- Email: [email protected]
New insights into the pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitis
Autoimmunity Reviews 1 (2002) 61–66
New insights into the pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitis Cees G.M. Kallenberga,*, Agnieszka Raroka, Coen A. Stegemanb, Pieter C. Limburga a
Department of Clinical Immunology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands b Department of Nephrology, University Hospital Groningen, The Netherlands Received 23 April 2001; accepted 10 October 2001
Abstract Antineutrophil cytoplasmic autoantibodies (ANCA) directed to proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA) are closely associated with the idiopathic systemic necrotizing vasculitides, in particular Wegener’s granulomatosis, microscopic polyangiitis and its renal limited manifestation, and Churg Strauss Syndrome. Many in vitro studies show that those ANCA have phlogistic potential, particularly at the interface of neutrophils and endothelial cells. A limited number of studies in experimental animals support their pathogenetic role. However, ANCA alone are not sufficient, as based on clinical and experimental data, and other, probably exogenous factors, seem necessary for disease induction and (re)activation. Among those silica and particularly, the carriage of Staphylococcus aureus have been proposed. Besides, various genetic factors are involved in disease susceptibility. The ANCA-associated vasculitides are systemic autoimmune diseases in which the interplay of autoimmunity with environmental and genetic factors determines their clinical expression. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Antineutrophil cytoplasmic antibodies (ANCA); Proteinase 3; Myeloperoxidase; Vasculitis; Wegener’s granulomatosis; Staphylococcus aureus Take-home messages ● ANCA directed to proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) are sensitive and specific markers for the idiopathic necrotizing small-vessel vasculitides. ● Changes in levels of PR3-ANCA as measured by ELISA, have predictive potential for ensuing relapses although this relation is far from absolute. ● In vitro and in vivo experimental data support, but do not prove the concept that ANCA are involved in the pathogenesis of the associated diseases. ● Various exogenous as well as genetic factors may be involved in disease expression of the ANCA-associated vasculitides.
* Corresponding author: Tel.: q31-50-3612945; fax: q31-50-3619069. E-mail address: [email protected] (C.G. Kallenberg).
1568-9972/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 8 - 9 9 7 2 Ž 0 1 . 0 0 0 0 8 - 8
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1. Introduction The idiopathic primary systemic vasculitides are defined, according to the Chapel Hill Consensus Conference w1x, on the basis of the size of the vessels involved, the histopathological findings, and characteristic clinical symptoms. Classification criteria for some of the diseases have been proposed by the American College of Rheumatology (ACR) w2x. Within the group of the small vessel vasculitides, Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA), Churg Strauss syndrome (CSS), and the renal limited form of those diseases, that is idiopathic necrotizing crescentic glomerulonephritis (NCGN), are closely associated with anti-neutrophil cytoplasmic autoantibodies (ANCA) w3x. The lesions in those diseases, particularly in the kidneys, lack immune deposits in most of the cases, and are described as ‘pauci-immune’. A number of observations suggest that ANCA, which are directed against proteinase 3 (PR3) or myeloperoxidase (MPO) w3x in those diseases, are involved in their pathogenesis. These data relate to clinical observations, in vitro experimental findings, and in vivo data from animal experiments. As will be discussed, the data point to a pathophysiological role of the autoantibodies, but also show that their presence is not sufficient to induce disease manifestations. Other, possibly extrinsic factors may be required as well. In this review, both the autoimmune background as well as the possible involvement of extrinsic factors in the ANCA-associated vasculitides will be discussed. 2. Role of ANCA 2.1. Clinical observations Longitudinal observations in patients point to a relationship between changes in level of the autoantibodies and changes in disease activity of the associated disorders. Rises in titres of ANCA appear to precede clinical disease activity w4,5x and treatment based on changes in ANCA titres resulted in the prevention of disease relapses w6x. These studies, generally based on small series of patients, used ANCA titration in the indirect
immunofluorescence test for quantification, which is not the most accurate way of quantitating levels of autoantibodies. Indeed other authors could not confirm a strong correlation between rising titres and ensuing relapses w7x. Recently, Boomsma et al. in a 2 year prospective study, have analyzed this relationship in 85 patients with anti-proteinase 3 (PR3)-associated glomerulonephritisyvasculitis using ELISA for quantification of the autoantibodies. They found that 27 out of the 33 relapses that occurred during the study period were preceded by a significant rise in PR3-ANCA levels, whereas 11 rises in PR3-ANCA occurred that were not followed by a relapse w8x. In addition to those data, it has been proven that patients with antiPR3 associated disease have an increased risk for development of a relapse once tests for ANCA are persistently or intermittently positive after induction of remission w9x. Taken together, all these in vivo data suggest that ANCA are involved in the pathophysiology of PR3-ANCAyMPO-ANCA associated vasculitides. 2.2. In vitro effects of ANCA Following the first observation by Falk et al. w10x that, in vitro, ANCA are able to activate neutrophils, an increasing number of studies have been published that further underscore the phlogistic potential of ANCA. Indeed, Falk et al. demonstrated that the antibodies, and even their F(ab9)2-fragments, although to a lesser extent, could induce the production of reactive oxygen species and the release of lytic enzymes such as elastase and PR3 by donor neutrophils. In order to get activated by ANCA, neutrophils must be in a state of pre-activation (‘primed’). Priming occurs in the presence of low amounts of pro-inflammatory cytokines such as tumor necrosis factor-a (TNFa) or interleukin-1. During priming the target antigens of ANCA, that is PR3 and MPO, are expressed at the cell surface and, so become accessible for interaction with ANCA. This interaction, which is followed by activation of neutrophils, only occurs when neutrophils are adherent to a surface, a process in which b2-integrins are involved w11x. In vivo, this process is assumed to occur at the endothelial surface. Indeed, activated
