophil activation is mediated by both engagement of Fc receptor (21,22} and binding of ANCA Fab'2 to antigens at the surface of neutrophils [23, 24, 25]. ANCA-activate neutrophils adherence to cultured endothelial cells, transmigration across the endothelial layer, and kill cultured endothelial cells [26, 27, 28, 29]. Monocytes (but not macrophages) also contain MPO and PR3 [30], and can be activated by ANCA IgG resulting in release of inflammatory mediators such as oxygen metabolites [31], monocyte chemoattractant protein-1 [32], and interleukin-8 [33]. Animal Models of ANCA Disease The pathogenicity of ANCA IgG is supported by a mouse model that has been developed by Xiao and her associates [34, 35, 36, 37]. Xiao et al have induced pauci-immune focal necrotizing and crescentic glomerulonephritis and systemic necrotizing vasculitis by intravenous injection of murine anti-MPO IgG into normal mice [34, 35, 36, 37]. The anti-MPO IgG is obtained form MPO knock out mice immunized with mouse MPO. All mice injected with a nephritogenic dose of anti-MPO IgG develop focal segmental fibrinoid necrosis and crescents in glomeruli. No mice injected with control anti-BSA IgG develop glomerulonephritis. Some mice also develop systemic small vessel vasculitis including leukocytoclastic angiitis, necrotizing arteritis, pulmonary capillaritis and necrotizing granulomatous inflammation. The critical role for neutrophils in this model is demonstrated by the complete protection against disease when mice first are injected with NIMP-R14, a rat monoclonal antibody that depletes mouse neutrophils [35]. The importance of neutrophil priming in this model was demonstrated by augmenting the glomerular disease by injection of LPS, which caused increased circulating TNF-alpha [36]. This effect was eliminated by injections antibodies to TNF-alpha. A surprising finding is that the alternative complement system is important in the induction of disease by anti-MPO [37]. Activation of neutrophils by ANCA causes the release of factors that activate complement, which in turn amplifies the inflammatory injury. A much more severe necrotizing and crescentic glomerulonephritis is induced by passive transfer of splenocytes (including anti-MPO B cells) from MPO-/- mice that have been immunized with murine MPO in Rag2-/- immune deficient mice [34]. This model is complicated by the induction of immune complex glomerulonephritis in the background, which is a nonspecific consequence of injection immune competent lymphocytes into immune deficient mice. Injection of anti-MPO splenocytes causes necrosis and crescents in approximately 80% of glomeruli [34]. Selective depletion of various lymphocytes from the transferred splenocytes demonstrates that this disease is caused primarily by the transfer of B cells and not T cells [38]. Injection of the unaltered anti-MPO splenocytes causes crescents and necrosis in approximately 80% of glomeruli whereas injection of preparations with 80% T cells caused necrosis and crescents in only 5% of glomeruli and injection of preparations with >99% pure T cell caused no crescents or necrosis. Little et al. have induced pauci-immune focal necrotizing and crescentic glomerulonephritis and pulmonary capillaritis in rats by immunization with human MPO, which induced anti-MPO antibodies that cross react with human and rat MPO [39]. They also used intravital microscopy of mesenteric vessels to demonstrate anti-MPO induced adherence of leukocytes to vessel walls with resultant injury and hemorrhage. Immunogenesis of the ANCA Autoimmune Response Pendergraft et al. have published data suggesting that peptides that are mimics of the anti-sense complementary peptides of PR3 can induce a pathogenic anti-PR3 autoimmune response [40]. Some patients with PR3-ANCA glomerulonephritis and small vessel vasculitis have antibodies that react with a PR3 complementary peptide (i.e. anti-sense peptide). These anti-complementary PR3 antibodies bind to anti-PR3 antibodies, and these two antibodies comprise an anti-idiotypic pair. Mice immunized with complementary PR3 peptide produce separate populations of antibodies that react with complementary PR3 and normal PR3, and are an anti-idiotypic pair. In patients, complementary PR3 could be endogenously produced or introduced exogenously, for example by an infectious pathogen. With respect to this latter possibility, ANCA disease is known to be associated with certain infections, such as Staphylococcus aureus and Ross River virus [41]. Interestingly, Staphylococcus aureus and Ross River virus pathogens contain peptides that are mimics of complementary peptides of PR3 [41]. References Jennette JC, Falk RJ. Small vessel vasculitis. N Engl J Med 1997;337:1512-23. 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