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Anti-neutrophil cytoplasmic antibodies and pathogenesis of small vessel vasculitides
Autoimmunity Reviews 2 (2003) 158–164
Anti-neutrophil cytoplasmic antibodies and pathogenesis of small vessel vasculitides Elena Csernok* ¨ Department of Rheumatology, University of Lubeck and Rheumaklinik Bad Bramstedt, Oskar-Alexander-Str. 26, 24576 Bad Bramstedt, Germany Received 22 November 2002; accepted 24 December 2002
Abstract Anti-neutrophil cytoplasmic antibodies (ANCA) are a heterogenous group of autoantibodies with a broad spectrum of clinically associated diseases. ANCA testing has been established as a useful tool for the diagnosis of small vessel vasculitides, especially of ‘ANCA-associated vasculitides’ (AAV), such as Wegener’s granulomatosis, microscopic polyangiitis and Churg–Strauss syndrome, in which circulating ANCA are commonly found. Within the last 20 years these antibodies were subject of intensive studies and a growing body of evidence arose for a distinct role of ANCA in the pathogenesis of the AAV. Our current concept of whether ANCA directly or indirectly contribute to vascular damage (ANCA-cytokine-sequence-theory) was mainly developed from in vitro studies and is supported by data from clinical investigations as well as animal models. Recently a direct causal link between ANCA and the development of glomerulonephritis and vasculitis has been demonstrated. We now know that a passive transfer of ANCA is sufficient to induce disease, but it remains to be discovered how the autoantibodies to neutrophil antigens might triggered. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Vasculitis; Anti-neutrophil cytoplasmic antibodies; Pathogenesis
1. Introduction The discovery that autoantibodies against cytoplasmic antigens of neutrophils (anti-neutrophil cytoplasmic antibodies (ANCA)) are closely associated with vasculitic disorders has improved diagnosis of patients with clinically suspected vasculitis andyor glomerulonephritis (GN). Like the introduction of ANA serology for systemic *Tel.: q49-4192-902195; fax: q49-4192-902381. E-mail address: [email protected] (E. Csernok).
lupus erythematosus, introduction of ANCA testing for vasculitis has revealed a myriad clinicopathological presentations beyond the previously recognized patterns of systemic disease. As a clinicopathological process, vasculitis occurs both as a primary process (primary or idiopathic vasculitis) and as a secondary feature of other diseases (secondary vasculitis) such as collagen vascular diseases, infectious disorders, malignancy, etc. The clinical spectrum of vasculitides is wide and varied. The vasculitic syndromes share a common histopathological substrate: inflammation within
1568-9972/03/$ - see front matter 䊚 2003 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 8 - 9 9 7 2 ( 0 3 ) 0 0 0 1 0 - 7
E. Csernok / Autoimmunity Reviews 2 (2003) 158–164
blood vessels resulting in vascular obstruction with tissue ischemia and infarction. Focal necrotizing lesions are the common vascular pathology that characterizes the ANCA-associated disorders: Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA) and Churg– Strauss syndrome (CSS). These lesions can affect many types of vessel and lead to a variety of symptoms and signs. Immunohistology shows little deposition of immune reactants, a feature which distinguishes lesions due to AAV from those of antiglomerular basement membrane disease, IgA nephropathy, and lupus nephritis. In contrast to WG and CSS, MPA is an AAV without granulomatous inflammation, asthma, or eosinophilia. ANCA were first described in the early 1980s as a cause of diffuse granular cytoplasmic immunofluorescence staining (C-ANCA) on ethanolfixed neutrophils in association with GN, vasculitis and WG. Proteinase 3 (PR3) was subsequently identified as the principal target antigen for these ANCA. At the same time, ANCA reacting with myeloperoxidase (MPO) and producing a perinuclear immunofluorescence staining pattern on ethanol-fixed neutrophils (P-ANCA) were found in patients with MPA or its renal-limited variant, with pauci-immune GN or, less frequently, with WG. Multiple other neutrophil granule constituents have since been identified as potential targets for ANCA in a variety of disorders. However, many large clinical studies have confirmed that only C-ANCA reacting with PR3 and P-ANCA reacting with MPO are highly specific for the autoimmune vasculitides WG and MPA, respectively (for review see Refs. w1,2x). The model of how ANCA directly and indirectly contribute to vascular injury is derived mostly from in vitro studies. This model has been shown to be consistent with clinical and animal model observations. Still, significant gaps in our knowledge remain and it has yet to be settled whether ANCA cause disease manifestations or are merely a marker of the disease. Answers to the following questions would represent major advances in our understanding of the pathogenesis of AAV. (1) What initiates the immune response that generates ANCA? (2) Are ANCA the consequence or the cause of the inflammatory response? (3) Why only
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PR3-ANCA and MPO-ANCA are linked to pauciimmune small-vessel vasculitis? (4) Why not every patient with PR3-ANCA or MPO-ANCA has an active vasculitis? The aim of the present review is to update current knowledge on the pathophysiology of vasculitides as revealed by basic and clinical research on ANCA. 2. Origin of the ANCA response The basis for the appearance of ANCA (immunogenesis) is not understood. One favored hypothesis is that environmental factors such infectious pathogen are required to activate the pre-existing potential autoimmune cellular repertoire. The proposed mechanisms by which infections break selftolerance can include bystander damage, unveiling of ‘hidden’ self epitopes, molecular mimicry and determinant molecular spreading. There is evidence that certain types of environmental exposure, to silica, for example, or to infectious pathogens (e.g. Staphylococcus aureus), are associated with AAV w3x. Current data demonstrates that ANCA directed against a variety of target antigens (mainly BPI) are present in infections involving bacterial, fungal, viral organisms. However, positivity for PR3- and MPO-ANCA is highly specific for smallvessel vasculitis and extremely uncommon in infections. Nevertheless, C-ANCAyPR3-ANCA have been reported in subacute bacterial endocarditis and disappeared with appropriate antimicrobial therapy w4x. It is therefore an attractive hypothesis that infections may trigger an initial ANCA response. If the response happens to be directed against the ‘right’ molecules, i.e. PR3 or MPO, and is allowed to persist, the stage is set for subsequent development of small-vessel vasculitis. It is currently unclear, however, why these two antigens—uniquely among all the known ANCA target antigens—are closely associated with small-vessel vasculitis. Recently, Voswinkel et al. detected a germinal center-like B-lymphoid infiltrate in endonasal WG granuloma. This finding raises the possibility that the antigen-driven immune response which ultimately leads to ANCA-associated generalized vasculitis is initiated in the upper respiratory tract. They also analyzed B lymphocytes from nasal
160
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tissue of patients with WG for the distribution and mutational pattern of antibody-encoding genes in order to draw the suspected antigen. Rearranged immunoglobulin genes were detected for all VH (VH1-6), indicating a polyclonal repertoire. Sequence analysis revealed a high frequency of mutations, with amino-acid substitution and a biased repertoire of represented genes indicating selection by an antigen. Three VH genes in cells producing PR3-ANCA were found to be overrepresented. The authors demonstrated that B cells are involved in the autoimmune mechanism of WG and this probably happens through the generation of high-affinity ANCAs by contact to PR3 or a cross-reacting microbial epitope in the inflamed endonasal tissue w5x.