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neutrophils, adherent to the endothelium, are observed in renal biopsies from patients with ANCA-associated NCGN w12x. ANCA induced neutrophil activation involves not only binding of the antibodies via their F(ab9)2-fragments to surface expressed PR3 or MPO, but also interaction of their Fc-fragments with Fc-receptors on neutrophils, particularly with the Fcg RIIa-receptor w13x, although F(ab9)2-fragments also have some activating potential w14x. Very recently, Ben-Smith et al. demonstrated that ligation of Fcg RIIa and Fcg RIIIb is necessary for ANCA-induced neutrophil activation, but that the signaling cascades used by ANCA were different from the signal pathways used by Fcg R engagement only w15x. They suggest that ANCA require other membrane cofactors for neutrophil activation than FcgR engagement alone. These other membrane cofactors have not been identified as yet w15x. The Fcg RIIa is the only Fc-receptor that interacts with IgG2 whereas this receptor also has a particular affinity for the IgG3-subclass. Interestingly, the increase in neutrophil activating capacity of serum IgG fractions from remission to relapse in patients with PR3-ANCA positive WG correlated with increases of levels of IgG3 subclass ANCA in those fractions and not with that of the other subclasses w16x. In addition, renal relapses of WG are particularly associated with increases of the IgG3 subclass of ANCA, although IgG1- and IgG4 subclasses of ANCA are present as well w17x. It should, however, be stated that measuring the IgG3 subclass of PR3ANCA concomitantly with the total IgG PR3ANCA, did not improve the predictive value of a rise in ANCA for ensuing relapses w8x. Nevertheless, these data suggest that the IgG3 subclass of ANCA may play a particular role in neutrophil activation via Fcg receptor interactions. Also, other in vitro effects, in particular of PR3ANCA, have been identified. PR3-ANCA can interfere with the proteolytic activity of PR3 w18x. These interfering antibodies may thus act as alternative inhibitors. However, at the site of inflammation PR3 can cleave these inhibiting ANCA leaving active PR3 w19x. PR3 can also interfere with the binding of PR3 to its physiological inhibitor a1-antitrypsin w20x. The different in vitro effects of PR3-ANCA may be related to differenc-
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es in epitope specificity of the antibodies. This area is still under investigation w21x. Besides neutrophils, also monocytes can be activated by ANCA. Affinity-purified PR3-ANCA and MPO-ANCA were able to induce monocytes to produce oxygen radicals. Blocking of the Fcg RIIa reduced the formation of oxygen radicals but F(ab9)2-fragments of ANCA were still able to activate monocytes suggesting that activation occurs along several lines w22x. In vitro studies using endothelial monolayers also have shown that neutrophils, in the presence of ANCA are able to adhere to and lyse endothelial cells w23x. Elegant studies by the group of Savage demonstrated that ANCA are able to induce stable adherence of rolling neutrophils to layers expressing adhesion molecules w24x. Whether endothelial cells themselves express ANCA-antigens such as PR3, has been a subject of controversy. Data from Mayet et al. suggest that endothelial cells express PR3, particularly when activated, and that subsequently, ANCA can bind to surface expressed PR3 resulting in upregulation of adhesion molecules and further activation of those cells w25x. Others, however, have not been able to confirm PR3 expression by endothelial cells but demonstrated that PR3 binds to endothelial cells via a specific receptor w26x. How do these in vitro effects of ANCA correlate with disease activity, particularly in comparison with titres of ANCA? Changes in various in vitro effects of (PR3-) ANCA have been suggested to follow changes in disease activity more accurately than changes in ANCA titres alone w18,20x, but this suggestion is based on observations of small numbers of patients only and so far remains to be proven. Further elucidation of the different epitopes on PR3 and MPO recognized by ANCA and their relation to disease activity may be one of the clues to this question. 2.3. Data from in vivo experimental models More definite evidence for a pathophysiological role of ANCA may come from animal models. Unfortunately, fully satisfactory models for ANCA-associated glomerulonephritis are not (yet)
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available. Passive transfer of ANCA in primates or inducing an autoimmune response to MPO in rats did not result in renal lesions w27x. A recent study, interestingly, showed that rats injected with syngeneic rat apoptotic neutrophils develop ANCA, but the specificity of these ANCA could not be established and did not seem to be PR3 or MPO w28x. When, however, the products of activated neutrophils, that is lytic enzymes, MPO, and its substrate H2O2, are perfused into the renal artery in rats immunized with MPO, severe pauciimmune NCGN develops w27x. The results of those studies suggest that, initially, cationic proteins such as MPO and PR3 from activated neutrophils, adhere to the glomerular capillary wall and are bound by their cognate antibodies. The in situ formed immune complexes activate the complement system resulting, amongst others, in attraction of neutrophils. Those neutrophils are subsequently activated by ANCA and degrade the immune complexes that were initially present. The potential of ANCA to augment in vivo an inflammatory reaction has been demonstrated by Heeringa et al. w29x in an animal model of anti-glomerular basement membrane (anti-GBM) disease. They injected a subclinical dose of heterologous anti-GBM antibodies in rats, which resulted in deposition of immunoglobulins along the GBM but did not induce severe glomerulonephritis. In rats immunized with human MPO, which led to the development of anti-MPO antibodies cross-reacting with their own MPO, however, severe necrotizing and crescentic glomerulonephritis developed after injection of this subclinical dose of anti-GBM antibodies. These experiments most convincingly show the phlogistic potential of ANCA. Nevertheless, a fully satisfying animal model for ANCAassociated vasculitisyglomerulonephritis does not yet exist. What can be deduced from the foregoing data? ANCA alone are not sufficient to induce disease activity in the ANCA-associated vasculitides. Both the in vitro and in vivo experimental data suggest that additional factors, with a pro-inflammatory character capable of initiating the activation of neutrophils, are necessary.
