Pathophysiology of ANCA-associated glomerulonephritis.

Nephrol Dial Transplant (1999) 14: 1366–1375

Nephrology Dialysis Transplantation

Molecular Basis of Renal Disease

Pathophysiology of ANCA-associated glomerulonephritis Anneke C. Muller Kobold1, Ymke M. van der Geld1, Pieter C. Limburg1,2, Jan Willem Cohen Tervaert1 and Cees G. M. Kallenberg1 Departments of 1Clinical Immunology and 2Rheumatology, University Hospital Groningen, The Netherlands

Key words: ANCA; glomerulonephritis; vasculitis; pathophysiology; neutrophil

Introduction Rapidly progressive glomerulonephritis (RPGN ) is a clinical syndrome characterized by rapid deterioration of renal function together with proteinuria and haematuria with cellular casts. The syndrome is histopathologically manifested as fibrinoid necrosis of the capillary wall with extracapillary proliferation and crescent formation [1]. The major categories of this, so-called necrotizing crescentic glomerulonephritis (NCGN ) are anti-glomerular basement membrane (GBM ) disease, immune complex-mediated crescentic glomerulonephritis and pauci-immune crescentic glomerulonephritis. Pauci-immune crescentic glomerulonephritis is characterized by paucity or absence of immunoglobulin deposits in the glomeruli and is often, but not always [2], associated with anti-neutrophil cytoplasmic antibodies (ANCA). It may occur as an idiopathic variety without systemic manifestations or as part of systemic vasculitis. The vasculitic syndromes associated with pauci-immune NCGN, i.e. Wegener’s granulomatosis ( WG), microscopic polyangiitis (MPA) and, rarely, Churg–Strauss syndrome (CSS), systemically affect small blood vessels and are usually associated with ANCA ( Table 1). The idiopathic variety of NCGN may be considered as a renal limited form of systemic vasculitis. The target antigens of ANCA in those diseases are proteinase 3 (Pr3), myeloperoxidase (MPO) [3,4] and, occasionally, human leukocyte elastase [5]. In patients with WG, the prevalence of anti-Pr3 antibodies is 80–90% [6 ]. The prevalence of either anti-Pr3 or antiMPO antibodies in patients with active MPA and in patients with idiopathic NCGN is 90% [3,7], whereas anti-MPO antibodies occur in 60–70% of patients with CSS [8–10]. Several in vivo observations suggest that antibodies Correspondence and offprint requests to: A. C. Muller Kobold, Department of Clinical Immunology, University Hospital Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands.

to Pr3 and MPO are involved in the pathophysiology of their associated disorders. ANCA titres decline or even become negative when remission is induced [6 ]. In addition, patients who are persistently or intermittently anti-Pr3 ANCA positive during remission are prone to develop relapses [11,12]. Furthermore, it has been demonstrated that titres of Pr3 ANCA rise prior to a relapse of WG in most patients [6,13,14], although these data have been disputed by others [15,16 ]. Finally, treatment based on changes in ANCA titres prevented the development of relapses in patients with WG [17]. In vivo experimental animal studies, reviewed recently by Heeringa et al. [18], also support a pathophysiological role for ANCA. The pathophysiological role of ANCA in ANCAassociated vasculitides is, however, not yet completely understood. Whereas autoantibodies to structural antigens of the GBM and immune complexes are involved in the pathophysiology of anti-GBM and immune complex-mediated crescentic glomerulonephritis, it is not fully clarified how ANCA induce pauci-immune crescentic glomerulonephritis. In vitro experimental work has extensively analysed the effects of ANCA on neutrophils and monocytes, as these cells contain the primary target antigens of ANCA. Little is known, however, about the in vivo relevance of these in vitro effects of ANCA and how these effects may finally result into in vivo systemic inflammation, damage of blood vessels and pauciimmune glomerulonephritis. In this review, we will Table 1. Disease associations of anti-Pr3 antibodies and anti-MPO antibodies [3] Disease entity

Wegener’s granulomatosis Microscopic polyangiitis Idiopathic crescentic glomerulonephritis Churg–Strauss syndrome

© 1999 European Renal Association–European Dialysis and Transplant Association

Sensitivity of Anti-Pr3 (%)

Anti-MPO (%)

85

10

45

45

25

65

10

60

Pathophysiology of ANCA-associated glomerulonephritis

connect in vitro data on biological effects of ANCA to in vivo observations in patients with ANCA-associated vasculitides.

1367

uclear leukocytes, as a result of cytoplasmic granular translocation during apoptosis. Interaction of ANCA with leukocytes

In vitro data: ANCA activates primed neutrophils and monocytes The most important in vitro finding relates to the potential of ANCA to activate primed neutrophils and monocytes. In the following section, we will discuss these findings in more detail. Intracellular antigens ANCA are directed primarily against constituents of neutrophils and monocytes which are stored in intracellular granules. This raises the question of how ANCA can interact with these intracellular antigens. In vitro, inflammatory mediators, such as tumour necrosis factor a ( TNFa), interleukin-1 (IL-1) or IL-8, are, even at low doses, capable of priming neutrophils or monocytes [19–22]. Priming or pre-activation leads to a state in which these cells are not yet fully activated. When, however, subsequently activated by a second stimulus, primed cells respond more strongly than nonprimed cells [23]. Upon in vitro priming, membrane expression of Pr3 and MPO can be observed [19–22]. During priming, translocation of azurophilic and specific granules may occur, resulting in the partial release of granule constituents. It remains to be determined how these antigens are actually expressed at the cell surface. One explanation might be that the cationic proteins Pr3 [isoelectric point (PI )] and MPO (PI >11) bind to the negatively charged cell membrane via charge interactions. Also another mechanism might explain binding of Pr3 or elastase to the outer surface of cells. Alpha-1-antitrypsin (a1-AT ) is the natural inhibitor of serine proteases, including Pr3 and elastase, and binding of this protein results in enzyme–inhibitor complexes. These complexes can interact with the serine proteinase inhibitor (serpin) enzyme complex (SEC ) receptor, which is present on a variety of cells, including neutrophils and mononuclear phagocytes [24]. Recently, other mechanisms for binding of ANCA antigens to the cell membrane of neutrophils have been suggested. Binding of MPO may be mediated by b2 integrins, especially the CD11b subunit [25], whereas binding of elastase to the cell membrane of activated neutrophils appears also to be mediated by b2 integrins, but here especially the CD11a subunit is involved [26 ]. Since elastase and Pr3 share 54% amino acid sequence homology [27], a similar mechanism might be expected for Pr3. Thus, upon priming of neutrophils, intracellularly stored ANCA antigens are released and bind to the cell membrane by charge interactions, or via membrane-bound molecules, such as the SEC receptor or b2 integrins. Finally, Gilligan et al. [28] demonstrated ANCA antigen surface expression on apoptotic polymorphon-

