Extended Abstracts Nephron 2015;129(suppl 2):1–44 DOI: 10.1159/000381120

Andrew J. Rees Clinical Institute of Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna Correspondence to: Andrew J. Rees E-Mail: [email protected]

The morphological appearances of vasculitis were described well over 150 years ago and clear descriptions of the clinical syndromes we recognise today are almost a century old, including those of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) – the subject of this brief review. Analogies to the Arthus reaction, and to clinical and experimental serum sickness, firmly established small vasculitis as immune complex diseases despite the scanty deposits of immunoglobulins and complement. However, the intensive search for suitable immune complex assays proved fruitless. It is ironic that Foko van der Woude was attempting to develop a novel immune complex assay when he made the serendipitous discovery that the uptake of immunoglobulins from patients’ serum were not immune complexes internalised by Fc receptors but autoantibodies specific for cytoplasmic antigens. Niels Rasmussen had independently discovered the same autoantibodies in Scandanavian patients in the seminal paper that linked antibodies to cytoplasmic antibodies (ACPA – now ANCA) to GPA [1]. In retrospect, antileukocyte antibodies were originally described in ‘polyarteritis nodosa’ in a paper that went unnoticed at the time and has been largely ignored since [2], and was followed by reports of ANCA in two small groups of Australian patients with focal necrotizing glomerulonephritis ‘indistinguishable from microscopic polyarteritis’. However, these were small and incomplete studies and it was the van der Woude/Rasmussen paper that transformed understanding of autoantibodies in small vessel vasculitis and was quickly followed by the identification of MPO and PR3 as their principal targets [3, 4]. It was rapidly shown that positive assays for ANCA and their target antigens have a better than 90% sensitivity and specificity for diagnosing MPA and GPA; in vitro studies showed antibodies to MPO and PR3 activated primed neutrophils; and experimental models proved that anti-MPO antibodies could cause FNGN in vivo [5]. This led to the current paradigm that MPA and GPA are autoimmune diseases caused by autoantibodies to MPO and PR3 and led to the collective term ANCA-associated vasculitis (AAV). Collaborations between those investigating AAV increased: genome wide association studies have provided further evidence for the importance of PR3 and MPO for pathogenesis [6], and well powered multinational controlled clinical trials have regularised treatment strategies. Unexpectedly, data from these trials, together with meta-analyses, revealed the association between autoantibodies to MPO and PR3 and AAV are not as straightforward as previ-

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ously supposed [7–9]. Indeed, quantifying of ANCA and antibodies to MPO or PR3 show they reflect disease activity poorly and cannot be used to predict relapses, either in those treated with traditional immunosuppressive drugs or with anti-B cell therapy rituximab. It is now important to determine whether this is simply due to technical deficiencies with the assays currently in use or reflects the underlying biology of the disease. The issue whether injury in AAV is predominantly due to the canonical ANCA is given added weight by data emerging from three different sources: increasingly sophisticated studies of the T cells from patients with AAV and murine models of FNGN induced by autoimmunity to MPO; the discovery of new autoantibodies with the potential to contribute to the pathogenesis of AAV; and clear descriptions of groups of patients with apparently typical AAV in the absence of detectable ANCA or autoantibodies to MPO and PR3. These data will now be reviewed briefly. Pathogenicity of autoantibodies to MPO. Passive transfer experiments in mice provide direct evidence of pathogenicity of anti-MPO antibodies [3, 5]. MPO is not expressed in normal glomeruli but can be released there from infiltrating neutrophils, either after degranulation or through the formation of neutrophil extracellular traps (NETs). Accordingly, injury is relatively mild except in strains of mice with a genetically determined increase in neutrophil sensitivity, unless high doses of anti-MPO antibodies are injected or additional neutrophil activating factors such as LPS, TNF or C5a are co-administered. In these, passive injury is Fc and complement dependent. Delayed type hypersensitivity in autoimmunity to MPO The passive transfer experiments established that anti-MPO antibodies could be pathogenic but active autoimmune models are needed to delineate the respective contributions of specific antibody and T cells. This has been analysed systematically by Holdsworth, Kitching and their colleagues in a series of papers with surprising results. They show that mice immunized with MPO develop autoantibodies to MPO together with delayed type hypersensitivity (DTH) to it, characterised by Th1 and Th17 cells [10]. The autoimmune mice remained healthy but focal necrotizing glomerulonephritis (FNGN) could be triggered by injection of a dose of anti-GBM antibody too low to cause detectabl injury but still sufficient to induce glomerular neutrophil recruitment and local release of MPO [11]. Unexpectedly the injury in this model was caused by DTH and not anti-MPO antibodies since it occurred normally in immunoglobulin μ chain deficient mice that have no antibodies and could be transferred by T cells and T cell clones [12]. Subsequent experiments showed that anti-GBM antibodies were not essential but could be replaced by other stimuli that caused release of MPO within glomeruli. These experiments establish that FNGN induced by autoimmunity to MPO in these mice is induced by DTH and not anti-MPO antibodies but it does not follow that the same will be true in man. Major differences in murine immune system make such extrapolations hazardous and the clinical effectiveness of rituximab in management provides proof of the importance of B cells to pathogenDownloaded by: Kainan University - 4/28/2015 11:50:22 AM

