Department of Rheumatology, Ipswich Hospital NHS Trust and School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, 1 Department of Rheumatology, Norfolk and Norwich University Hospital NHS Trust and School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, 2 Renal Section, Imperial College London, Hammersmith Hospitals NHS Trust, London, 3 Division of Immunity and Infection, Medical School, University of Birmingham, Birmingham and 4 Renal Medicine, Addenbrooke's Hospital, Cambridge, UK
Correspondence to: R. Watts, Department of Rheumatology, Ipswich Hospital NHS Trust, Heath Road, Ipswich IP4 5PD, UK. E-mail: Richard.watts{at}ipswichhospital.nhs.uk
Despite significant advances in treatment, the systemic vasculitides remain something of an enigma as the aetiology remains obscure. Five years ago a meeting VasculitisAims of Therapy was held in Cambridge to discuss the then state of the art in the management of vasculitis [1]. Since then there have been major advances in their treatment with the completion of the first wave of European Vasculitis Study Group (EUVAS) series of trials (NORAM, CYCAZREM and MEPEX) (see Table 1 for a summary). The results of these trials now provide a firm evidence base for the first line treatment of most patients presenting with systemic vasculitis. A major triumph has been the transformation of these diseases from conditions with an appalling prognosis to chronic relapsing and remitting diseases [2, 3].
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The CYCAZAREM trial compared azathioprine (AZA) and CYC for maintenance of remission in patients with moderate renal involvement (creatinine <500 µmol/l) [5]. One hundred and fifty-five patients with AAV were studied. Oral CYC (2 mg/kg/day) and prednisolone (1 mg/kg/day tapering to 0.25 mg/kg/day by 12 weeks) resulted in remission in 144 patients. One hundred and nineteen (77%) achieved remission by 3 months. There were seven deaths during the induction phase and one withdrawal. After remission induction at 36 months, patients were randomized to either continue CYC (1.5 mg/kg/day) for 12 months or switch to AZA (2 mg/kg/day), with the same prednisolone dose (10 mg/day). At 12 months both groups received AZA (1.5 mg/kg/day) and prednisolone (7.5 mg/day). There was no difference in relapse rates (15.5% in the AZA group and 13.7% in the CYC group) [P = 0.65; 95% confidence interval (CI) 9.9 to +13.0%] up to the end of the study at 18 months after treatment outset. This suggests that CYC can be safely withdrawn following induction of remission.
The MEPEX trial compared plasma exchange and pulsed methylprednisolone as adjunctive therapy in biopsy-proven AAV with acute renal failure (creatinine >500 µmol/l) [abstract, 6]. One hundred and fifty-one patients received either seven plasma exchanges (60 ml/kg) or three pulses of methylprednisolone (15 mg/kg) in addition to standard therapy with cyclophosphamide and tapering prednisolone. Preliminary data suggest that renal outcome was better in the plasma exchange-treated group, when expressed as either dialysis independence in surviving patients or overall rate of dialysis-free survival. Death rates were similar in the two groups. These results were maintained at the 1-yr follow-up. Recruitment is now complete and data analysis is taking place.
The second wave of studies started about 5 yr ago and is now nearing completion. CYCLOPS was designed to address the question whether pulse CYC was as effective as and less toxic than conventional oral low-dose daily CYC. A meta-analysis of 143 patients in three randomized controlled trials of ANCA-associated vasculitis concluded that pulsed CYC was less likely to fail to induce remission than continuous oral CYC and had a significantly lower risk of infection and leucopenia [7]. Relapses occurred slightly more frequently with pulsed CYC treatment. The 11 non-randomized studies comprised 202 patients receiving pulse CYC. Pulses of CYC were given at doses of 3751000 mg/m2 per pulse at 1- to 4-week intervals with variable steroid and adjuvant therapy regimens. Remission was achieved in 112/191 evaluable patients. Relapse occurred in 68/135 patients. Leucopenia, infection, haemorrhagic cystitis and death were rare [reviewed in 7]. The regimen for intravenous CYC in CYCLOPS was designed on the basis of a consensus on the best available evidence and is not identical to the regimens reviewed above.
An analysis of four prospective trials conducted in France concluded that, in 215 patients with polyarteritis nodosa, microscopic polyangiitis or ChurgStrauss syndrome the overall survival was the same in patients receiving CYC and corticosteroids as in those that only received corticosteroids [8]. Stratification for disease severity suggested that patients with more severe disease (five-factor score 2) had prolonged survival when treated with CYC. CYC did not, however, appear to reduce the relapse rate. They concluded that CYC should be used for patients with severe disease at presentation.
Thus, we now have evidence on which to base the initial therapy for most patients presenting with AAV. Toxicity has already been reduced by the use of lower cumulative doses of CYC. The challenge now is to develop less toxic treatments for use as first-line therapies to replace CYC but also to treat those refractory or intolerant to CYC, and to reduce the toxicity of long-term maintenance therapy.