crucial question of whether this patients express PR3yMPO on the cell surface, placing them at risk for the development of ANCA-mediated disease, or whether neutrophils in patients with ANCA are primed in the circulation, thus causing PR3 and MPO plasma membrane expression. A recent report introduced the concept that subsets of neutrophils express PR3 molecules on their surface and that the proportion of neutrophils presenting PR3 is genetically controlled and highly stable w10x. Indeed, the authors found that the phenotype of increased PR3 surface expression was significantly increased in patients with AAV. These data confirmed early reports that have demonstrated the presence of PR3 at the surface of activated neutrophils from patients with AAV w11,12x.
3. Characterization of ANCA antigens Another area of substantial research in this field involves the roles of the target antigens MPO and PR3 in the development of vascular injury. PR3 and MPO are known to have numerous effects other than that of being just a destructive enzyme. Both MPO and PR3 are capable of entering endothelial cells of many types. This process of antigen entry into cells is probably a consequence of receptor-mediated endocytosis. The exact nature of the receptor remains controversial, although Daha et al. suggested that the 111-kb protein is an important ligand w6x. It was demonstrated that PR3 cleaves p65 NF-kB at a site in the vicinity of a caspase 3 site, rendering it dysfunctional w7x. These results suggest that PR3 may function in a manner analogous to granzyme B released from lymphocytes. While the substrates are different, the killing effect of these proteases is quite similar w8x. MPO and PR3 are cationic proteins and bind on the endothelial cell surface. In doing so, they may be the source of ANCA binding that results in local vascular injury. Whether the antibody–antigen interaction on the surface of the endothelial cell is a prime effector arm of endothelial cell injury remains controversial w8x. Harper et al. report that neutrophils from ANCA patients display a higher level of apoptosis and that this increase correlates with higher concentrations of surface PR3 w9x. These findings raise a
4. Influence of PR3- and MPO-ANCA-subtypes on disease phenotype Studies comparing the clinical and histopathological associations between patients with PR3ANCA and MPO-ANCA are scarce. Although it is currently unclear why among all the described ANCA target antigens only ANCA against these two antigens (PR3 and MPO) are closely associated with small vessel vasculitis, their specificities do appear to have a bearing on clinical disease manifestations. Whereas WG is mostly associated with C-ANCAyPR3-ANCA, the majority of MPA patients have P-ANCAyMPO-ANCA. A direct comparison of clinical features of patients categorized by their ANCA-status revealed that extrarenal manifestations, granuloma formation and relapse were more frequent in patients with PR3ANCA than in those with MPO-ANCA w13,14x. ¨ Schonermarck et al. compared MPO-ANCA-positive WG patients with matched PR3-ANCA-positive WG patients and found the former to have less extensive disease than the latter. There was no difference regarding respiratory tract involvement, but the kidneys, eyes and the peripheral nervous system of the MPO-ANCA-positive WG patients tended to be less often affected w15x. Analysis of renal biopsy specimens from 173 patients obtained at the time of diagnosis suggested that active and chronic renal lesions are more
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Table 1 Clinical and pathological evidence of ANCA pathogenetic role (1) The high frequency of ANCA in patients with a distinctive pattern of small vessels vasculitis (systemic vasculitis and pauciimmune GN) that are shared by AAV (WG, MPA, CSS) (2) Changes in titers of ANCA screen to reflect disease activity (3) WG responds to immunosupressive therapy (4) WG are characterized by the absence of immunohistlogical evidence for vascular wall localization of immune-complex or anti-GBM antibodies (5) IgM-ANCA are associated with pulmonary hemorrhage (6) Plasma exchange and intravenous immunglobulins are effective in relieving the clinical symptoms (7) Recurrences are associated with IgG3-ANCA, which are more effective in activating neutrophlis than IgG1- and IgG4-ANCA (8) The severety of renal lesions correlates with the number of accumulated neutrophils and their degree of activation; the activation of neutrophils correlated with the ANCA Titer (9) Active WG is associated with widespread neutrophil activation and the presence neutrophilic alveolitis is the initial pulmonary in patients with recent onset disease (10) ANCA-target antigens (i.e. PR3, elastase) can be detected with glomeruli, crescents, in glomerulonephritic conditions that are associated with neutrophilic infiltrates (11) Drug-induced ANCA are associated with pauci-immune GN and vasculitis that disappear with discontinuation of the drug
common in MPO-ANCA-positive patients than in PR3-ANCA-positive patients, confirming previous observations made by others in a smaller cohort of patients w16x. Thus, despite substantial overlap there appear to be clinical and pathologic differences between patients with PR3-ANCA and patients with MPO-ANCA that may reflect different pathogenic interactions between ANCA, their target antigens, and organ envolvement at the molecular level w17x.