Small Vessel Vasculitis – To ANCA and Beyond

Published online: April 15, 2015


Nephron 2015;129(suppl 2):1–44 DOI: 10.1159/000381120

ognised an epitope that was cryptic in the extensively glycosylated LAMP-2 expressed by human neutrophils but was still accessible in the less complexly glycosylated LAMP-2 expressed in glomeruli. Autoantibodies from these patients bound to glomerular endothelium in tissue sections and to human glomerular endothelial cells in culture. This suggests they can cause vasculitis in the absence of antibodies to MPO and PR3, as is the case of rodent models. Conclusion

The discovery of ANCA and their close relation to MPA and GPA transformed understanding of these devastating syndromes and identified them as autoimmune diseases. Clinical and experimental studies have established an overwhelming case for the involvement of autoantibodies to MPO and PR3 in pathogenesis but recent data suggest they may need to operate in concert with other factors, but more data are needed to confirm whether or not this is so and whether novel autoantibodies and DTH are involved. Disclosure Statement

The author has no financial relationship with a commercial entity that has an interest in the subject of this paper. References 1 van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es LA, van der Giessen M, van der Hem GK, The TH: Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis. Lancet. 1985;1:425–129. 2 Yust I, Schwartz J, Dreyfuss F: A cytotoxic serum factor in polyarteritis nodosa and related conditions. Am J Med 1970:472–472. 3 Jennette JC, Falk RJ, Hu P, Xiao H: Pathogenesis of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Annu Rev Pathol 2013;8:139–160. 4 Schönermarck U, Csernok E, Gross WL: Pathogenesis of anti-neutrophil cytoplasmic antibody-associated vasculitis: challenges and solutions 2014. Nephrol Dial Transplant. 2014. pii: gfu398. [Epub ahead of print] Review. PMID: 25540095. 5 Heeringa P, Little MA: In vivo approaches to investigate ANCA-associated vasculitis: lessons and limitations. Arthritis Res Ther 2011;13:204. 6 Lyons PA, Rayner TF, Trivedi S, Holle JU, Watts RA, Jayne DR, et al: Genetically distinct subsets within ANCA-associated vasculitis. N Engl J Med 2012;367:214–223. 7 Miloslavsky EM, Specks U, Merkel PA, Seo P, Spiera R, Langford CA, et al: Clinical outcomes of remission induction therapy for severe antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Rheum 2013;65:2441–2449. 8 Tomasson G, Grayson PC, Mahr AD, Lavalley M, Merkel PA: Value of ANCA measurements during remission to predict a relapse of ANCAassociated vasculitis – a meta-analysis. Rheumatology (Oxford). 2012; 51:100–109. 9 Specks U, Merkel PA, Seo P, Spiera R, Langford CA, Hoffman GS, et al: Efficacy of remission-induction regimens for ANCA-associated vasculitis. N Engl J Med 2013;369:417–427. 10 Ooi JD, Gan PY, Odobasic D, Holdsworth SR, Kitching AR: T cell mediated autoimmune glomerular disease in mice. Curr Protoc Immunol. 2014;107:15.27.1–15.27.19. doi: 10.1002/0471142735.im1527s107. 11 Ruth AJ, Kitching AR, Kwan RY, Odobasic D, Ooi JD, Timoshanko JR, Hickey MJ, Holdsworth SR: Anti-neutrophil cytoplasmic antibodies and effector CD4+ cells play nonredundant roles in anti-myeloperoxidase