Since primed neutrophils and monocytes express the ANCA antigens Pr3 and MPO on their cell surface, ANCA can bind to these cells via the antigen-binding site of the antibody. It has been shown by Kettritz et al. that ANCA-induced neutrophil activation is dependent on cross-linking of surface molecules, as ANCA IgG and F(ab)2 fragments, but not Fab fragments, were capable of stimulating the production of oxygen radicals by primed neutrophils [29]. Upon binding of ANCA to their antigens at the cell membrane, an activating signal has to be transduced into the cell. Since Pr3 and MPO themselves do not contain a transmembrane domain and cytoplasmic tail for signal transduction, other molecules or receptors must function as such. Possible candidates are, as mentioned earlier, the b2 integrins, which have been demonstrated to bind MPO and elastase, or the SEC receptor, which binds a-1-AT–Pr3 complexes. Fcc receptors should, however, not be excluded from this list of ‘signal transducers’, since these receptors are also capable of transducing an activating signal into the cell. Three classes of Fcc receptors have been identified in man, i.e. FccRI (CD64), FccRII (CD32) and FccRIII (CD16) ( Table 2). Neutrophils predominantly express FccRIIa and FccRIIIb, whereas monocytes express FccRI, FccRIIa and FccRIIIa. These receptors differ in their ability to bind monomer or complexed IgG and interact differently with the various subclasses of IgG. The IgG class of immunoglobulins consists of four subclasses, which differ in biological functions such as the ability to activate complement or the affinity for certain Fc receptors [30]. The FccRIIa is the only Fc receptor that interacts with IgG2. In addition, this receptor has a particularly affinity for the IgG3 subclass [31]. Interestingly, the increase in neutrophil-activating capacity of serum IgG fractions from remission to relapse in patients with WG correlated with increases in levels of IgG3 subclass ANCA in those fractions and not with that of the other subclasses [32]. In addition, renal exacerbations of WG are particularly associated with increases of IgG3 subclass of ANCA [33], although IgG1 and IgG4 subclasses of ANCA are present as well [33]. These data suggest that the IgG3 subclass of ANCA plays an important role in in vitro neutrophil activation via Fcc receptor interactions. Although some studies demonstrated that incubation of primed neutrophils with IgG F(ab)2 ANCA alone also results in neutrophil activation [19,29], other studies suggest that both antigen binding and Fc receptor interaction are involved [34–36 ]. In these in vitro experiments, ANCA-induced neutrophil activation did not occur when F(ab)2 fragments of ANCA IgG were used and/or was strongly inhibited when blocking monoclonal antibodies against the second Fcc receptor were used.

1368

A. C. Muller Kobold et al.

Table 2. FccR distribution and ligand specificity [30] FccR class

FccRI (CD64) FccRII (CD32)

FccRIII (CD16)

Expression

Functional polymorphisms

Constitutive

Induced

monocyte macrophage monocyte, neutrophil, macrophage, eosinophil monocyte macrophage, neutrophil

neutrophil, eosinophil

monocyte eosinophil

Thus, ANCA bind to their respective antigens at the cell surface and may, at the same time, interact via the Fc portion of the antibody with Fcc receptors on the same or other cells [37]. Cross-linking of molecules ( b2 integrins, SEC receptors or Fcc receptors) takes place and an activating signal is transduced over the plasma membrane into the cell. The signal transduction pathway in neutrophils stimulated with IgG ANCA has only been investigated recently. Activation by ANCA IgG results in translocation of multiple protein kinase C (PKC ) isoenzymes and tyrosine phosphorylation of a number of proteins [38]. The transduction route does not involve G-proteins, is dependent on phosphatidylinositol 3-kinase (PI -K ) and is sensitive to calcium fluxes [39]. 3 The signal for F-actin polymerization seen after addition of ANCA to neutrophils is most likely transduced via the FcRcIIa route [40,41]. Translocation of PKC, tyrosine phosphorylation, PI -K activation and cal3 cium sensitivity are all part of very common signal transduction pathways, among which is the pathway of FccRIIa [41]. Signal transduction via b2 integrins also involves the above-mentioned pathways. Furthermore, b2 integrins on neutrophils are involved in the Fc-mediated phagocytosis and respiratory burst. FccRIII interacts directly with b2 integrins leading to an enhanced respiratory burst by a mechanism that involves tyrosine phosphorylation of FccRII and its association with the actin cytoskeleton [42,43]. Signal transduction pathways switched on in neutrophils upon activation by ANCA include the pathway of FccRII and pathways from other receptors, which still have to be defined (see Table 3) [42–45]. Reumeax et al. [36 ] demonstrated that, besides priming and binding of ANCA, a third factor is needed before neutrophils are activated by ANCA. ANCAinduced activation of neutrophils could not be demonstrated when adherence of the cells was prevented by continuous stirring of the suspension or by the addition of blocking antibodies directed against FccRIIa or, interestingly, against b2 integrins, in particular CD18. From this study, it appears that b2 integrin–mediated outside-in signalling [46 ] is instrumental in ANCAinduced neutrophil activation. Therefore, we postulate that ANCA-induced neutrophil activation only occurs when primed cells are bound to a surface, e.g. endothel-