3. Exogenous factors involved in disease expression in the ANCA-associated vasculitides Although various exogenous factors such as silica exposure w30x have been suggested to be associated with ANCA-associated vasculitis, most intriguing data have come from studies on the role of microbial agents, in particular Staphylococcus aureus. Nasal carriage of S. aureus was found to be a significant risk factor for relapses in Wegener’s granulomatosis w9x, and maintenance treatment with co-trimoxazole was able to prevent relapses of the disease w31x. How S. aureus may induce (re)activation of disease activity has not been fully established. Various mechanisms may be operative. S. aureus produces a cationic protein, staphylococcal acid phosphatase, which can bind to endothelial cells, by charge interaction, in vitro w32x, and has been demonstrated in glomeruli of some patients with Wegener’s granulomatosis. Antibodies to this phosphatase are present in the sera of Wegener’s patients and controls, albeit at higher levels in the patients w33x. It has been hypothesized that focal immune complex formation, possibly consisting, at least in part, of the phosphatase and its cognate antibodies, occurs in the vessel wall which attracts neutrophils. In the presence of ANCA, this focal inflammatory reaction is strongly enhanced resulting in necrotizing lesions and degradation of the immune complexes that were initially present. Indeed, immune complexes have been demonstrated in early skin lesions of patients with Wegener’s granulomatosis w34x. Other S. aureus-related mechanisms of disease activation have been suggested as well, in particular lymphocyte activation resulting from stimulation with S. aureus-derived superantigens w9x. Presence of S. aureus was indeed, found to be associated with presence of ANCA in patients who were in remission during immunosuppressive treatment w9x 4. Genetic factors involved in ANCA-associated vasculitis Although only weak associations of ANCAassociated vasculitis with HLA-antigens were observed w35x, several other genetic factors appear
C.G. Kallenberg et al. / Autoimmunity Reviews 1 (2002) 61–66
to be involved in those diseases. First, the natural inhibitor of PR3, alpha-1 antitrypsine (a1-AT), is highly polymorphic resulting in functional differences between the several phenotypes. Alpha1-AT phenotypes leading to decreased levels of functional a1-AT have been associated with an increased risk of Wegener’s granulomatosis as well as with increased morbidity w36x. Secondly, Fcgreceptor polymorphism seems to be involved in disease expression w37x. Thirdly, the PR3 gene itself shows polymorphism and an association was demonstrated for the A-564G polymorphism in the PR3 promoter, affecting a putative transcription factor binding site, with Wegener’s granulomatosis w38x. Finally, it has been shown that, in some persons, PR3 is expressed on the cell surface of a varying percentage of resting peripheral blood neutrophils. This so-called bimodal expression of PR3 was more frequently seen in patients with Wegener’s granulomatosis as well as in their healthy relatives w39x. The functional significance of this putatively genetically based and constitutively present membrane expression of PR3 awaits further study. 5. Summary Accumulating evidence suggests that ANCA directed to PR3 and MPO, are involved in the pathophysiology of the associated vasculitic disorders. However, other concomitant factors, possibly of microbial origin, appear necessary for disease expression as well. Multiple genetic factors seem to be involved in disease susceptibility. The ANCA-associated vasculitides are systemic autoimmune diseases in which the interplay of autoimmunity with environmental and genetic factors determines their clinical expression. References w1x Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides: the proposal of an international consensus conference. Arthritis Rheum 1994;37:187 – 92. w2x Hunder GG, Arend WP, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of vasculitis. Arthritis Rheum 1990;33:1088 –93.