Extended Abstracts

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esis in AAV. However, it does not necessarily follow that this is due to the reduction of autoantibodies: anti-MPO and PR3 concentrations do not correlate with treatment responses after its use. B cells are not only precursors of antibody producing plasma cells but have other functions including antigen presentation and regulation of the ensuing response, and depleting B cells with rituximab in rheumatoid arthritis has profound effects on the T cell compartment [13]. MPO and PR3 specific CD4 T cells have long been recognized in AAV but little is known about their contribution to injury beyond providing help for autoantibody production [14]. However, T cells do infiltrate injured tissues in AAV and the numbers of effector memory T cells increase rapidly in the urine of patients with active renal disease [15], perhaps suggesting a contribution analogous to the mouse model. The most suggestive data about the involvement of effector T cells has been the identification of a CD8 transcriptomic signature that predicts a worse outcome (ie more frequent relapses) [17]. Novel autoantibodies in ANCA-associated vasculitis. ANCA specific for MPO and PR3 are not the only autoantibodies found in AAV. Antibodies to poorly characterised antigens on endothelial cells and particularly glomerular endothelial cells have long been known to occur AAV. More recently autoantibodies to an increasing number of specific proteins have been described, including plasminogen, moesin and lysosome-associated membrane protein-2 (LAMP-2) [17]. Anti-plasminogen antibodies are found in approximately of a quarter of patients with AAV, especially when active, and have been associated with more severe glomerular pathology and with a higher incidence of thromboembolic disease. Moesin is a protein expressed on the neutrophil surface and critical for locomotion and autoantibodies to it have been associated with higher inflammatory markers in patients with AAV but their presence did not correlate with disease activity [18]. We consistently find autoantibodies to LAMP-2 in 80–90% of European patients presenting with pauci-immune FNGN – although a lower frequency has been reported by others [19]. The autoantibodies rapidly become undetectable after the start of immunosuppressive treatment and are rare during remission but can again be detected during relapses. Injection of antibodies to LAMP-2 causes vasculitis in rodent models, and specifically bind glomerular endothelium and cause FNGN when injected into WKY rats. However, the strongest evidence for their pathogenicity comes from the molecular mimicry between LAMP-2 and FimH, a bacterial adhesin expressed by E.coli, Klebsiella and Proteus. Patients’ autoantibodies recognise a common LAMP-2 epitope (P41–49) with 100% homology to FimH and cross-react with it. Rats immunized with FimH develop antibodies that bind native human and rat LAMP-2 in neutrophils and glomerular endothelium confirming the molecular mimicry [19]. ANCA-negative patients.The recent characterisation of patients apparently with AAV but without detectable ANCA or autoantibodies to MPO and PR3 presents a challenge. The cause of injury in these ANCA-negative patients is uncertain but four possible explanations have been identified: low titres of anti-PR3 antibodies that can only be detected using extra sensitive immunoassays; caeruloplasmin fragments acting as circulating inhibitors of assays for autoantibodies to MPO [21]; podocyte specific nonimmune triggers to crescent formation that have been identified in murine models; and autoantibodies to LAMP-2 similar to those in ANCA-positive disease [22]. We detected such autoantibodies in eight of eleven ANCA-negative patients and showed that they rec-