5. ANCA and pathogenesis of small vessel vasculitides The clinical, pathological and experimental evidence regarding the role of ANCA in the pathogenesis of systemic vasculitis and GN are summarized in Tables 1 and 2. How these in vitro effects of ANCA finally lead to in vivo systemic inflammation, blood vessel damage and GN is still a matter of speculation. Several mechanisms have
Table 2 Experimental evidence for the pathogenetic role of ANCA Effect of ANCA on neutroplils (1) Priming and apoptosis of neutrophils results in cell membrane expression of target antigens, making them accesible for ANCA (2) Cause degranulation and an oxidative burst of normal neutrophils primed with TNFa by binding simultaneously to Fcg-RII receptor and to the corresponding antigen ex pressed on the cell surface (3) Activation of neutrophils by ANCA insolves 5-lypoxygenase pathway inducing the production of leukotrine (LTB4), a chemoattract for neutophils (4) Induce expression interleukin-1b and IL-8 in neutrophils Effect of ANCA on monocytes (5) Activate monocytes (induce ROI release and the production of monocytes chemoattract-1, a potent of neutrophils; induce IL8 production) Effect of ANCA on endothelial cells (EC) (6) Induce expression o adhesion molecules and may enhance the adhesion of neutrophils and mononuclear cells to EC Effect of ANCA on PR3 (7) Prevent inactivation of PR3 by natural inhibitor a1-antitrypsin
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been proposed to explain how the interaction of ANCA with their target antigens results in necrotizing vasculitis. In inflammatory conditions leukocytes have to migrate by means of a complex process across the endothelial wall to arrive at the site of inflammation. This process is not accompanied by vessel wall injury (necrosis). If ANCA cause the vasculitis in vasculitic conditions, however, the autoantibodies must interact with neutrophils and monocytes in the circulation, resulting in activation, microvascular adherence of leukocytes, and subsequent vascular inflammation and necrosis (ANCA-cytokine-sequence-theory) w18x. One main conclusion from recent studies investigating the potential pathogenic role of ANCA is that ANCA in combination with exogenous factors are able to aggravate a clinical inflammatory process and may result in systemic vasculitis and GN. Many of the basic features of the mechanisms underlying the ANCA response, such as the factors responsible for the generation and perpetuation of these autoantibodies and for the shaping of the ANCA immune response, remain unknown. 6. Apoptosis, ANCA and vasculitis Neutrophils play an important role in the pathogenesis of vasculitis: they predominate at the site of tissue injury (necrotizing vasculitis and granuloma) and they are the main target cell of the ANCA antigens. The mechanisms by which ANCA alter neutrophils and monocytes seem to require ANCA binding to the antigen. What happens with the ANCA target antigens in dying cells? Only a few studies have addressed the fate of ANCA and their target antigens during apoptosis. Gilligan et al. described the presence of ANCA target antigens on the surface of apoptotic neutrophils w19x. The demonstration that ANCA interact with primary granule constituents (i.e. PR3, MPO) on the surface of apoptotic neutrophils and that ingestion of opsonized apoptotic neutrophils modulates macrophage behaviour, raises the question of whether ingestion of ANCA-opsonized apoptotic neutrophils may influence the uptake process and functions of macrophages. To investigate the above interactions, we analyzed the effects of PR3-ANCA-opsonized apoptotic neutrophils on
the uptake process and production of TNFa, IL10 and IL-12, as well on the secretion of the lipid inflammatory mediators, TxB2, LTB4 and PGE2 by human monocyte-derived macrophages. We could show that opsonization of apoptotic neutrophils by PR3-ANCA substantially enhanced recognition and binding by scavenger macrophages. Moreover, phagocytosis of ANCA-opsonized apoptotic neutrophils by macrophages triggered production of TNFa and TxB2. These findings indicate that PR3-ANCA opsonization of apoptotic neutrophils results in effective phagocytosis by human monocyte-derived macrophages and leads to upregulation of proinflammatory mediator production. These results point to a novel mechanism by which ANCA actively participate in apoptotic neutrophil clearance and modulate macrophage function. They may also help to explain the amplification and perpetuation of inflammation associated with severe necrotizing vascular injury. Furthermore, our observations emphasize the key role of TNFa in the pathogenesis of vasculitides and indicate a possible avenue for therapeutic interventions w20x. There is new evidence that after activation neutrophils are driven down an accelerated apoptotic death pathway by reactive oxygen species. These neutrophils develop the morphologic features of apoptosis, but there is a lack of the cell surface changes that normally accompany apoptosis, including phosphatidylserine expression w21x. After ANCA stimulation, phagocytic macrophages are less able to clear apoptotic neutrophils. The failure of safe removal means that apoptotic neutrophils eventually disintegrate, releasing cytotoxic contents. This process may explain the leukocytoclasia often seen in vasculitic lesions. Finally, recent experimental data from animal studies suggest that the persistence of apoptotic cells in an inflammatory environment, possibly enhanced by their opsonization, may result in the presentation of autoantigens to the immune system, with consequent autoantibody formation. A recent study showed the development of ANCA, albeit of undefined specificity, in rats after multiple injections of apoptotic syngeneic neutrophils w22x. So, theoretically, accumulation of apoptotic neutrophils may boost the PR3-specific autoimmune response.
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7. Experimental models of AAV Several animal studies have been conducted on ANCA and vasculitisyGN using various experimental approaches w23x. Establishment of an animal model has proven extremely difficult. There are few animal models for AAV that resemble models for MPA. The systemic autoimmune response to MPO in these models led to severe necrotizing vasculitis with extracapillary GN and, in some animals, to extravascular granuloma formation. It is noteworthy that induction of MPOANCA alone did not cause disease manifestations. Additional factors (infections?) were required that induced primingyactivation of neutrophils, monocytes and endothelial cells with subsequent release of toxic cellular products w24x. Xiao et al. recently made use of a model in which MPO knockout mice are immunized with murine MPO. As reported, these mice developed a brisk anti-MPO-antibody response. When splenocytes from these animals are transferred to Rag2 mice, small vessel vasculitis and a necrotizing and crescentic GN develops. When mice are immunized with control antigens such as bovine serum albumin and splenocytes transferred into them, no lesion occurs. Interestingly, both experimental and control mice have a baseline immune complex deposition in their glomeruli. The authors showed that transfer only of anti-MPO antibodies derived from immunized MPO knockout mice into Rag2 mice results in a necrotizing GN and crescentic GN. These data indicate that the anti-MPO antibody alone is capable of creating a necrotizing GN and vasculitis w25x. This model is the first convincing animal model for MPO-ANCA-vasculitis and provides the first evidence that ANCA are sufficient to cause systemic pauci-immune vasculitis and GN in vivo. In summary, we now know that a passive transfer of MPO-ANCA is sufficient to induce disease, but it remains to be discovered how the autoantibodies to neutrophil antigens might triggered. Furthermore, a satisfactory animal model for PR3-ANCA induced vasculitis has yet to be developed.