Over the past 5 yr, there have been rapid developments in the treatment of other forms of autoimmune disease and inevitably these treatments are beginning to be used in vasculitis patients. Anti-TNF- therapy, initially developed for treatment of RA and inflammatory bowel disease, has proved extremely successful and is now widely used to treat these patients. Other disease, such as ankylosing spondylitis and psoriasis, are being treated using this approach. B-cell depletion therapy with monoclonal antibodies is proving useful in many autoimmune diseases, especially RA and lupus [9, 10]. At present most reports of TNF-
blockade in vasculitis relate to case series or small uncontrolled trials. In the USA, the WGET placebo-controlled trial of etanercept in Wegener's granulomatosis reported that in 180 patients etanercept was not effective at either induction or maintenance of disease remission when used in addition to conventional therapy [11]. In the UK, infliximab has been used in active disease both at first presentation and relapse, as well in persistent disease. Remission was induced in 88% of 32 patients, but there was a significant infection rate (21%) and 20% patients relapsed [12]. Similarly, there are several promising small-scale studies looking at B cell depletion using rituximab. Antithymocyte globulin may be effective in an open trial of patients with refractory Wegener's granulomatosis; 13 of 15 patients responded with either a partial or complete response, but seven patients relapsed during a mean follow-up of 8.4 months [13].
Translational research is the process by which developments in basic science are developed to provide novel therapies in the clinic. The aetiology of AAV remains unknown. However, understanding of disease pathogenesis has improved. It has become increasingly clear that ANCA are important in disease pathogenesis. These autoantibodies recognize proteinase 3 (PR3) or myeloperoxidase (MPO) presented on the surface of cytokine-primed neutrophils or monocytes, allowing cell activation with release of reactive oxygen species, cytokines and degranulation. Activation of neutrophils requires antigen engagement via the Fab portion of the antibody with surface-expressed PR3 or MPO and ligation of Fc receptors, both Fc
RIIa and Fc
RIIIb, for full activation. ANCA signalling differs from that described for most immune complex signalling via Fc receptors. ANCA F(ab')2 fragments stimulate G-protein-coupled pathways; this alone is necessary but not sufficient to generate a respiratory burst. Fc receptor signalling activates Syk kinase, protein kinase Cß and calcium release, and both F(ab')2 and Fc engagement are required for protein kinase B activity, culminating in the generation of a respiratory burst. For full activation, engagement of ß2 integrins is also important, as neutrophils are unable to generate a respiratory burst without ß2 integrin ligation [for review see 14].
ANCA are predominantly of the immunoglobulin G (IgG) isotype. The IgG subclass of ANCA may also be important in neutrophil activation. Renal relapses of Wegener's granulomatosis are correlated with increases in the level of IgG3, although other studies suggest that pathogenicity is correlated with IgG1 and/or IgG4. IgG4 is reported to bind only FcRI receptors, which are not constitutively expressed on neutrophils. However, recent studies have suggested that isolated IgG4 with ANCA activity can activate neutrophils via Fc
RIIIB [15]. Other properties in addition to IgG subclass may also influence the biological outcome of activation of neutrophils by ANCA. Polyclonal IgG isolated from ANCA-positive patients has a high proportion of hypogalactosylated IgG. In other autoimmune diseases, such as RA, increased hypogalactosylated IgG is also present, and can activate the complement system by via mannose-binding lectin activation.
ANCA recognize conformational epitopes on MPO and PR3 [for review see 16]. Despite the apparent requirement for a tertiary structure, there are reports of PR3-ANCA binding to linear peptides and MPO-ANCA binding to the heavy chain of MPO, using recombinant deletion mutants. The epitopes recognized by ANCA are restricted, as competition studies of PR3-ANCA or MPO-ANCA with monoclonal antibodies to PR3 or MPO inhibit binding by patient ANCA. The most antigenic site of PR3 appears to be around or in the catalytic site. Biosensor technology studies suggest an immunodominant epitope common to all PR3-ANCA from patients at initial presentation, as these antibodies recognize overlapping regions on PR3. The epitopes recognized by these antibodies may change over time or depending on disease activity. However unlike in SLE, where epitope spreading may involve different autoantigens, epitope spreading in ANCA-associated vasculitis is only observed within MPO or PR3.
Immunohistochemistry studies of renal biopsies from patients have shown neutrophils adherent to the endothelium and trapped within the glomerulus. In vitro, activation of neutrophils by ANCA induces actin polymerization, with increased cell rigidity [17] and cell adhesion to TNF-primed endothelial cells potentially resulting in trapping of neutrophils within the microvasculature. Unlike the generation of a respiratory burst, actin polymerization does not depend on TNF priming and correlates with the expression of PR3 on the cell surface [18]. In static assays, ANCA activation of neutrophils results in adhesion and endothelial cell lysis. However, under flow conditions, which mimic the shear stresses present in vessels, ANCA promotes the adherence of neutrophils to primed endothelial cells and promotes transmigration. This requires ß2 integrin and the chemokine receptor CXCR2, suggesting synergy between signals from CXCR2 and those provided by ANCA to promote neutrophil activation [19]. Ongoing studies are addressing endothelial damage in this model.