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w3x Kallenberg CGM, Brouwer E, Weening JJ, Cohen Tervaert JW. Anti-neutrophil cytoplasmic antibodies: current diagnostic and pathophysiological potential. Kidney Int 1994;46:1 –15. w4x Cohen Tervaert JW, van der Woude FJ, Fauci AS, et al. Association between active Wegener’s Granulomatosis and anticytoplasmic antibodies. Arch Intern Med 1989;149:2461 –5. w5x Jayne DRW, Gaskin G, Pusey CD, Lockwood CM. ANCA and predicting relapse in systemic vasculitis. Q J M 1995;88:127 –33. w6x Cohen Tervaert JW, Huitema MG, Hene´ RJ, et al. Prevention of relapses in Wegener’s granulomatosis by treatment based on antineutrophil cytoplasmic antibody titre. Lancet 1990;336:709 –11. w7x Kerr GR, Fleischer THA, Hallahan CD, et al. Limited prognostic value of changes in antineutrophil cytoplasmic antibody titer in patients with Wegener’s granulomatosis. Arthritis Rheum 1993;36:365 –71. w8x Boomsma MM, Stegeman CA, van der Leij MJ, et al. Prediction of relapses in Wegener’s granulomatosis by measurement of anti neutrophil cytoplasmic antibody levels; a prospective study. Arthritis Rheum 2000;43:2025 –33. w9x Stegeman CA, Cohen Tervaert JW, Sluiter WJ, Manson W, de Jong PE, Kallenberg CGM. Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener’s granulomatosis. Ann Intern Med 1994;113:12 –7. w10x Falk RJ, Terrell RS, Charles LA, Jennette JC. Antineutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA 1990;87:4115 –9. w11x Reumaux D, Vossebeld PJ, Roos D, Verhoeven AJ. Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies. Blood 1995;86:3189 –95. w12x Brouwer E, Huitema MG, Mulder AHL, et al. Neutrophil activation in vitro and in vivo in Wegener’s granulomatosis. Kidney Int 1994;45:1120 –31. w13x Porges AJ, Redecha PB, Kimberly WT, Csernok E, Gross WL, Kimberly RP. Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa. J Immunol 1994;153:1271 –80. w14x Kettritz R, Jennette JC, Falk RJ. Crosslinking of ANCAantigens stimulates superoxide release by human neutrophils. J Am Soc Nephrol 1997;8:386 –94. w15x Ben-Smith A, Dove SK, Martin A, Wakelam MJ, Savage CO. Antineutrophil cytoplasm autoantibodies from patients with systemic vasculitis activate neutrophils through distinct signaling cascades: comparison with conventional Fc-gamma receptor ligation. Blood 2001;98:1448 –55. w16x Mulder AH, Stegeman CA, Kallenberg CGM. Activation of granulocytes by anti-neutrophil cytoplasmic anti-
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C.G. Kallenberg et al. / Autoimmunity Reviews 1 (2002) 61–66 bodies (ANCA) in Wegener’s granulomatosis: a predominant role for the IgG3 subclass of ANCA. Clin Exp Immunol 1995;101:227 –32. Brouwer E, Cohen Tervaert JW, Horst G, et al. Predominance of IgG4 subclass of anti-neutrophil cytoplasmic autoantibodies in patients with Wegener’s granulomatosis and clinically-related disorders. Clin Exp Immunol 1991;83:379 –86. Daouk GH, Palsson R, Arnaout MA. Inhibition of proteinase 3 by ANCA and its correlation with disease activity in Wegener’s granulomatosis. Kidney Int 1995;47:1528 –36. Dolman KM, Jager A, Sonnenberg A, von dem Borne AEGKr, Goldschmeding R. Proteolysis of classic antineutrophil cytoplasmic autoantibodies by neutrophil proteinase 3. Clin Exp Immunol 1995;101:8 –12. Dolman KM, Stegeman CA, van de Wiel BA, et al. Relevance of classic anti-neutrophil cytoplasmic autoantibody-mediated inhibition of proteinase 3-alpha 1 antitrypsin complexation to disease activity in Wegener’s granulomatosis. Clin Exp Immunol 1993;93:404 – 10. van der Geld YM, Simpelaar A, van der Zee R, et al. Anti-neutrophil cytoplasmic antibodies to proteinase 3 in Wegener’s granulomatosis: epitope analysis using synthetic peptides. Kidney Int 2001;59:147 –59. Weidner S, Neupert W, Goppelt-Struebe M, Rupprecht HD. Antineutrophil cytoplasmic antibodies induce human monocytes to produce oxygen radicals in vitro. Arthritis Rheum 2001;44:1698 –706. Savage CO, Pottinger BE, Gaskin G, Pusey CD, Pearson JD. Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial cells. Am J Pathol 1992;141:335 –42. Radford DJ, Savage COS, Nash GB. Treatment of rolling neutrophils with antineutrophil cytoplasmic antibodies causes conversion to firm integrin-mediated adhesion. Arthritis Rheum 2000;43:1337 –45. Mayet WJ, Schwarting A, Orth T, Duchmann R, ¨ Meyer zum Buschenfelde KH. Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1). Clin Exp Immunol 1996;103:259 – 67. Taekema-Roelvink ME, Van Kooten C, Heemskerk E, Schroeijers W, Daha MR. Proteinase 3 interacts with a III-KD membrane molecule of human umbilical vein endothelial cells. J Am Soc Nephrol 2000;11:640 –8. Brouwer E, Huitema MG, Klok PA, Cohen Tervaert JW, Weening JJ, Kallenberg CGM. Anti-myeloperoxidase