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Take-home messages ● Small vessel vasculitides such as WG, MPA and CSS are tightly correlated with the presence of antineutrophil cytoplasmic antibodies (ANCA) (so called ‘ANCA-associated vasculitides’) ● ANCA are more than a serologic marker of disease and a direct causal link between ANCA and the development of glomerulonephrtis and vasculitis has been demonstrated. ● There appear to be clinical and pathologic differences between patients with PR3-ANCA and patients with MPO-ANCA that may reflect different pathogenic interactions between ANCA, their target antigens, and organ envolvement at the molecular level. ● Many of the basic features of the mechanisms underlying the ANCA response, such as the factors responsible for the generation and perpetuation of these autoantibodies and for the shaping of the ANCA immune response, remain unknown.
References w1x Hoffman GS, Specks U. Antineutrophil cytoplasmic antibodies. Arthritis Rheum 1998;41:1521 –37. w2x Hagen EC, Daha MR, Hermans J, et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. ECy BCR project for ANCA assay standardization. Kidney Int 1998;53:743 –53. w3x Kallenberg CG, Tervaert JW. What is new with antineutrophil cytoplasmic antibodies: diagnostic, pathogenetic and therapeutic implications. Curr Opin Nephrol Hypertens 1999;8:307 –15. w4x Choi HK, Lamprecht P, Niles JL, Gross WL, Merkel PA. Subacute bacterial endocarditis with positive cytoplasmic antineutrophil cytoplasmic antibodies and antiproteinase 3 antibodies. Arthritis Rheum 2000;43(1):226 –31. w5x Voswinkel J, Muller ¨ A, Lamprecht P, Gross WL, Gause A. Does ANCA formation result from an antigentriggered immune response in Wegener’s endonasal inflamed tissue. Clev Clinic J Med 2002;69(SII159)38–129. 10th International Vasculitis and ANCA Workshop, Cleveland, April 25–28, 2002. w6x van der Geld YM, Limburg PC, Kallenberg CG. Proteinase 3, Wegener’s autoantigen: from gene to antigen. J Leukoc Biol 2001;69:177 –90.
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w7x Yang JJ, Preston GA, Pendergraft WF, et al. Internalization of proteinase 3 is concomitant with endothelial cell apoptosis and internalization of myeloperoxidase with generation of intracellular oxidants. Am J Pathol 2001;158(2):581 –92. w8x Preston GA, Yang JJ, Xiao H, Falk RJ. Understanding the pathogenesis of ANCA: where are we today? Clev Clin J Med 2002;69(Suppl. 2):SII51 –SII54. w9x Harper L, Cockwell P, Dwoma A, Savage C. Neutrophil priming and apoptosis in ANCA-associated vasculitis. Kidney Int 2001;59:1729 –38. w10x 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 rheumatoid arthritis. J Am Soc Nephrol 1999;10:1224 –33 (in process citation). w11x Csernok E, Ernst M, Schmitt W, Bainton DF, Gross WL. Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo. Clin Exp Immunol 1994;95:244 –50. w12x Muller-Kobold AC, Mesander G, Stegemann CA, Kallenberg CGM, Cohen Tervaert JW. Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmatic antibody associated vasculitides? Clin Exp Immunol 1998;114(3):491 –9. w13x Franssen CF, Gans RO, Arends B, et al. Differences between anti-myeloperoxidase and anti-proteinase 3 associated renal disease. Kidney Int 1995;47:193 –9. w14x Franssen C, Gans R, Kallenberg C, Hageluken C, Hoorntje S. Disease spectrum of patients with antineutrophil cytoplasmic autoantibodies of defined specificity: distinct differences between patients with anti-proteinase 3 and anti-myeloperoxidase autoantibodies. J Int Med 1998;244:209 –16. w15x Schonermarck ¨ U, Lamprecht P, Csernok E, Gross WL. Prevalence and spectrum of rheumatic diseases associated with proteinase 3-antineutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase-ANCA. Rheumatology (Oxford) 2001;40:178 –84. w16x Hauer HA, Bajema IM, van Houwelingen HC, et al. Renal histology in ANCA-associated vasculitis: differ-
w17x w18x
w19x
w20x
w21x
w22x
w23x w24x
w25x
ences between diagnostic and serologic subgroups. Kidney Int 2002;61:80 –9. Specks U. ANCA subsets: influence on disease phenotype. Clev Clin J Med 2002;69(Suppl. 2):SII56 –SII59. ¨ Csernok E, Muller A, Gross WL. Immunopathology of ANCA-associated vasculitis. Int Med 1999;38(10):759 – 65. Gilligan HM, Bredy B, Brady HR, et al. Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming. J Exp Med 1996;184:2231 –41. Moosig F, Csernok E, Kumanovics G, Gross WL. Opsonization of apoptotic neutrophils by anti-neutrophil cytoplasmic antibodies (ANCA) leads to enhanced uptake by macrophages and increased release of TNFalpha. Clin Exp Immunol 2000;122(3):499 –503. Harper JM, Thiru S, Lockwood CM, Cooke A. Myeloperoxidase autoantibodies distinguish vasculitis mediated by anti-neutrophil cytoplasmatic antibdies from immune complex disease in MRLyMp-IpryIpr mice: a spontaneous model for microscopic angiitis. Eur J Immunol 1998;28:2217 –26. Patry YC, Trewick DC, Gregoire M, et al. Rats injected with syngenic rat apoptotic neutrophils develop antineutrophil cytoplasmic antibodies. J Am Soc Nephrol 2001;12(8):1764 –8. Specks U. Are animal models of vasculitis suitable tools? Curr Opin Rheumatol 2000;12:11 –9. Heeringa P, Brouwer E, Cohen Tervaert JW, Weening JJ, Kallenberg CG. Animal models of anti-neutrophil cytoplasmic antibody associated vasculitis. Kidney Int 1998;53(2):253 –63. Xiao H, Heeringa P, Hu P, et al. Antineutrophil cytoplasmatic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest 2002;110:955 –63.