ANCA may also be important in frustrating the normal anti-inflammatory pathways that promote the resolution of inflammation. As neutrophils constitutively undergo apoptosis (programmed cell death), there is increased expression of PR3 and MPO on the cell surface, allowing binding by ANCA. This does not result in activation, but these opsonized apoptotic cells show increased phagocytosis by macrophages in a proinflammatory manner, with release of IL-8 [20]. Macrophages generally phagocytose apoptotic cells in a non-phlogistic manner with release of the immune-suppressing cytokine TGF-ß.
ANCA may also frustrate the process of neutrophils becoming apoptotic. Pathologically, ANCA-associated vasculitis is characterized by leucocytoclasis, and electron microscopy studies have suggested that there may be a defect in the clearance of apoptotic neutrophils. There is evidence of leucocytes with degraded nuclear material undergoing disintegration in tissues [21], and apoptotic cells have been observed in ANCA-positive renal vasculitis [22]. In vitro, neutrophils activated by ANCA show accelerated nuclear changes of apoptosis in a manner dependent on reactive oxygen species. However, the surface changes of apoptosis that allow macrophages to recognize apoptotic cells (i.e. surface expression of phosphatidylserine) are uncoupled from the nuclear changes. This allows neutrophils to undergo secondary necrosis as they are not phagocytosed by macrophages [23]. This is interesting, as pyocyanin, a toxin released by Pseudomonas infection, accelerates neutrophil apoptosis in a manner which is also dependent on reactive oxygen species, frustrating the resolution of infection and inflammation in patients with cystic fibrosis [24].
Despite the considerable evidence that ANCA have important biological effects in vitro, demonstrating this in vivo has proved more difficult. In early studies from Brouwer et al., immunization of rats with MPO did not by itself lead to vasculitis. However, when the products of activated neutrophils, including MPO and its substrate, H2O2, were perfused into the renal artery in these animals, they developed a severe pauci-immune crescentic nephritis [25]. Subsequently, Yang et al. reported the presence of immune complexes in the glomeruli of a similar model, and suggested that the mechanism involved may therefore be different from that in the human disease [26]. In a further study, the in vivo effects of ANCA were demonstrated in rats immunized with MPO and then given a small subnephritic dose of anti-glomerular basement membrane (GBM) antibodies. These animals developed a severe crescentic nephritis, whereas controls immunized with MPO alone or given anti-GBM antibodies alone did not [27].
More recently, two new animal models have provided stronger evidence that ANCA are directly pathogenic. Xiao and colleagues immunized MPO-knockout mice with mouse MPO to achieve a strong autoimmune response. Splenocytes from these mice, when transferred to Rag2 knockout mice, led to the development of crescentic nephritis and systemic vasculitis in the recipients. Anti-MPO antibodies, purified from the serum of the immunized MPO knockout mice, were injected intravenously into both Rag2-knockout and wild-type recipients. The recipient mice developed pauci-immune focal necrotizing crescentic nephritis, demonstrating that anti-MPO antibodies alone were sufficient to cause disease [28]. The second new model, described by Pusey's group, has so far only been reported in abstracts [29, 30]. WKY rats immunized with human MPO developed a high titre of MPO antibodies, together with pauci-immune crescentic nephritis and lung haemorrhage. This model has been termed experimental autoimmune vasculitis (EAV). The use of intravital microscopy allowed in vivo observation of mesenteric vessels in these rats. Animals with EAV showed enhanced adhesion and transmigration of leucocytes in response to GRO- (CXCL-1), compared with BSA-immunized controls. Furthermore, transfer of anti-MPO antibodies from rats with EAV to naive recipients led to increased leucocyte adhesion, even without an inflammatory stimulus, and increased transmigration and microvascular haemorrhage following GRO-
. This work provides further compelling evidence for the pathogenicity of ANCA.
The developments described above highlight potential novel therapeutic targets. For example, the role of ß2 integrin and the chemokine receptor CXCR2 in promoting ANCA-associated neutrophil activation and that of ANCA in frustrating the resolution of inflammation and neutrophil apoptosis suggest that modulation of these processes might be useful therapeutic avenues to explore in the future.
It seemed appropriate with these recent developments in our understanding of the immunopathogenesis of AAV to hold a second meeting with the objective of discussing recent scientific developments and how these could be moved forward into the clinic. This meeting was held recently in Cambridge, and over the next few months we will be publishing a series of review articles arising from this symposium, which hopefully will provoke discussion and stimulate the development of new protocols to treat this fascinating group of patients.
J. Levy received support from Schering Plough for the pilot study of infliximab in vasculitis.
The other authors have declared no conflicts of interest.
References
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