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associated proliferative glomerulonephritis: an animal model. J Exp Med 1993;177:905 –14. Patry YC, Trewick DC, Gregoire M, et al. Rats injected with syngeneic rat apoptotic neutrophils develop antineutrophil cytoplasmic antibodies. J Am Soc Nephrol 2001;12:1764 –8. Heeringa P, Brouwer E, Klok PA, et al. Autoantibodies to myeloperoxidase aggravate mild anti-glomerularbasement – membrane-mediated glomerular injury in the rat. Am J Pathol 1996;149:1695 –706. Hogan SL, Satterly KK, Dooley MA, Nachman PH, Jennette JC, Falk RJ. Silica exposure in anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and lupus nephritis. J Am Soc Nephrol 2001;12:134 – 42. Stegeman CA, Cohen Tervaert JW, de Jong PE, Kallenberg CGM. Trimethoprim – sulfamethoxazole for the prevention of relapses of Wegener’s granulomatosis. N Engl J Med 1996;335:16 –20. Brons RH, Bakker HI, van Wijk NW, et al. Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener’s granulomatosis? Clin Exp Immunol 2000;19:566 –73. Brons RH, Kallenberg CGM, Cohen Tervaert JW. Are ANCA-associated vasculitides pauci-immune? Rheum Dis Clin N Am 2001;27:833 –48. Brons RH, de Jong MCJM, de Boer NK, Stegeman CA, Kallenberg CGM, Cohen Tervaert JW. Detection of immune deposits in skin lesions of patients with Wegener’s granulomatosis. Ann Rheum Dis 2001;60:1097 – 102. Hagen EC, Stegeman CA, D’Amaro J, et al. Decreased frequency of HLA-DR13yDR6 in Wegener’s granulomatosis. Kidney Int 1995;48:801 –5. Esnault VL, Audrain MA, Sesboue R. Alpha-1-antitrypsin phenotyping in ANCA-associated diseases: one of several arguments for proteaseyantiprotease imbalance in systemic vasculitis. Exp Clin Immunogenet 1997;14:206 –13. Dijstelbloem HM, Scheepers RH, Oost WW, et al. Fcgamma receptor polymorphisms in Wegener’s granulomatosis: risk factors for disease relapse. Arthritis Rheum 1999;42:1823 –7. Gencik M, Muller S, Borgmann S, Fricke H. Proteinase 3 gene polymorphisms and Wegener’s granulomatosis. Kidney Int 2000;58:2473 –7. Witko-Sarsat V, Lesavre P, Lopez S, et al. A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatic arthritis. J Am Soc Nephrol 1999;10:1224 –33.
New insights into the pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitis Cees G.M. Kallenberga,*, Agnieszka Raroka, Coen A. Stegemanb, Pieter C. Limburga a
Department of Clinical Immunology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands b Department of Nephrology, University Hospital Groningen, The Netherlands Received 23 April 2001; accepted 10 October 2001
Abstract Antineutrophil cytoplasmic autoantibodies (ANCA) directed to proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA) are closely associated with the idiopathic systemic necrotizing vasculitides, in particular Wegener’s granulomatosis, microscopic polyangiitis and its renal limited manifestation, and Churg Strauss Syndrome. Many in vitro studies show that those ANCA have phlogistic potential, particularly at the interface of neutrophils and endothelial cells. A limited number of studies in experimental animals support their pathogenetic role. However, ANCA alone are not sufficient, as based on clinical and experimental data, and other, probably exogenous factors, seem necessary for disease induction and (re)activation. Among those silica and particularly, the carriage of Staphylococcus aureus have been proposed. Besides, various genetic factors are involved in disease susceptibility. The ANCA-associated vasculitides are systemic autoimmune diseases in which the interplay of autoimmunity with environmental and genetic factors determines their clinical expression. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Antineutrophil cytoplasmic antibodies (ANCA); Proteinase 3; Myeloperoxidase; Vasculitis; Wegener’s granulomatosis; Staphylococcus aureus Take-home messages ● ANCA directed to proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) are sensitive and specific markers for the idiopathic necrotizing small-vessel vasculitides. ● Changes in levels of PR3-ANCA as measured by ELISA, have predictive potential for ensuing relapses although this relation is far from absolute. ● In vitro and in vivo experimental data support, but do not prove the concept that ANCA are involved in the pathogenesis of the associated diseases. ● Various exogenous as well as genetic factors may be involved in disease expression of the ANCA-associated vasculitides.
* Corresponding author: Tel.: q31-50-3612945; fax: q31-50-3619069. E-mail address: [email protected] (C.G. Kallenberg).