Ligand specificity (IgG subclasses) 3>1>4>>>2

IIa, R131 IIa, H131

3>1>>>2,4 3>1=2>>>4

IIIb, NA1 IIIb, NA2

1=3>>>2,4

ial cells, a process which appears to be dependent on adhesion molecules, especially the b2 integrins. In vitro effects of ANCA-induced cell activation Upon activation by ANCA, activated neutrophils or monocytes do what all activated neutrophils and monocytes are capable of: they produce and secrete reactive oxygen species (ROS) [19,29,35,47], they degranulate lysosomal enzymes, including Pr3, MPO and elastase [19,20], and they produce inflammatory mediators, such as TNFa, IL-1, IL-8 and leukotriene B4 [22,47–49], which may in turn prime or attract other cells. Finally, upon activation by ANCA, these cells express increased levels of adhesion molecules, including b2 integrins, that facilitate binding to and transmigration through the endothelial monolayer [50]. As discussed above, b2 integrin binding is required for ANCA-induced leukocyte activation [36 ]. Interaction between activated leukocytes, ANCA and activated endothelial cells can result in damage to the latter cells by toxic products released from the activated leukocytes [51–54]. In addition, lysosomal products, including Pr3 and MPO, are being released and may bind to the endothelial cell surface, serving as ‘planted’ antigens and new targets for ANCA. Indeed, endothelial cells can be coated in vitro with these antigens [55,56 ]. Furthermore, in vitro incubation of endothelial cells with Pr3 induces production of IL-8 [57], endothelial cell apoptosis [58] and endothelial cell detachment and lysis [59]. These effects were independent of serine protease activity of Pr3. Several studies have suggested that ANCA are also capable of directly activating endothelial cells in vitro. Incubation of endothelial cells with IgG preparations from patients positive for ANCA resulted in endothelial cell activation as demonstrated by the increased expression of adhesion molecules ( E-selectin, VCAM-1 and ICAM-1) or tissue factor [60–64]. However, it is unclear how ANCA can interact with and, subsequently, directly activate endothelial cells, as the expression of ANCA antigens, such as Pr3 and MPO, has been disputed [65–67], and no specific signal transducing transmembrane receptors are identified in relation to Pr3 and MPO on endothelial cells.


1369

Table 3. Signal transduction pathways of FccRIIa, b integrins and the SEC receptor 2

Protein tyrosine phosphorylation Activation PLC PKC translocation Intracellular Ca2+ mobilization PI -kinase activation 3 Actin polymerization G-protein involvement Effect Reference

FccRIIa

b2 integrins

SEC receptor

Yes PLCb/c Yes Yes Yes Yes No Respiratory burst Phagocytosis [41]

Yes PLCc Yes Yes

? ? ? Yes ? ? Pertusis toxin-sensitive G protein Increase a1-AT synthesis [24] Neutrophil chemotaxis [44] [45]

In vivo relevance of in vitro experimental data for ANCA-associated vasculitis How do these in vitro effects of ANCA finally result into in vivo systemic inflammation, damage of blood vessels and pauci-immune glomerulonephritis? The following section focuses on connecting the in vitro data discussed above to in vivo manifestations of ANCAassociated vasculitides. From those in vitro data, we extracted four prerequisites for endothelial cell damage by ANCA: (i) the presence of ANCA; (ii) expression of the target antigens for ANCA on primed neutrophils and monocytes; (iii) the necessity for an interaction between primed neutrophils and endothelium via b2 integrins; and, finally, (iv) activation of endothelial cells. What in vivo data substantiate these prerequisites for endothelial injury, extracted from in vitro data? These prerequisites are represented schematically in Figure 2. Presence of ANCA, their IgG subclasses and epitope specificities In patients with idiopathic small vessel vasculitides, the prevalence of ANCA is high and these autoantibodies are in most cases directed against Pr3 and MPO ( Table 1). As discussed earlier, titres of Pr3 ANCA rise prior to a relapse of WG in most patients [6,13,14], and treatment based on changes in these ANCA titres prevented the development of relapses in patients with WG [17]. However, not all patients are ANCA positive during active disease, and high levels of ANCA can be observed in patients without disease activity [68], indicating that other mechanisms independent of ANCA may lead to (pauci-immune) vasculitis or glomerulonephritis. These ANCA-dependent and -independent mechanisms, which will be discussed in the following section, may influence, both quantitatively and qualitatively, the mechanisms involved in disease (re)activation. Differences in neutrophil-activating capacity have been demonstrated for different IgG subclasses of ANCA. ANCA antibodies are usually of the IgG isotype, although ANCA of other isotypes have been described as well [69]. As discussed in the previous section, the increase in neutrophil-activating capacity

No Respiratory burst Phagocytosis [42,43]

of serum IgG fractions from remission to relapse in patients with WG correlated with increases in levels of IgG3 subclass ANCA and not with those of the other subclasses [32]. These in vitro data suggest a specific role for the IgG3 subclass of ANCA. Does in vivo evidence exist to support such a role? Jayne and co-workers demonstrated an over-representation of the IgG3 subclass during active disease in patients with ANCA-associated vasculitis, whereas during remission IgG3 levels decreased [70]. In addition, Brouwer et al. showed that the presence of the IgG3 subclass of ANCA is associated with renal involvement [33]. Furthermore, preliminary data from our group suggest that increases of IgG3 anti-Pr3 levels in combination with a rise in Pr3 ANCA titre is a better marker to predict relapses than a rise in Pr3 ANCA titre alone [71]. The importance of the IgG3 subclass of ANCA in in vitro neutrophil activation and in vivo disease activity, together with the high affinity of the second Fcc receptor for IgG3, suggests a significant role for IgG3 and FccRIIa in the pathophysiology of ANCAassociated vasculitides. Besides their capacity to activate primed neutrophils, Pr3 ANCA from patients with WG can interfere with Pr3 proteolytic activity as well as with the binding of a1-AT to Pr3 [72–74]. Since Pr3 ANCA interferes with the complexation of a1-AT and Pr3, Pr3 is not fully inactivated, leaving active Pr3 in the circulation [75]. Furthermore, since there are fewer Pr3–a1-AT complexes, less Pr3 is cleared [24]. Data from a relatively small group of patients show that the above-mentioned functional characteristics of Pr3 ANCA have a stronger correlation with disease activity than the ANCA titre alone [74]. Those and other data suggest that differences in the pathogenic potential of ANCA are related to differences in epitope specificity of these antibodies. However, little is known about the epitopes of Pr3 that are recognized by Pr3 ANCA. The majority of Pr3 ANCA sera recognize conformational epitopes, which makes epitope mapping of Pr3 ANCA difficult [76 ]. Some groups have tried to elucidate the epitopes recognized by Pr3 ANCA using linear peptides. In 1994, Williams et al. [77] identified various antigenic sites on Pr3 which were surface accessible. Background binding was, however, high, and some of the peptides also bound control antibodies. Using the same test