Genetic Analysis of Large Vessel Vasculitis F. David Carmona1, Miguel A. González-Gay2,3, Javier Martín1 1 Instituto de Parasitología y Biomedicina ‘López-Neyra’, CSIC, Parque Tecnológico Ciencias de la Salud. Avda del Conocimiento s/n 18016-Armilla, Granada, Spain; 2 Department of Rheumatology, Hospital Universitario Marqués de Valdecilla, IDIVAL, Santander, Spain; 3 University of the Witwatersrand, Johannesburg, South Africa Correspondence to: F. David Carmona, PhD E-Mail: [email protected]

Novel approaches for the genetic analysis of complex diseases are being very useful in the elucidation of their genetic background. Although the large vessel vasculitides, including Takayasu arteritis (TA) and giant cell arteritis (GCA), have benefitted considerably from the golden age of the large-scale genetic analy-

Continuing Progress in Vasculitis Research

ses, the current knowledge of their aetiology is still far from that of other immune-mediated diseases like rheumatoid arthritis, from which much larger sample collections are available. Despite this, great advances have been made during the last years, particularly in the clarification of the strong HLA association with their predisposition but also in the identification of other firm susceptibility loci involved in the immune response such as IL12B, FCGR2A/FCGR3A, IL6, RPS9/LILRB3 and the 21q22 genomic region for TA, and PTPN22 for GCA. Further collaborative efforts may allow more powerful studies that will definitively shed light into the obscure pathophysiology of these severe disorders, something crucial for the development of more effective therapeutic strategies. Introduction

The vasculitides comprise a heterogeneous group of immune-mediated disorders which major hallmark is a self-sustaining inflammatory damage of the vessel wall. They show a wide range of clinical manifestations that depend on the affected blood vessel, which make it difficult an accurate classification of them. The large vessel vasculitides, which include Takayasu arteritis (TA) [MIM 207600] and giant cell arteritis (GCA) [MIM 187360], represent one of the seven main vasculitis groups that have been recently established by the Chapel Hill Consensus Conference [1]. In these types of vasculitides, largesized vessels, such as the aorta, are affected by intimal hyperplasia and narrowing of the lumen [2]. Both TA and GCA develop predominantly in women, with GCA affecting generally people over 50 years of age in Western countries and TA affecting younger patients with a higher incidence in Asia and Latin America [3, 4]. Like most immune-mediated diseases, the large vessel vasculitides show a complex aetiology in which both environmental and genetic factors may interact for their development and progression. The use of novel techniques for the genetic studies has produced a substantial advance in the elucidation of their genetic background during the last years [5]. In this review we will summarise the recent findings on the genetic susceptibility of TA and GCA. We will also discuss the perspective and possible approaches that may shed light into the complex genetic network underlying these severe disorders. Candidate Gene Studies

Early candidate gene studies in TA and GCA focused on the HLA region, as this genomic region harbours the strongest association signals with both types of vasculitis. Although different studies reported HLA associations with TA and GCA within both class I and II regions, the only well established HLA risk factors were HLA-B*52:01 for TA and DRB1*04 (mainly DRB1*0401 and DRB1*0404) for GCA [6, 7]. Regarding the non-HLA region, until recently very few consistent associations with these large vessel vasculitides were described, mainly due to the low statistical power of the studies performed and the lack of replication in independent populations. Evidences of common genetic associations between TA and GCA were observed within some loci encoding immune molecules, such us the interleukins IL-2/IL-21 and IL-6 [6, 7]. Other non-