1568-9972/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 8 - 9 9 7 2 Ž 0 1 . 0 0 0 0 8 - 8
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C.G. Kallenberg et al. / Autoimmunity Reviews 1 (2002) 61–66
1. Introduction The idiopathic primary systemic vasculitides are defined, according to the Chapel Hill Consensus Conference w1x, on the basis of the size of the vessels involved, the histopathological findings, and characteristic clinical symptoms. Classification criteria for some of the diseases have been proposed by the American College of Rheumatology (ACR) w2x. Within the group of the small vessel vasculitides, Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA), Churg Strauss syndrome (CSS), and the renal limited form of those diseases, that is idiopathic necrotizing crescentic glomerulonephritis (NCGN), are closely associated with anti-neutrophil cytoplasmic autoantibodies (ANCA) w3x. The lesions in those diseases, particularly in the kidneys, lack immune deposits in most of the cases, and are described as ‘pauci-immune’. A number of observations suggest that ANCA, which are directed against proteinase 3 (PR3) or myeloperoxidase (MPO) w3x in those diseases, are involved in their pathogenesis. These data relate to clinical observations, in vitro experimental findings, and in vivo data from animal experiments. As will be discussed, the data point to a pathophysiological role of the autoantibodies, but also show that their presence is not sufficient to induce disease manifestations. Other, possibly extrinsic factors may be required as well. In this review, both the autoimmune background as well as the possible involvement of extrinsic factors in the ANCA-associated vasculitides will be discussed. 2. Role of ANCA 2.1. Clinical observations Longitudinal observations in patients point to a relationship between changes in level of the autoantibodies and changes in disease activity of the associated disorders. Rises in titres of ANCA appear to precede clinical disease activity w4,5x and treatment based on changes in ANCA titres resulted in the prevention of disease relapses w6x. These studies, generally based on small series of patients, used ANCA titration in the indirect
immunofluorescence test for quantification, which is not the most accurate way of quantitating levels of autoantibodies. Indeed other authors could not confirm a strong correlation between rising titres and ensuing relapses w7x. Recently, Boomsma et al. in a 2 year prospective study, have analyzed this relationship in 85 patients with anti-proteinase 3 (PR3)-associated glomerulonephritisyvasculitis using ELISA for quantification of the autoantibodies. They found that 27 out of the 33 relapses that occurred during the study period were preceded by a significant rise in PR3-ANCA levels, whereas 11 rises in PR3-ANCA occurred that were not followed by a relapse w8x. In addition to those data, it has been proven that patients with antiPR3 associated disease have an increased risk for development of a relapse once tests for ANCA are persistently or intermittently positive after induction of remission w9x. Taken together, all these in vivo data suggest that ANCA are involved in the pathophysiology of PR3-ANCAyMPO-ANCA associated vasculitides. 2.2. In vitro effects of ANCA Following the first observation by Falk et al. w10x that, in vitro, ANCA are able to activate neutrophils, an increasing number of studies have been published that further underscore the phlogistic potential of ANCA. Indeed, Falk et al. demonstrated that the antibodies, and even their F(ab9)2-fragments, although to a lesser extent, could induce the production of reactive oxygen species and the release of lytic enzymes such as elastase and PR3 by donor neutrophils. In order to get activated by ANCA, neutrophils must be in a state of pre-activation (‘primed’). Priming occurs in the presence of low amounts of pro-inflammatory cytokines such as tumor necrosis factor-a (TNFa) or interleukin-1. During priming the target antigens of ANCA, that is PR3 and MPO, are expressed at the cell surface and, so become accessible for interaction with ANCA. This interaction, which is followed by activation of neutrophils, only occurs when neutrophils are adherent to a surface, a process in which b2-integrins are involved w11x. In vivo, this process is assumed to occur at the endothelial surface. Indeed, activated
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neutrophils, adherent to the endothelium, are observed in renal biopsies from patients with ANCA-associated NCGN w12x. ANCA induced neutrophil activation involves not only binding of the antibodies via their F(ab9)2-fragments to surface expressed PR3 or MPO, but also interaction of their Fc-fragments with Fc-receptors on neutrophils, particularly with the Fcg RIIa-receptor w13x, although F(ab9)2-fragments also have some activating potential w14x. Very recently, Ben-Smith et al. demonstrated that ligation of Fcg RIIa and Fcg RIIIb is necessary for ANCA-induced neutrophil activation, but that the signaling cascades used by ANCA were different from the signal pathways used by Fcg R engagement only w15x. They suggest that ANCA require other membrane cofactors for neutrophil activation than FcgR engagement alone. These other membrane cofactors have not been identified as yet w15x. The Fcg RIIa is the only Fc-receptor that interacts with IgG2 whereas this receptor also has a particular affinity for the IgG3-subclass. Interestingly, the increase in neutrophil activating