1370

system, Chang et al. could not reproduce these results [78]. Recently, Griffith et al. found that Pr3 ANCA from different patients with active vasculitis bound to linear peptides of Pr3 in a highly restricted manner [79]. Peptides were surface accessible and one peptide even coincided with the catalytic site. However, these peptides did not correspond to those identified by Williams [79]. Furthermore, Griffith found, in one patient, epitope spreading, which occurred following remission, and which may have led to a change in the pathogenicity of ANCA (personal communication). Therefore, anti-Pr3 antibodies from patients with WG may recognize a restricted number of epitopes, some of which may relate to functional characteristics and disease activity. Recent observations suggest that sera from patients with anti-MPO-associated vasculitis also recognize a restricted number of epitopes on MPO [80,81,83]. In one patient, an epitope shift was seen between sequential relapses [82]. In addition, a similar interference to that seen for Pr3 ANCA and a1-AT has been proposed for MPO ANCA and the complexation of MPO with ceruloplasmin, the possible physiological inhibitor of MPO [84,85]. These data may indicate that a change in epitope specificity of Pr3 ANCA and/or MPO ANCA may lead to a change in the pathogenicity of ANCA in ANCA-associated vasculitides. The specificity of the antigen recognized by ANCA, i.e. proteinase 3 or MPO, has clinical relevance, since differences in clinical manifestations between patients with anti-Pr3 antibodies and patients with anti-MPO antibodies have been reported [86,87]. Patients with NCGN and anti-MPO antibodies have more chronic lesions in renal biopsies and a slower decline in renal function than patients with NCGN and anti-Pr3 antibodies, who have more active lesions and a much faster decline in renal function [86 ]. Only recently could these results be linked to in vitro differences in neutrophil-activating capacity between these antibody specificities. By using IgG preparations from anti-Pr3 and anti-MPO-positive patients, Franssen et al. [88] demonstrated that anti-Pr3 antibodies had a greater capacity to activate neutrophils in vitro than anti-MPO antibodies, as demonstrated by the amount of oxygen radicals produced and the amount of b-glucuronidase degranulated, thus probably explaining some of the observed clinical differences. Priming and ANCA antigen expression The second prerequisite for endothelial damage is priming of leukocytes. Leukocyte priming is induced by low concentrations of inflammatory mediators which in vivo can be released when processes such as infection or tissue damage occur. Several clinical observations suggest an important role for infections in the induction of the disease and/or disease re-activation [89]. Firstly, an increased incidence of ANCA-associated vasculitides has been noted during winter months [90]. Secondly, we demonstrated that chronic nasal carriage of Staphyloccocus aureus is an important risk


factor for the occurrence of relapses in WG [12] and thirdly, anti-microbial treatment reduces the incidence of relapses [91]. These data suggest that infections may be the origin of proinflammatory cytokines that prime circulating neutrophils. This state of priming, or pre-activation, is in vitro associated with the membrane expression of Pr3 and MPO [21]. We and others demonstrated that in patients with WG, circulating neutrophils and monocytes express Pr3 and MPO [21,92]. Furthermore, Pr3 expression on circulating neutrophils correlated with disease activity [92], which may indicate that the extent of antigen expression on leukocytes plays an important pathophysiological role in the disease process in patients with WG. Previously, Halbwachs-Mecarelli [93] demonstrated in vivo a bimodal distribution of Pr3 surface expression on neutrophils from healthy individuals. Furthermore, we demonstrated that this bimodal distribution occurs more frequently in patients with WG than in healthy controls [92]. It is, however, unclear whether or not this phenomenon results from a genetic background. Neutrophil and monocyte activation in vivo Activated endothelial cells express increased levels of adhesion molecules, facilitating the adherence of leukocytes and the subsequent activation of these cells by ANCA. These activated adherent leukocytes can be detected in biopsies from patients with ANCA-associated vasculitides (Figure 1) by demonstrating the production of hydrogen peroxide by these cells [94]. Interestingly, these fully activated, oxygen radicalproducing neutrophils adhered to endothelial cells, confirming the in vitro findings of Reumeaux et al. that adherence to a surface is a prerequisite for ANCAinduced neutrophil activation [36 ]. Thus, locally activated neutrophils are present at the endothelial monolayer, whereas in normal situations these cells, once activated, transmigrate into the underlying tissue, leaving the endothelial monolayer undamaged. In ANCAassociated vasculitides, neutrophils are stimulated by ANCA at the endothelial lining, where these cells release toxic products, which subsequently injure endothelial cells. Importantly, the presence of oxygen radical-producing neutrophils correlated with the extent of impairment of renal function, indicating their role in the destructive process [94]. Are these neutrophils already activated in the circulation? Haller and co-workers found increased expression of CD11b on circulating neutrophils in patients with active WG [95]. Their results suggest that these neutrophils may have already been intravascularly activated. Using a non-activating whole blood method, we could not confirm these findings, since we could not find increased expression of adhesion molecules in patients with active vasculitis, in contrast to patients with sepsis [96 ]. We concluded that during active disease, patients with vasculitis have circulating neutrophils that are primed intravascularly but not fully activated. These in vivo findings once again correspond to the hypothesis


1371

Fig. 1. Activated neutrophils, as demonstrated by precipitation of diamiobenzidine (DAB) (black staining) by endogenously produced H O in neutrophils in renal biopsies from patients with Wegener’s granulomatosis. A venule within the interstitium is shown with H O 2 2 2 2 producing neutrophils. Note the close association between these activated neutrophils and the endothelial lining.

that activation of primed neutrophils by ANCA only occurs when these cells adhere to a surface. As discussed above, ANCA are also capable of inducing monocyte activation. Monocytes/macrophages play a pivotal role in lesion development in WG. An important histopathological feature in patients with WG is granulomatous inflammation. Granuloma formation is thought to result from activated monocytes/macrophages. Since activated monocytes/macrophages are present in renal biopsies [97] and since these cells participate in granuloma formation by synthesizing and secreting a variety of chemoattractants, growth factors and cytokines [98], we recently evaluated in vivo monocyte activation in patients with WG [99]. We found that plasma levels of neopterin as well as surface expression of CD64 on circulating monocytes were increased in patients with active disease compared with healthy controls. In addition, levels of neopterin and IL-6, both released by activated monocytes/macrophages, and the surface expression of CD63 on monocytes correlated with disease activity. Thus, like neutrophils, circulating monocytes in vivo express Pr3 and MPO [92] and may be fully activated by ANCA, once they adhere to the endothelial surface. These activated monocytes may eventually participate in granuloma formation in patients with WG. Endothelial cell activation and adhesion molecule expression The fourth prerequisite for endothelial cell damage is endothelial cell activation. Activated endothelial cells express increased levels of endothelial adhesion molecules, which facilitate leukocyte adherence and transmigration to underlying tissues. In biopsies from patients

with ANCA-associated vasculitis, endothelial cells from different sites demonstrate an up-regulation of VCAM-1 and ICAM-1, which suggests (glomerular) endothelial cell activation [97,100–102]. Endothelial cell adhesion molecules also occur in soluble form in plasma, due to enzymatic cleavage of the molecule from the membrane or due to alternative splicing [103]. In patients with ANCA-associated vasculitis, levels of these soluble adhesion molecules are increased during active disease and correlate with disease activity [102,104,105].