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crescentic glomerulonephritis. J Am Soc Nephrol 2006;17:1940–1949. 12 Ooi JD, Chang J, Hickey MJ, Borza DB, Fugger L, Holdsworth SR, Kitching AR: The immunodominant myeloperoxidase T-cell epitope induces local cell-mediated injury in antimyeloperoxidase glomerulonephritis. Proc Natl Acad Sci U S A 2012;109:E2615–E2624. 13 Mélet J, Mulleman D, Goupille P, Ribourtout B, Watier H, Thibault G: Rituximab-induced T cell depletion in patients with rheumatoid arthritis: association with clinical response. Arthritis Rheum 2013;65:2783–2790. 14 Lintermans LL, Stegeman CA, Heeringa P, Abdulahad WH: T cells in vascular inflammatory diseases. Front Immunol 2014;5:504. doi: 10.3389/fimmu.2014.00504. 15 Abdulahad WH, Kallenberg CG, Limburg PC, Stegeman CA: UrinaryCD4+ effectormemory Tcells reflect renal disease activity in antineutrophil cyto-plasmic antibody-associated vasculitis. Arthritis Rheum 2009;60:2830–2838. 16 McKinney EF, Lyons PA, Carr EJ, Hollis JL, Jayne DR, Willcocks LC, Koukoulaki M, Brazma A, Jovanovic V, Kemeny DM, Pollard AJ, Macary PA, Chaudhry AN, Smith KG: A CD8+ T cell transcription signature predicts prognosis in autoimmune disease Nat Med 2010;16:586– 591. 17 Salama AD, Rees AJ: Autoantibodies in anti-neutrophil cytoplasm antibody-associated vasculitis. Nephrol Dial Transplant 2014;29:1105– 1107. 18 Susuki K, Nagao T, Itabashi M, Hamano Y, Sugamata R, Yamazaki Y, et al: A novel autoantibody in the serum of patients with MPO-ANCA associated vasculitis. Nephrol Dial Transplant 2014;29:1168–1177. 19 Kain R, Tadema H, McKinney EF, Benharkou A, Brandes R, Peschel A, et al: High prevalence of autoantibodies to hLAMP-2 in anti-neutrophil cytoplasmic antibody-associated vasculitis. J Am Soc Nephrol 2012; 23:556–566. 20 Chen M, Kallenberg CGM, Zhao MH: ANCA-negative pauci-immune crescentic glomerulonephritis. Nat Rev Nephrol 2009;5:313–318. 21 Roth AJ, Ooi JD, Hess JJ, van Timmeren MM, Berg EA, Poulton CE, McGregor J, Burkart M, Hogan SL, Hu Y, Winnik W, Nachman PH, Stegeman CA, Niles J, Heeringa P, Kitching AR, Holdsworth S, Jennette JC, Preston GA, Falk RJ: Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis. J Clin Invest 2013; 123:1773–1783. 22 Peschel A, Basu N, Benharkou A, Brandes R, Brown M, Dieckmann R, Rees AJ, Kain R: Autoantibodies to hLAMP-2 in ANCA-negative pauciimmune focal necrotizing GN. J Am Soc Nephrol 2014;25:455–463.

Table 1. Main associations of classical HLA alleles, HLA amino acids and non-HLA genetic variants with large vessel vasculitides

Takayasu arteritis Variation



p value



N (case/control) Reference

Classical HLA allele Classical HLA allele HLA amino acid HLA amino acid SNP SNP SNP SNP SNP SNP


52:01 52:01 His171 Phe67 rs6871626 rs56167332 rs10919543 rs2069837 rs11666543 rs2836878

1.00E-16 8.98E-10 1.80E-09 3.80E-05 1.70E-13 2.18E-08 5.89E-12 6.70E-09 2.34E-08 3.62E-10

2.82 4.81 >1.00

Continuing Progress in Vasculitis Research. Abstracts of the 17th International Vasculitis & ANCA WorkshopLondon, April 19-22, 2015: Abstracts.

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