The World of Autoimmunity; Literature Synopsis Anti-recoverin antibodies in cancer-associated retinopathy Cancer-associated retinopathy (CAR) is a paraneoplastic disease associated with anti-recoverin antibodies. Shiraga and Adamus (J Neuroimmunol 2002;132:72) investigated the mechanisms by which these antibodies induce apoptosis of photoreceptor cells. Using an in-vitro model, they show that internalization of antirecoverin antibodies or their Fab fragment by retinal cells- leads to cytotoxicity. Apoptosis results through the mitochondrial pathway involving caspases 9 and 3. As patients having CAR possess high levels of circulating anti-recoverin antibodies, this mechanism of action can explain the pathogenic potential of these autoantibodies.
Anti-neutrophil cytoplasmic antibodies and pathogenesis of small vessel vasculitides Elena Csernok* ¨ Department of Rheumatology, University of Lubeck and Rheumaklinik Bad Bramstedt, Oskar-Alexander-Str. 26, 24576 Bad Bramstedt, Germany Received 22 November 2002; accepted 24 December 2002
Abstract Anti-neutrophil cytoplasmic antibodies (ANCA) are a heterogenous group of autoantibodies with a broad spectrum of clinically associated diseases. ANCA testing has been established as a useful tool for the diagnosis of small vessel vasculitides, especially of ‘ANCA-associated vasculitides’ (AAV), such as Wegener’s granulomatosis, microscopic polyangiitis and Churg–Strauss syndrome, in which circulating ANCA are commonly found. Within the last 20 years these antibodies were subject of intensive studies and a growing body of evidence arose for a distinct role of ANCA in the pathogenesis of the AAV. Our current concept of whether ANCA directly or indirectly contribute to vascular damage (ANCA-cytokine-sequence-theory) was mainly developed from in vitro studies and is supported by data from clinical investigations as well as animal models. Recently a direct causal link between ANCA and the development of glomerulonephritis and vasculitis has been demonstrated. We now know that a passive transfer of ANCA is sufficient to induce disease, but it remains to be discovered how the autoantibodies to neutrophil antigens might triggered. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Vasculitis; Anti-neutrophil cytoplasmic antibodies; Pathogenesis
1. Introduction The discovery that autoantibodies against cytoplasmic antigens of neutrophils (anti-neutrophil cytoplasmic antibodies (ANCA)) are closely associated with vasculitic disorders has improved diagnosis of patients with clinically suspected vasculitis andyor glomerulonephritis (GN). Like the introduction of ANA serology for systemic *Tel.: q49-4192-902195; fax: q49-4192-902381. E-mail address: [email protected] (E. Csernok).
lupus erythematosus, introduction of ANCA testing for vasculitis has revealed a myriad clinicopathological presentations beyond the previously recognized patterns of systemic disease. As a clinicopathological process, vasculitis occurs both as a primary process (primary or idiopathic vasculitis) and as a secondary feature of other diseases (secondary vasculitis) such as collagen vascular diseases, infectious disorders, malignancy, etc. The clinical spectrum of vasculitides is wide and varied. The vasculitic syndromes share a common histopathological substrate: inflammation within
1568-9972/03/$ - see front matter 䊚 2003 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 8 - 9 9 7 2 ( 0 3 ) 0 0 0 1 0 - 7
E. Csernok / Autoimmunity Reviews 2 (2003) 158–164
blood vessels resulting in vascular obstruction with tissue ischemia and infarction. Focal necrotizing lesions are the common vascular pathology that characterizes the ANCA-associated disorders: Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA) and Churg– Strauss syndrome (CSS). These lesions can affect many types of vessel and lead to a variety of symptoms and signs. Immunohistology shows little deposition of immune reactants, a feature which distinguishes lesions due to AAV from those of antiglomerular basement membrane disease, IgA nephropathy, and lupus nephritis. In contrast to WG and CSS, MPA is an AAV without granulomatous inflammation, asthma, or eosinophilia. ANCA were first described in the early 1980s as a cause of diffuse granular cytoplasmic immunofluorescence staining (C-ANCA) on ethanolfixed neutrophils in association with GN, vasculitis and WG. Proteinase 3 (PR3) was subsequently identified as the principal target antigen for these ANCA. At the same time, ANCA reacting with myeloperoxidase (MPO) and producing a perinuclear immunofluorescence staining pattern on ethanol-fixed neutrophils (P-ANCA) were found in patients with MPA or its renal-limited variant, with pauci-immune GN or, less frequently, with WG. Multiple other neutrophil granule constituents have since been identified as potential targets for ANCA in a variety of disorders. However, many large clinical studies have confirmed that only C-ANCA reacting with PR3 and P-ANCA reacting with MPO are highly specific for the autoimmune vasculitides WG and MPA, respectively (for review see Refs. w1,2x). The model of how ANCA directly and indirectly contribute to vascular injury is derived mostly from in vitro studies. This model has been shown to be consistent with clinical and animal model observations. Still, significant gaps in our knowledge remain and it has yet to be settled whether ANCA cause disease manifestations or are merely a marker of the disease. Answers to the following questions would represent major advances in our understanding of the pathogenesis of AAV. (1) What initiates the immune response that generates ANCA? (2) Are ANCA the consequence or the cause of the inflammatory response? (3) Why only
159
PR3-ANCA and MPO-ANCA are linked to pauciimmune small-vessel vasculitis? (4) Why not every patient with PR3-ANCA or MPO-ANCA has an active vasculitis? The aim of the present review is to update current knowledge on the pathophysiology of vasculitides as revealed by basic and clinical research on ANCA. 2. Origin of the ANCA response The basis for the appearance of ANCA (immunogenesis) is not understood. One favored hypothesis is that environmental factors such infectious pathogen are required to activate the pre-existing potential autoimmune cellular repertoire. The proposed mechanisms by which infections break selftolerance can include bystander damage, unveiling of ‘hidden’ self epitopes, molecular mimicry and determinant molecular spreading. There is evidence that certain types of environmental exposure, to silica, for example, or to infectious pathogens (e.g. Staphylococcus aureus), are associated with AAV w3x. Current data demonstrates that ANCA directed against a variety of target antigens (mainly BPI) are present in infections involving bacterial, fungal, viral organisms. However, positivity for PR3- and MPO-ANCA is highly specific for smallvessel vasculitis and extremely uncommon in infections. Nevertheless, C-ANCAyPR3-ANCA have been reported in subacute bacterial endocarditis and disappeared with appropriate antimicrobial therapy w4x. It is therefore an attractive hypothesis that infections may trigger an initial ANCA response. If the response happens to be directed against the ‘right’ molecules, i.e. PR3 or MPO, and is allowed to persist, the stage is set for subsequent development of small-vessel vasculitis. It is currently unclear, however, why these two antigens—uniquely among all the known ANCA target antigens—are closely associated with small-vessel vasculitis. Recently, Voswinkel et al. detected a germinal center-like B-lymphoid infiltrate in endonasal WG granuloma. This finding raises the possibility that the antigen-driven immune response which ultimately leads to ANCA-associated generalized vasculitis is initiated in the upper respiratory tract. They also analyzed B lymphocytes from nasal