Conclusion Based on the data presented, we propose the following hypothesis. Patients with ANCA-associated vasculitis frequently suffer from infections (see also Figure 2, no. 1). As a result of these infections, proinflammatory cytokines, such as TNFa or IL-1 are released, which prime neutrophils and monocytes ( Figure 2, no. 2). These circulating leukocytes express the target antigens for ANCA, Pr3 and MPO. Once these cells roll over the endothelium, they can bind via their b2 integins to locally activated endothelium, where they can be activated by ANCA ( Figure 2, no. 3). Upon stimulation by ANCA at the endothelial surface, these activated neutrophils and monocytes release toxic products such as ROS and lytic enzymes, including Pr3 and MPO ( Figure 2, no. 3). Pr3 itself may cause endothelial cell injury, as manifested by endothelial cell lysis, detachment from basement membrane or even apoptosis. In addition, degranulated Pr3 and MPO may bind to the endothelial monolayer and serve as planted antigens, resulting in in situ immune complex formation ( Figure 2, no. 4). These in situ immune complexes may

1372


Fig. 2. Schematic representation of the immune mechanisms supposedly involved in the pathophysiology of ANCA-associated vasculitides. (1) Cytokines released due to ( local ) infection cause up-regulation of adhesion molecules on the endothelium and priming of neutrophils and/or monocytes. (2) Circulating primed neutrophils and/or monocytes express the ANCA antigens on the cell surface. (3) Adherence of primed neutrophils and/or monocytes to the endothelium, followed by activation of these cells by ANCA. Activated neutrophils and/or monocytes release reactive oxygen species (ROS) and lysosomal enzymes, which leads to endothelial cell injury and eventually to necrotizing inflammation. (4) Degranulation of proteinase 3 by these ANCA-activated neutrophils and/or monocytes results in endothelial cell activation, endothelial cell injury or even endothelial cell apoptosis. Furthermore, bound Pr3 and MPO serve as planted antigens, resulting in in situ immune complexes, which in turn attract other neutrophils. (5) ANCA-induced monocyte activation leads to production of monocyte chemoattractant protein-1 (MCP-1) and interleukin 8 (IL-8) production by these cells. The release of these chemoattractants by these cells amplifies monocyte and neutrophil recruitment possibly leading to granuloma formation (6). (A)–(D) represent the four prerequisites for endothelial cell damage by ANCA: (A) the presence of ANCA; (B) expression of the target antigens for ANCA on primed neutrophils and monocytes; (C ) the necessity of an interaction between primed neutrophils and endothelium via b2 integrins; and, finally, (D) activation of endothelial cells.

bind to and subsequently activate the complement cascade, but will eventually disappear, probably due to degradation by proteolytic enzymes, such as Pr3 and elastase. The in situ immune complexes also attract other neutrophils, which may then become activated, resulting in further endothelial cell activation, endothelial cell injury and, eventually, vascular damage and glomerulonephritis. Presumably attracted by tissue

damage, activated neutrophils and monocytes transmigrate through the endothelial lining towards the inflammatory focus (Figure 2, no. 5). Once there, these activated cells contribute to the production of monocyte chemoattractant protein-1 (MCP-1), IL-8 and other chemoattractants and cytokines ( Figure 2, no. 6). These locally produced inflammatory mediators attract other inflammatory cells, and at the same time


activate nearby endothelial cells. The inflammatory response is intensified and will eventually lead to chronic granulomatous inflammation. Although we are now beginning to understand the pathophysiological mechanisms involved in ANCA-associated vasculitides, and pauci-immune NCGN in particular, more experimental work in combination with clinical observations is needed to substantiate this hypothesis further.

References 1. Couser WG. Rapidly progressive glomerulonephritis: classification, pathogenetic mechanism and therapy. Am J Kidney Dis 1988; 11: 449–464 2. Harris AA, Falk RJ, Jennette JC. Crescentic glomerulonephritis with a paucity of glomerular immunoglobulin localisation: renal biopsy teaching case. Am J Kidney Dis 1998; 32: 179–184 3. Kallenberg CGM, Brouwer E, Weening JJ, Cohen Tervaert JW. Anti-neutrophil cytoplasmic antibodies: current diagnostic and pathophysiological potential. Kidney Int 1994; 46: 1–15 4. Jennette JC, Falk RJ. Small vessel vasculitis. N Engl J Med 1997; 337: 1512–1523 5. Cohen Tervaert JW, Mulder L, Stegeman C et al. Occurrence of autoantibodies to human leucocyte elastase in Wegener’s granulomatosis and other inflammatory disorders. Ann Rheum Dis 1993; 52: 115–120 6. Cohen Tervaert JW, van der Woude FJ, Fauci AS et al. Association between active Wegener’s granulomatosis and antineutrophil cytoplasmic antibodies. Arch Intern Med 1989; 149: 2461–2465 7. Falk RJ, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxydase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988; 318: 1651–1657 8. Cohen Tervaert JW, Goldschmeding R, Elema JD et al. Association of autoantibodies to myeloperoxidase with different forms of vasculitis. Arthritis Rheum 1990; 33: 1264–1272 9. Cohen Tervaert JW, Limburg PC, Elema JD et al. Detection of autoantibodies against myeloid lysosomal enzymes: a useful adjunct to classification of patients with biopsy-proven necrotizing arteritis. Am J Med 1991; 91: 59–66 10. Guillevin L, Visser H, Noel LH et al. Antineutrophil cytoplasm antibodies in systemic polyarteritis nodosa with and without hepatitis B virus infection and Churg–Strauss syndrome—62 patients. J Rheumatol 1993; 20: 1345–1349 11. Gaskin G, Savage CO, Ryan JJ et al. Anti-neutrophil cytoplasmic antibodies and disease activity during long-term followup of 70 patients with systemic vasculitis. Nephrol Dial Transplant 1991; 6: 689–694 12. Stegeman CA, Cohen Tervaert JW, Sluiter WJ, Manson WL, de Jong PE, Kallenberg CGM. Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener granulomatosis. Ann Intern Med 1994; 120: 12–17 13. Jayne DR, Gaskin G, Pusey CD, Lockwood CM. ANCA and predicting relapse in systemic vasculitis. Q J Med 1995; 88: 127–133 14. Egner W, Chapel HM. Titration of antibodies against neutrophil cytoplasmic antigens is useful in monitoring disease activity in systemic vasculitides. Clin Exp Immunol 1990; 82: 244–249 15. Kerr GS, Fleisher TA, Hallahan CW, Leavitt RY, Fauci AS, Hoffman GS. Limited prognostic value of changes in antineutrophil cytoplasmic antibody titer in patients with Wegener’s granulomatosis. Arthritis Rheum 1993; 36: 365–371 16. Hoffman GS, Kerr GS. Rise in ANCA titer: to treat or not to treat [ letter]. Am J Med 1995; 98: 102–103 17. 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: 709–711 18. Heeringa P, Brouwer E, Cohen Tervaert JW, Weening JJ, Kallenberg CGM. Animal models of anti-neutrophil cyto-

1373

19.