capacity of serum IgG fractions from remission to relapse in patients with PR3-ANCA positive WG correlated with increases of levels of IgG3 subclass ANCA in those fractions and not with that of the other subclasses w16x. In addition, renal relapses of WG are particularly associated with increases of the IgG3 subclass of ANCA, although IgG1- and IgG4 subclasses of ANCA are present as well w17x. It should, however, be stated that measuring the IgG3 subclass of PR3ANCA concomitantly with the total IgG PR3ANCA, did not improve the predictive value of a rise in ANCA for ensuing relapses w8x. Nevertheless, these data suggest that the IgG3 subclass of ANCA may play a particular role in neutrophil activation via Fcg receptor interactions. Also, other in vitro effects, in particular of PR3ANCA, have been identified. PR3-ANCA can interfere with the proteolytic activity of PR3 w18x. These interfering antibodies may thus act as alternative inhibitors. However, at the site of inflammation PR3 can cleave these inhibiting ANCA leaving active PR3 w19x. PR3 can also interfere with the binding of PR3 to its physiological inhibitor a1-antitrypsin w20x. The different in vitro effects of PR3-ANCA may be related to differenc-
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es in epitope specificity of the antibodies. This area is still under investigation w21x. Besides neutrophils, also monocytes can be activated by ANCA. Affinity-purified PR3-ANCA and MPO-ANCA were able to induce monocytes to produce oxygen radicals. Blocking of the Fcg RIIa reduced the formation of oxygen radicals but F(ab9)2-fragments of ANCA were still able to activate monocytes suggesting that activation occurs along several lines w22x. In vitro studies using endothelial monolayers also have shown that neutrophils, in the presence of ANCA are able to adhere to and lyse endothelial cells w23x. Elegant studies by the group of Savage demonstrated that ANCA are able to induce stable adherence of rolling neutrophils to layers expressing adhesion molecules w24x. Whether endothelial cells themselves express ANCA-antigens such as PR3, has been a subject of controversy. Data from Mayet et al. suggest that endothelial cells express PR3, particularly when activated, and that subsequently, ANCA can bind to surface expressed PR3 resulting in upregulation of adhesion molecules and further activation of those cells w25x. Others, however, have not been able to confirm PR3 expression by endothelial cells but demonstrated that PR3 binds to endothelial cells via a specific receptor w26x. How do these in vitro effects of ANCA correlate with disease activity, particularly in comparison with titres of ANCA? Changes in various in vitro effects of (PR3-) ANCA have been suggested to follow changes in disease activity more accurately than changes in ANCA titres alone w18,20x, but this suggestion is based on observations of small numbers of patients only and so far remains to be proven. Further elucidation of the different epitopes on PR3 and MPO recognized by ANCA and their relation to disease activity may be one of the clues to this question. 2.3. Data from in vivo experimental models More definite evidence for a pathophysiological role of ANCA may come from animal models. Unfortunately, fully satisfactory models for ANCA-associated glomerulonephritis are not (yet)
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available. Passive transfer of ANCA in primates or inducing an autoimmune response to MPO in rats did not result in renal lesions w27x. A recent study, interestingly, showed that rats injected with syngeneic rat apoptotic neutrophils develop ANCA, but the specificity of these ANCA could not be established and did not seem to be PR3 or MPO w28x. When, however, the products of activated neutrophils, that is lytic enzymes, MPO, and its substrate H2O2, are perfused into the renal artery in rats immunized with MPO, severe pauciimmune NCGN develops w27x. The results of those studies suggest that, initially, cationic proteins such as MPO and PR3 from activated neutrophils, adhere to the glomerular capillary wall and are bound by their cognate antibodies. The in situ formed immune complexes activate the complement system resulting, amongst others, in attraction of neutrophils. Those neutrophils are subsequently activated by ANCA and degrade the immune complexes that were initially present. The potential of ANCA to augment in vivo an inflammatory reaction has been demonstrated by Heeringa et al. w29x in an animal model of anti-glomerular basement membrane (anti-GBM) disease. They injected a subclinical dose of heterologous anti-GBM antibodies in rats, which resulted in deposition of immunoglobulins along the GBM but did not induce severe glomerulonephritis. In rats immunized with human MPO, which led to the development of anti-MPO antibodies cross-reacting with their own MPO, however, severe necrotizing and crescentic glomerulonephritis developed after injection of this subclinical dose of anti-GBM antibodies. These experiments most convincingly show the phlogistic potential of ANCA. Nevertheless, a fully satisfying animal model for ANCAassociated vasculitisyglomerulonephritis does not yet exist. What can be deduced from the foregoing data? ANCA alone are not sufficient to induce disease activity in the ANCA-associated vasculitides. Both the in vitro and in vivo experimental data suggest that additional factors, with a pro-inflammatory character capable of initiating the activation of neutrophils, are necessary.