160
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tissue of patients with WG for the distribution and mutational pattern of antibody-encoding genes in order to draw the suspected antigen. Rearranged immunoglobulin genes were detected for all VH (VH1-6), indicating a polyclonal repertoire. Sequence analysis revealed a high frequency of mutations, with amino-acid substitution and a biased repertoire of represented genes indicating selection by an antigen. Three VH genes in cells producing PR3-ANCA were found to be overrepresented. The authors demonstrated that B cells are involved in the autoimmune mechanism of WG and this probably happens through the generation of high-affinity ANCAs by contact to PR3 or a cross-reacting microbial epitope in the inflamed endonasal tissue w5x.
crucial question of whether this patients express PR3yMPO on the cell surface, placing them at risk for the development of ANCA-mediated disease, or whether neutrophils in patients with ANCA are primed in the circulation, thus causing PR3 and MPO plasma membrane expression. A recent report introduced the concept that subsets of neutrophils express PR3 molecules on their surface and that the proportion of neutrophils presenting PR3 is genetically controlled and highly stable w10x. Indeed, the authors found that the phenotype of increased PR3 surface expression was significantly increased in patients with AAV. These data confirmed early reports that have demonstrated the presence of PR3 at the surface of activated neutrophils from patients with AAV w11,12x.
3. Characterization of ANCA antigens Another area of substantial research in this field involves the roles of the target antigens MPO and PR3 in the development of vascular injury. PR3 and MPO are known to have numerous effects other than that of being just a destructive enzyme. Both MPO and PR3 are capable of entering endothelial cells of many types. This process of antigen entry into cells is probably a consequence of receptor-mediated endocytosis. The exact nature of the receptor remains controversial, although Daha et al. suggested that the 111-kb protein is an important ligand w6x. It was demonstrated that PR3 cleaves p65 NF-kB at a site in the vicinity of a caspase 3 site, rendering it dysfunctional w7x. These results suggest that PR3 may function in a manner analogous to granzyme B released from lymphocytes. While the substrates are different, the killing effect of these proteases is quite similar w8x. MPO and PR3 are cationic proteins and bind on the endothelial cell surface. In doing so, they may be the source of ANCA binding that results in local vascular injury. Whether the antibody–antigen interaction on the surface of the endothelial cell is a prime effector arm of endothelial cell injury remains controversial w8x. Harper et al. report that neutrophils from ANCA patients display a higher level of apoptosis and that this increase correlates with higher concentrations of surface PR3 w9x. These findings raise a
4. Influence of PR3- and MPO-ANCA-subtypes on disease phenotype Studies comparing the clinical and histopathological associations between patients with PR3ANCA and MPO-ANCA are scarce. Although it is currently unclear why among all the described ANCA target antigens only ANCA against these two antigens (PR3 and MPO) are closely associated with small vessel vasculitis, their specificities do appear to have a bearing on clinical disease manifestations. Whereas WG is mostly associated with C-ANCAyPR3-ANCA, the majority of MPA patients have P-ANCAyMPO-ANCA. A direct comparison of clinical features of patients categorized by their ANCA-status revealed that extrarenal manifestations, granuloma formation and relapse were more frequent in patients with PR3ANCA than in those with MPO-ANCA w13,14x. ¨ Schonermarck et al. compared MPO-ANCA-positive WG patients with matched PR3-ANCA-positive WG patients and found the former to have less extensive disease than the latter. There was no difference regarding respiratory tract involvement, but the kidneys, eyes and the peripheral nervous system of the MPO-ANCA-positive WG patients tended to be less often affected w15x. Analysis of renal biopsy specimens from 173 patients obtained at the time of diagnosis suggested that active and chronic renal lesions are more
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Table 1 Clinical and pathological evidence of ANCA pathogenetic role (1) The high frequency of ANCA in patients with a distinctive pattern of small vessels vasculitis (systemic vasculitis and pauciimmune GN) that are shared by AAV (WG, MPA, CSS) (2) Changes in titers of ANCA screen to reflect disease activity (3) WG responds to immunosupressive therapy (4) WG are characterized by the absence of immunohistlogical evidence for vascular wall localization of immune-complex or anti-GBM antibodies (5) IgM-ANCA are associated with pulmonary hemorrhage (6) Plasma exchange and intravenous immunglobulins are effective in relieving the clinical symptoms (7) Recurrences are associated with IgG3-ANCA, which are more effective in activating neutrophlis than IgG1- and IgG4-ANCA (8) The severety of renal lesions correlates with the number of accumulated neutrophils and their degree of activation; the activation of neutrophils correlated with the ANCA Titer (9) Active WG is associated with widespread neutrophil activation and the presence neutrophilic alveolitis is the initial pulmonary in patients with recent onset disease (10) ANCA-target antigens (i.e. PR3, elastase) can be detected with glomeruli, crescents, in glomerulonephritic conditions that are associated with neutrophilic infiltrates (11) Drug-induced ANCA are associated with pauci-immune GN and vasculitis that disappear with discontinuation of the drug
common in MPO-ANCA-positive patients than in PR3-ANCA-positive patients, confirming previous observations made by others in a smaller cohort of patients w16x. Thus, despite substantial overlap there appear to be clinical and pathologic differences between patients with PR3-ANCA and patients with MPO-ANCA that may reflect different pathogenic interactions between ANCA, their target antigens, and organ envolvement at the molecular level w17x.