20. 21.

22.

23. 24.

25.

26.

27. 28.

29. 30. 31. 32.

33.

34.

35.

36.

37.

plasmic antibody associated vasculitis. Kidney Int 1998; 53: 253–263 Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA 1990; 87: 4115–4119 Charles LA, Caldas ML, Falk RJ, Terrell RS, Jennette JC. Antibodies against granule proteins activate neutrophils in vitro. J Leukocyte Biol 1991; 50: 539–546 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: 244–250 Ralston DR, Marsh CB, Lowe MP, Wewers MD. Antineutrophil cytoplasmic antibodies induce monocyte IL-8 release. Role of surface proteinase 3, alphal-antitrypsin, and Fcgamma receptors. J Clin Invest 1997; 100: 1416–1424 Hallett MB, Lloyds D. Neutrophil priming: the cellular signals that say ‘amber’ but not ‘green’. Immunol Today 1995; 16: 264–268 Perlmutter DH, Joslin G, Nelson P, Schasteen C, Adams SP, Fallon RJ. Endocytosis and degradation of alpha-1 antitrypsin–protease complexes is mediated by the serpin–enzyme complex receptor. J Biol Chem 1990; 265: 16713–16716 Johansson MW, Patarroyo M, Oberg F, Siegbahn A, Nilsson K. Myeloperoxidase mediates cell adhesion via the alpha M/beta 2 integrin (Mac-1, CD11b, CD18). J Cell Sci 1997; 110: 1133–1139 Zimmerman F, Andrassy K, Hansch M. Surface expression of polymorphonuclear neutrophil (PMN ) elastase: beta 2 integrins may function as binding site. Clin Exp Immunol 1998; 112 [Suppl 1]: 40 (abstract) Campanelli D, Melchior M, Fu Y et al. Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils. J Exp Med 1990; 172: 1709–1715 Gilligan HM, Bredy B, Brady HR et al. Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apototic neutrophils in the apsence of neutrophil priming. J Exp Med 1996; 184: 2231–2241 Kettritz R, Jennette JC, Falk RJ. Crosslinking of ANCA antigens stimulates superoxide release by human neutrophils. J Am Soc Nephrol 1997; 8: 386–394 van de Winkel JGJ, Capel PJA. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol Today 1993; 14: 215–221 van de Winkel JGJ, Capel JA. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol Today 1993; 14: 215–221 Mulder AH, Stegeman CA, Kallenberg CGM. Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA) in Wegener’s granulomatosis: a predominant role for the IgG3 subclass of ANCA. Clin Exp Immunol 1995; 101: 227–232 Brouwer E, Cohen Tervaert JW, Horst G et al. Predominance of IgG1 and IgG4 subclasses of anti-neutrophil cytoplasmic autoantibodies (ANCA) in patients with Wegener’s granulomatosis and clinically related disorders. Clin Exp Immunol 1991; 83: 379–386 Mulder AH, Heeringa P, Brouwer E, Limburg PC, Kallenberg CGM. Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA): a Fc gamma RII-dependent process. Clin Exp Immunol 1994; 98: 270–278 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: 1271–1280 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: 3189–3195 Kurlander RJ. Blockade of Fc receptor mediated binding to U-937 cells by murine monoclonal antibodies directed against a variety of surface antigens. J Immunol 1983; 131: 140–145

1374 38. Radford DJ, Lord JM, Savage COS. ANCA activation of human neutrophils induces tyrosine phosphorylation and translocation of protein kinase C. Clin Exp Immunol 1998; 112: 39 (Abstract) 39. Radford DJ, Ben-Smith A, Savage COS. Further determination of the signaling pathways involved in the ANCA activated respiratory burst. Clin Exp Immunol 1998; 112: 39 (Abstract) 40. Tse WY, Radford DJ, Nash GB, Savage COS. Signal pathways of ANCA-induced actin reorganisation in neutrophils. Clin Exp Immunol 1998; 112: 39 (Abstract) 41. Daeron M. Fc receptor biology. Annu Rev Immunol 1997; 15: 203–234 42. Downey GP, Fukushima T, Fialkow L, Waddell TK. Intracellular signaling in neutrophil priming and activation. Semin Cell Biol 1995; 6: 345–356 43. Berton G, Yan SR, Fumagalli L, Lowell CA. Neutrophil activation by adhesion: mechanisms and pathophysiological implications. Int J Clin Lab Res 1996; 26: 160–177 44. Joslin G, Griffin GL, August AM et al. The serpin–enzyme complex (SEC ) receptor mediates the neutrophil chemotactic effect of alpha-1 antitrypsin–elastase complexes and amyloidbeta peptide. J Clin Invest 1992; 90: 1150–1154 45. Takenouchi T, Munekata E. Beta amyloid peptide, substance P, and SEC receptor ligand activate cytoplasmic Ca2+ in neutrophil-like HL-60 cells: effect of chemotactic peptide antagonist BocMLF. Peptides 1995; 16: 1019–1024 46. Lub M, van Kooyk Y, Figdor CG. Ins and outs of LFA-1. Immunol Today 1995; 16: 479–483 47. Grimminger F, Hattar K, Papavassilis C, Temmesfeld B, Csernok E, Gross WL. Neutrophil activation by anti-proteinase 3 antibodies in Wegener’s granulomatosis: role of arachidonic acid and leukotriene B4 generation. J Exp Med 1996; 184: 1567–1572 48. Brooks CJ, King WJ, Radford DJ, Adu D, McGrath M, Savage COS. IL-1 beta production by human polymorphonuclear leukocytes stimulated by anti-neutrophil cytoplasmic autoantibodies: relevance to systemic vasculitis. Clin Exp Immunol 1996; 106: 273–279 49. Casselman BL, Kilgore KS, Miller BF, Warren JS. Antibodies to neutrophil cytoplasmic antigens induce monocyte chemoattractant protein-1 secretion from human monocytes. J Lab Clin Med 1995; 126: 495–502 50. Johnson PA, Alexander HD, McMillan SA, Maxwell AP. Up-regulation of the granulocyte adhesion molecule Mac-1 by autoantibodies in autoimmune vasculitis. Clin Exp Immunol 1997; 107: 513–519 51. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989; 320: 365–376 52. Ewert BH, Jennette JC, Falk RJ. Anti-myeloperoxidase antibodies stimulate neutrophils to damage human endothelial cells. Kidney Int 1992; 41: 375–383 53. 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: 335–342 54. Mayet WJ, Schwarting A, Meyer zum Buschenfelde KH. Cytotoxic effects of antibodies to proteinase 3 (C-ANCA) on human endothelial cells. Clin Exp Immunol 1994; 97: 458–465 55. Savage CO, Gaskin G, Pusey CD, Pearson JD. Anti-neutrophil cytoplasm antibodies can recognize vascular endothelial cellbound anti-neutrophil cytoplasm antibody-associated autoantigens. Exp Nephrol 1993; 1: 190–195 56. Ballieux BE, Zondervan KT, Kievit P et al. Binding of proteinase 3 and myeloperoxidase to endothelial cells: ANCAmediated endothelial damage through ADCC? Clin Exp Immunol 1994; 97: 52–60 57. Berger SP, Seelen MA, Hiemstra PS et al. Proteinase 3, the major autoantigen of Wegener’s granulomatosis, enhances IL-8 production by endothelial cells in vitro. J Am Soc Nephrol 1996; 7: 694–701 58. Yang JJ, Kettritz R, Falk RJ, Jennette JC, Gaibo ML. Apoptosis of endothelial cells induced by neutrophil serine proteases proteinase 3 and elastase. Am J Pathol 1996; 149: 1617–1626