3. Exogenous factors involved in disease expression in the ANCA-associated vasculitides Although various exogenous factors such as silica exposure w30x have been suggested to be associated with ANCA-associated vasculitis, most intriguing data have come from studies on the role of microbial agents, in particular Staphylococcus aureus. Nasal carriage of S. aureus was found to be a significant risk factor for relapses in Wegener’s granulomatosis w9x, and maintenance treatment with co-trimoxazole was able to prevent relapses of the disease w31x. How S. aureus may induce (re)activation of disease activity has not been fully established. Various mechanisms may be operative. S. aureus produces a cationic protein, staphylococcal acid phosphatase, which can bind to endothelial cells, by charge interaction, in vitro w32x, and has been demonstrated in glomeruli of some patients with Wegener’s granulomatosis. Antibodies to this phosphatase are present in the sera of Wegener’s patients and controls, albeit at higher levels in the patients w33x. It has been hypothesized that focal immune complex formation, possibly consisting, at least in part, of the phosphatase and its cognate antibodies, occurs in the vessel wall which attracts neutrophils. In the presence of ANCA, this focal inflammatory reaction is strongly enhanced resulting in necrotizing lesions and degradation of the immune complexes that were initially present. Indeed, immune complexes have been demonstrated in early skin lesions of patients with Wegener’s granulomatosis w34x. Other S. aureus-related mechanisms of disease activation have been suggested as well, in particular lymphocyte activation resulting from stimulation with S. aureus-derived superantigens w9x. Presence of S. aureus was indeed, found to be associated with presence of ANCA in patients who were in remission during immunosuppressive treatment w9x 4. Genetic factors involved in ANCA-associated vasculitis Although only weak associations of ANCAassociated vasculitis with HLA-antigens were observed w35x, several other genetic factors appear
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to be involved in those diseases. First, the natural inhibitor of PR3, alpha-1 antitrypsine (a1-AT), is highly polymorphic resulting in functional differences between the several phenotypes. Alpha1-AT phenotypes leading to decreased levels of functional a1-AT have been associated with an increased risk of Wegener’s granulomatosis as well as with increased morbidity w36x. Secondly, Fcgreceptor polymorphism seems to be involved in disease expression w37x. Thirdly, the PR3 gene itself shows polymorphism and an association was demonstrated for the A-564G polymorphism in the PR3 promoter, affecting a putative transcription factor binding site, with Wegener’s granulomatosis w38x. Finally, it has been shown that, in some persons, PR3 is expressed on the cell surface of a varying percentage of resting peripheral blood neutrophils. This so-called bimodal expression of PR3 was more frequently seen in patients with Wegener’s granulomatosis as well as in their healthy relatives w39x. The functional significance of this putatively genetically based and constitutively present membrane expression of PR3 awaits further study. 5. Summary Accumulating evidence suggests that ANCA directed to PR3 and MPO, are involved in the pathophysiology of the associated vasculitic disorders. However, other concomitant factors, possibly of microbial origin, appear necessary for disease expression as well. Multiple genetic factors seem to be involved in disease susceptibility. The ANCA-associated vasculitides are systemic autoimmune diseases in which the interplay of autoimmunity with environmental and genetic factors determines their clinical expression. References w1x Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides: the proposal of an international consensus conference. Arthritis Rheum 1994;37:187 – 92. w2x Hunder GG, Arend WP, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of vasculitis. Arthritis Rheum 1990;33:1088 –93.
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w3x Kallenberg CGM, Brouwer E, Weening JJ, Cohen Tervaert JW. Anti-neutrophil cytoplasmic antibodies: current diagnostic and pathophysiological potential. Kidney Int 1994;46:1 –15. w4x Cohen Tervaert JW, van der Woude FJ, Fauci AS, et al. Association between active Wegener’s Granulomatosis and anticytoplasmic antibodies. Arch Intern Med 1989;149:2461 –5. w5x Jayne DRW, Gaskin G, Pusey CD, Lockwood CM. ANCA and predicting relapse in systemic vasculitis. Q J M 1995;88:127 –33. w6x Cohen Tervaert JW, Huitema MG, Hene´ RJ, et al. Prevention of relapses in Wegener’s granulomatosis by treatment based on antineutrophil cytoplasmic antibody titre. Lancet 1990;336:709 –11. w7x Kerr GR, Fleischer THA, Hallahan CD, et al. Limited prognostic value of changes in antineutrophil cytoplasmic antibody titer in patients with Wegener’s granulomatosis. Arthritis Rheum 1993;36:365 –71. w8x Boomsma MM, Stegeman CA, van der Leij MJ, et al. Prediction of relapses in Wegener’s granulomatosis by measurement of anti neutrophil cytoplasmic antibody levels; a prospective study. Arthritis Rheum 2000;43:2025 –33. w9x Stegeman CA, Cohen Tervaert JW, Sluiter WJ, Manson W, de Jong PE, Kallenberg CGM. Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener’s granulomatosis. Ann Intern Med 1994;113:12 –7. w10x Falk RJ, Terrell RS, Charles LA, Jennette JC. Antineutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA 1990;87:4115 –9. w11x Reumaux D, Vossebeld PJ, Roos D, Verhoeven AJ. Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies. Blood 1995;86:3189 –95. w12x Brouwer E, Huitema MG, Mulder AHL, et al. Neutrophil activation in vitro and in vivo in Wegener’s granulomatosis. Kidney Int 1994;45:1120 –31. w13x Porges AJ, Redecha PB, Kimberly WT, Csernok E, Gross WL, Kimberly RP. Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa. J Immunol 1994;153:1271 –80. w14x Kettritz R, Jennette JC, Falk RJ. Crosslinking of ANCAantigens stimulates superoxide release by human neutrophils. J Am Soc Nephrol 1997;8:386 –94. w15x Ben-Smith A, Dove SK, Martin A, Wakelam MJ, Savage CO. Antineutrophil cytoplasm autoantibodies from patients with systemic vasculitis activate neutrophils through distinct signaling cascades: comparison with conventional Fc-gamma receptor ligation. Blood 2001;98:1448 –55. w16x Mulder AH, Stegeman CA, Kallenberg CGM. Activation of granulocytes by anti-neutrophil cytoplasmic anti-
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