5. ANCA and pathogenesis of small vessel vasculitides The clinical, pathological and experimental evidence regarding the role of ANCA in the pathogenesis of systemic vasculitis and GN are summarized in Tables 1 and 2. How these in vitro effects of ANCA finally lead to in vivo systemic inflammation, blood vessel damage and GN is still a matter of speculation. Several mechanisms have
Table 2 Experimental evidence for the pathogenetic role of ANCA Effect of ANCA on neutroplils (1) Priming and apoptosis of neutrophils results in cell membrane expression of target antigens, making them accesible for ANCA (2) Cause degranulation and an oxidative burst of normal neutrophils primed with TNFa by binding simultaneously to Fcg-RII receptor and to the corresponding antigen ex pressed on the cell surface (3) Activation of neutrophils by ANCA insolves 5-lypoxygenase pathway inducing the production of leukotrine (LTB4), a chemoattract for neutophils (4) Induce expression interleukin-1b and IL-8 in neutrophils Effect of ANCA on monocytes (5) Activate monocytes (induce ROI release and the production of monocytes chemoattract-1, a potent of neutrophils; induce IL8 production) Effect of ANCA on endothelial cells (EC) (6) Induce expression o adhesion molecules and may enhance the adhesion of neutrophils and mononuclear cells to EC Effect of ANCA on PR3 (7) Prevent inactivation of PR3 by natural inhibitor a1-antitrypsin
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been proposed to explain how the interaction of ANCA with their target antigens results in necrotizing vasculitis. In inflammatory conditions leukocytes have to migrate by means of a complex process across the endothelial wall to arrive at the site of inflammation. This process is not accompanied by vessel wall injury (necrosis). If ANCA cause the vasculitis in vasculitic conditions, however, the autoantibodies must interact with neutrophils and monocytes in the circulation, resulting in activation, microvascular adherence of leukocytes, and subsequent vascular inflammation and necrosis (ANCA-cytokine-sequence-theory) w18x. One main conclusion from recent studies investigating the potential pathogenic role of ANCA is that ANCA in combination with exogenous factors are able to aggravate a clinical inflammatory process and may result in systemic vasculitis and GN. Many of the basic features of the mechanisms underlying the ANCA response, such as the factors responsible for the generation and perpetuation of these autoantibodies and for the shaping of the ANCA immune response, remain unknown. 6. Apoptosis, ANCA and vasculitis Neutrophils play an important role in the pathogenesis of vasculitis: they predominate at the site of tissue injury (necrotizing vasculitis and granuloma) and they are the main target cell of the ANCA antigens. The mechanisms by which ANCA alter neutrophils and monocytes seem to require ANCA binding to the antigen. What happens with the ANCA target antigens in dying cells? Only a few studies have addressed the fate of ANCA and their target antigens during apoptosis. Gilligan et al. described the presence of ANCA target antigens on the surface of apoptotic neutrophils w19x. The demonstration that ANCA interact with primary granule constituents (i.e. PR3, MPO) on the surface of apoptotic neutrophils and that ingestion of opsonized apoptotic neutrophils modulates macrophage behaviour, raises the question of whether ingestion of ANCA-opsonized apoptotic neutrophils may influence the uptake process and functions of macrophages. To investigate the above interactions, we analyzed the effects of PR3-ANCA-opsonized apoptotic neutrophils on
the uptake process and production of TNFa, IL10 and IL-12, as well on the secretion of the lipid inflammatory mediators, TxB2, LTB4 and PGE2 by human monocyte-derived macrophages. We could show that opsonization of apoptotic neutrophils by PR3-ANCA substantially enhanced recognition and binding by scavenger macrophages. Moreover, phagocytosis of ANCA-opsonized apoptotic neutrophils by macrophages triggered production of TNFa and TxB2. These findings indicate that PR3-ANCA opsonization of apoptotic neutrophils results in effective phagocytosis by human monocyte-derived macrophages and leads to upregulation of proinflammatory mediator production. These results point to a novel mechanism by which ANCA actively participate in apoptotic neutrophil clearance and modulate macrophage function. They may also help to explain the amplification and perpetuation of inflammation associated with severe necrotizing vascular injury. Furthermore, our observations emphasize the key role of TNFa in the pathogenesis of vasculitides and indicate a possible avenue for therapeutic interventions w20x. There is new evidence that after activation neutrophils are driven down an accelerated apoptotic death pathway by reactive oxygen species. These neutrophils develop the morphologic features of apoptosis, but there is a lack of the cell surface changes that normally accompany apoptosis, including phosphatidylserine expression w21x. After ANCA stimulation, phagocytic macrophages are less able to clear apoptotic neutrophils. The failure of safe removal means that apoptotic neutrophils eventually disintegrate, releasing cytotoxic contents. This process may explain the leukocytoclasia often seen in vasculitic lesions. Finally, recent experimental data from animal studies suggest that the persistence of apoptotic cells in an inflammatory environment, possibly enhanced by their opsonization, may result in the presentation of autoantigens to the immune system, with consequent autoantibody formation. A recent study showed the development of ANCA, albeit of undefined specificity, in rats after multiple injections of apoptotic syngeneic neutrophils w22x. So, theoretically, accumulation of apoptotic neutrophils may boost the PR3-specific autoimmune response.