A. C. Muller Kobold et al. 59. Ballieux BE, Hiemstra PS, Klar Mohamad N et al. Detachment and cytolysis of human endothelial cells by proteinase 3. Eur J Immunol 1994; 24: 3211–3215 60. Mayet WJ, Meyer zum Buschenfelde KH. Antibodies to proteinase 3 increase adhesion of neutrophils to human endothelial cells. Clin Exp Immunol 1993; 94: 440–446 61. 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: 259–267 62. Johnson PA, Alexander HD, McMillan SA, Maxwell AP. Up-regulation of the endothelial cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) by autoantibodies in autoimmune vasculitis. Clin Exp Immunol 1997; 108: 234–242 63. Sibelius U, Hattar K, Schenkel A et al. Wegener’s granulomatosis: anti-proteinase 3 antibodies are potent inducters of human endothelial cell signaling and leakage response. J Exp Med 1998; 187: 497–503 64. De Bandt M, Ollivier V, Meyer O et al. Induction of interleukin 1 and subsequent tissue factor expression by anti-proteinase 3 antibodies in human umbilical vein endothelial cells. Arthritis Rheum 1997; 40: 2030–2038 65. Mayet WJ, Csernok E, Szymkowiak C, Gross WL, Meyer zum Buschenfelde KH. Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener’s granulomatosis. Blood 1993; 82: 1221–1229 66. King WJ, Adu D, Daha MR et al. Endothelial cells and renal epithelial cells do not express the Wegener’s autoantigen, proteinase 3. Clin Exp Immunol 1995; 102: 98–105 67. Pendergraft WF, Yang JJ, Tuttle R et al. Myeloperoxydase (MPO) and proteinase 3 (Pr3) are not expressed by endothelial cells. Clin Exp Immunol 1998; 112 [Suppl ]: 19 (Abstract) 68. Cohen Tervaert JW. The value of serial ANCA testing during follow-up studies in patients with ANCA associated vasculitides. A review. J Nephrol 1996; 9: 232–240 69. Kallenberg CGM, Mulder AH, Cohen Tervaert JW. Antineutrophil cytoplasmic antibodies: a still-growing class of autoantibodies in inflammatory disorders. Am J Med 1992; 93: 675–682 70. Jayne DR, Weetman AP, Lockwood CM. IgG subclass distribution of autoantibodies to neutrophil cytoplasmic antigens in systemic vasculitis. Clin Exp Immunol 1991; 84: 476–481 71. Cohen Tervaert JW, Mulder AHL, Kallenberg CGM, Stegeman CA. Measurement of IgG3 levels of anti-proteinase 3 antibodies is useful in monitoring disease activity in Wegener’s granulomatosis. J Am Soc Nephrol 1994; 5: 828 (Abstract) 72. van de Wiel BA, Dolman KM, van der Meer Gerritsen CH, Hack CE, von dem Borne AE, Goldschmeding R. Interference of Wegener’s granulomatosis autoantibodies with neutrophil proteinase 3 activity. Clin Exp Immunol 1992; 90: 409–414 73. 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: 1528–1536 74. Dolman KM, Stegeman CA, van de Wiel BA et al. Relevance of classic anti-neutrophil cytoplasmic autoantibody (C-ANCA)-mediated inhibition of proteinase 3–alpha 1-antitrypsin complexation to disease activity in Wegener’s granulomatosis. Clin Exp Immunol 1993; 93: 405–410 75. Henshaw TJ, Malone CC, Gabay JE, Williams RC, Jr. Elevations of neutrophil proteinase 3 in serum of patients with Wegener’s granulomatosis and polyarteritis nodosa. Arthritis Rheum 1994; 37: 104–112 76. Bini P, Gabay JE, Teitel A, Melchior M, Zhou JL, Elkon KB. Antineutrophil cytoplasmic autoantibodies in Wegener’s granulomatosis recognize conformational epitope(s) on proteinase 3. J Immunol 1992; 149: 1409–1415 77. Williams RC, Jr., Staud R, Malone CC, Payabyab J, Byres L, Underwood D. Epitopes on proteinase-3 recognized by antibodies from patients with Wegener’s granulomatosis. J Immunol 1994; 152: 4722–4737 78. Chang L, Binos S, Savige J. Epitope mapping of anti-proteinase 3 and anti-myeloperoxidase antibodies. Clin Exp Immunol 1995; 102: 112–119 79. Griffith ME, Coulthart A, Pemberton S, Pusey CD. c-ANCA


80. 81. 82. 83.