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7. Experimental models of AAV Several animal studies have been conducted on ANCA and vasculitisyGN using various experimental approaches w23x. Establishment of an animal model has proven extremely difficult. There are few animal models for AAV that resemble models for MPA. The systemic autoimmune response to MPO in these models led to severe necrotizing vasculitis with extracapillary GN and, in some animals, to extravascular granuloma formation. It is noteworthy that induction of MPOANCA alone did not cause disease manifestations. Additional factors (infections?) were required that induced primingyactivation of neutrophils, monocytes and endothelial cells with subsequent release of toxic cellular products w24x. Xiao et al. recently made use of a model in which MPO knockout mice are immunized with murine MPO. As reported, these mice developed a brisk anti-MPO-antibody response. When splenocytes from these animals are transferred to Rag2 mice, small vessel vasculitis and a necrotizing and crescentic GN develops. When mice are immunized with control antigens such as bovine serum albumin and splenocytes transferred into them, no lesion occurs. Interestingly, both experimental and control mice have a baseline immune complex deposition in their glomeruli. The authors showed that transfer only of anti-MPO antibodies derived from immunized MPO knockout mice into Rag2 mice results in a necrotizing GN and crescentic GN. These data indicate that the anti-MPO antibody alone is capable of creating a necrotizing GN and vasculitis w25x. This model is the first convincing animal model for MPO-ANCA-vasculitis and provides the first evidence that ANCA are sufficient to cause systemic pauci-immune vasculitis and GN in vivo. In summary, we now know that a passive transfer of MPO-ANCA is sufficient to induce disease, but it remains to be discovered how the autoantibodies to neutrophil antigens might triggered. Furthermore, a satisfactory animal model for PR3-ANCA induced vasculitis has yet to be developed.
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Take-home messages ● Small vessel vasculitides such as WG, MPA and CSS are tightly correlated with the presence of antineutrophil cytoplasmic antibodies (ANCA) (so called ‘ANCA-associated vasculitides’) ● ANCA are more than a serologic marker of disease and a direct causal link between ANCA and the development of glomerulonephrtis and vasculitis has been demonstrated. ● There appear to be clinical and pathologic differences between patients with PR3-ANCA and patients with MPO-ANCA that may reflect different pathogenic interactions between ANCA, their target antigens, and organ envolvement at the molecular level. ● Many of the basic features of the mechanisms underlying the ANCA response, such as the factors responsible for the generation and perpetuation of these autoantibodies and for the shaping of the ANCA immune response, remain unknown.
References w1x Hoffman GS, Specks U. Antineutrophil cytoplasmic antibodies. Arthritis Rheum 1998;41:1521 –37. w2x Hagen EC, Daha MR, Hermans J, et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. ECy BCR project for ANCA assay standardization. Kidney Int 1998;53:743 –53. w3x Kallenberg CG, Tervaert JW. What is new with antineutrophil cytoplasmic antibodies: diagnostic, pathogenetic and therapeutic implications. Curr Opin Nephrol Hypertens 1999;8:307 –15. w4x Choi HK, Lamprecht P, Niles JL, Gross WL, Merkel PA. Subacute bacterial endocarditis with positive cytoplasmic antineutrophil cytoplasmic antibodies and antiproteinase 3 antibodies. Arthritis Rheum 2000;43(1):226 –31. w5x Voswinkel J, Muller ¨ A, Lamprecht P, Gross WL, Gause A. Does ANCA formation result from an antigentriggered immune response in Wegener’s endonasal inflamed tissue. Clev Clinic J Med 2002;69(SII159)38–129. 10th International Vasculitis and ANCA Workshop, Cleveland, April 25–28, 2002. w6x van der Geld YM, Limburg PC, Kallenberg CG. Proteinase 3, Wegener’s autoantigen: from gene to antigen. J Leukoc Biol 2001;69:177 –90.
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w7x Yang JJ, Preston GA, Pendergraft WF, et al. Internalization of proteinase 3 is concomitant with endothelial cell apoptosis and internalization of myeloperoxidase with generation of intracellular oxidants. Am J Pathol 2001;158(2):581 –92. w8x Preston GA, Yang JJ, Xiao H, Falk RJ. Understanding the pathogenesis of ANCA: where are we today? Clev Clin J Med 2002;69(Suppl. 2):SII51 –SII54. w9x Harper L, Cockwell P, Dwoma A, Savage C. Neutrophil priming and apoptosis in ANCA-associated vasculitis. Kidney Int 2001;59:1729 –38. w10x 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 rheumatoid arthritis. J Am Soc Nephrol 1999;10:1224 –33 (in process citation). w11x Csernok E, Ernst M, Schmitt W, Bainton DF, Gross WL. Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo. Clin Exp Immunol 1994;95:244 –50. w12x Muller-Kobold AC, Mesander G, Stegemann CA, Kallenberg CGM, Cohen Tervaert JW. Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmatic antibody associated vasculitides? Clin Exp Immunol 1998;114(3):491 –9. w13x Franssen CF, Gans RO, Arends B, et al. Differences between anti-myeloperoxidase and anti-proteinase 3 associated renal disease. Kidney Int 1995;47:193 –9. w14x Franssen C, Gans R, Kallenberg C, Hageluken C, Hoorntje S. Disease spectrum of patients with antineutrophil cytoplasmic autoantibodies of defined specificity: distinct differences between patients with anti-proteinase 3 and anti-myeloperoxidase autoantibodies. J Int Med 1998;244:209 –16. w15x Schonermarck ¨ U, Lamprecht P, Csernok E, Gross WL. Prevalence and spectrum of rheumatic diseases associated with proteinase 3-antineutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase-ANCA. Rheumatology (Oxford) 2001;40:178 –84. w16x Hauer HA, Bajema IM, van Houwelingen HC, et al. Renal histology in ANCA-associated vasculitis: differ-
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The World of Autoimmunity; Literature Synopsis Anti-recoverin antibodies in cancer-associated retinopathy Cancer-associated retinopathy (CAR) is a paraneoplastic disease associated with anti-recoverin antibodies. Shiraga and Adamus (J Neuroimmunol 2002;132:72) investigated the mechanisms by which these antibodies induce apoptosis of photoreceptor cells. Using an in-vitro model, they show that internalization of antirecoverin antibodies or their Fab fragment by retinal cells- leads to cytotoxicity. Apoptosis results through the mitochondrial pathway involving caspases 9 and 3. As patients having CAR possess high levels of circulating anti-recoverin antibodies, this mechanism of action can explain the pathogenic potential of these autoantibodies.