84. 85.

86. 87. 88.

89. 90.

91.

92.

recognise a restricted number of linear epitopes of proteinase 3 which involve the catalytic site. Sarcoidosis Vasc Diff Lung Dis 1996; 13: 256 (Abstract) Audrain MA, Baranger TA, Moguilevski N et al. Anti-native and recombinant myeloperoxidase monoclonals and human autoantibodies. Clin Exp Immunol 1997; 107: 127–134 Short AK, Lockwood CM. Studies of epitope restriction on myeloperoxidase (MPO), an important antigen in systemic vasculitis. Clin Exp Immunol 1997; 110: 270–276 Chapman PT, Short AK, Lockwood CM. The use of Biosensor technology in epitope mapping studies on human myeloperoxidase. The Immunologist 1997; (Abstract) Tomizawa K, Mine E, Fujii A et al. A panel set for epitope analysis of myeloperoxidase (MPO)-specific antineutrophil cytoplasmic antibody MPO-ANCA using recombinant hexamer histidine-tagged MPO deletion mutants. J Clin Immunol 1998; 18: 142–152 Segelmark M, Persson B, Hellmark T, Wieslander J. Binding and inhibition of myeloperoxidase (MPO): a major function of ceruloplasmin? Clin Exp Immunol 1997; 108: 167–174 Griffin SV, Lockwood CM. Different potential of antimyeloperoxidase (MPO) antibodies from patients with polyangiitis compared to Wegener’s granulomatosis to interfere with the inhibition of MPO by caeroloplasmin. Clin Exp Immunol 1998; 112: 54 (Abstract) Franssen CF, Gans RO, Arends B et al. Differences between anti-myeloperoxidase- and anti-proteinase 3-associated renal disease. Kidney Int 1995; 47: 193–199 Cohen Tervaert JW, Elema JD, Kallenberg CGM. Clinical and histopathological association of 29kD-ANCA and MPOANCA. APMIS Suppl. 1990; 19: 35 Franssen CF, Huitema MG, Muller Kobold AC et al. In vitro neutrophil activation by antibodies to proteinase 3 and myeloperoxydase from patients with crescentic glomerulonephritis. Kidney Int 1998; in press Pinching AJ, Rees AJ, Pussell BA, Lockwood CM, Mitchison RS, Peters DK. Relapses in Wegener’s granulomatosis: the role of infection. Br Med J 1980; 281: 836–838 Falk RJ, Hogan S, Carey TS, Jennette JC. Clinical course of anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and systemic vasculitis. The Glomerular Disease Collaborative Network. Ann Intern Med 1990; 113: 656–663 Stegeman CA, Cohen Tervaert JW, de Jong PE, Kallenberg CGM. Trimethoprim-sulfamethoxazole (co-trimoxazole) for the prevention of relapses of Wegener’s granulomatosis. Dutch Co-Trimoxazole Wegener Study Group. N Engl J Med 1996; 335: 16–20 Muller Kobold AC, Kallenberg CGM, Cohen Tervaert JW. Leukocyte membrane expression of proteinase 3 correlates with disease activity in patients with Wegener’s granulomatosis. Br J Rheumatol 1998; 37: 901–907

1375 93. Halbwachs Mecarelli L, Bessou G, Lesavre P, Lopez S, Witko Sarsat V. Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool. FEBS Lett 1995; 374: 29–33 94. Brouwer E, Huitema MG, Mulder AH et al. Neutrophil activation in vitro and in vivo in Wegener’s granulomatosis. Kidney Int 1994; 45: 1120–1131 95. Haller H, Eichhorn J, Pieper K, Goebel U, Luft FC. Circulating leukocyte integrin expression in Wegener’s granulomatosis. J Am Soc Nephrol 1996; 7: 40–48 96. Muller Kobold AC, Mesander G, Stegeman CA, Kallenberg CGM, Cohen Tervaert JW. Are circulating neutrophils intravascularly activated in patients with anti-neutrophil cytoplasmic antibody associated vasculitis? Clin Exp Immunol 1998; 114: 491–499 97. Rastaldi MP, Ferrario F, Tunesi S, Yang L, D’Amico G. Intraglomerular and interstitial leukocyte infiltration, adhesion molecules, and interleukin-1a expression in 15 cases of antineutrophil cytoplasmic autoantibody-associated renal vasculitis. Am J Kidney Dis 1996; 27: 48–57 98. Pike PC. The role of the monocyte and macrophage in the pathogenisis of vasculitis. In: LeRoy EC, ed. Systemic Vasculitis. The Biological Basis. Marcel Dekker, New York; 1992: 129–147 99. Muller Kobold AC, Kallenberg CGM, Cohen Tervaert JW. Monocyte activation in patients with Wegener’s granulomatosis. Ann Rheum Dis 1998; in press 100. Bruyn JA, Dinklo NJCM. Distinct patterns of expression of intracellular adhesion molecule-1, vascular adhesion molecule-1 and endothelial–leukocyte adhesion molecule-1 in renal disease. Lab Invest 1993; 69: 329–335 101. Brouwer E, Huitema MG, Heeringa P, Cohen Tervaert JW, Weening JJ, Kallenberg CGM. Upregulation of adhesion molecules in renal biopsies from patients with Wegener’s granulomatosis. Clin Exp Immunol 1993; 93 [Suppl 1]: 22 (Abstract) 102. Cohen Tervaert JW, Kallenberg CGM. Cell adhesion molecules in vasculitis. Curr Opin Rheumatol 1997; 9: 16–25 103. Gearing AJH, Hemingway I, Pigott R, Hughes J, Rees AJ, Cashman SJ. Soluble forms of vascular adhesion molecules, E-selectin, ICAM-1, and VCAM-1: pathological significance. Ann NY Acad Sci 1992; 667: 324–331 104. Stegeman CA, Cohen Tervaert JW, Huitema MG, de Jong PE, Kallenberg CGM. Serum levels of soluble adhesion molecules intercellular adhesion molecule 1, vascular adhesion molecule 1, and E-selectin in patients with Wegener’s granulomatosis. Arthritis Rheum 1994; 37: 1228–1235 105. Wang CR, Liu MF, Tsai RT, Chuang CY, Chen CY. Circulating intercellular adhesion molecules-1 and autoantibodies including anti-endothelial cell, anti-cardiolipin, and antineutrophil cytoplasma antibodies in patients with vasculitis. Clin Rheumatol 1993; 12: 375–380