Matthew David Morgan,
Lorraine Harper,
Julie Williams and
Caroline Savage
Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham, United Kingdom
Address correspondence to: Prof. Caroline Savage, Division of Immunity and Infection, The Medical School, University of Birmingham, Vincent Drive, Birmingham, B15 2TT, United Kingdom. Phone: +21-414-6841; Fax: +21-414-6794; E-mail: c.o.s.savage{at}bham.ac.uk
Wegener’s granulomatosis, microscopic polyangiitis, andrenal limited vasculitis are associated with circulating anti-neutrophilcytoplasm antibodies and are an important cause of rapidly progressiveglomerulonephritis. This review gives an account of recent advancesin the understanding of the pathogenesis underlying these conditionsand how these may lead to future treatments. Consideration isgiven to recent clinical trials in the management of anti-neutrophilcytoplasm antibodies (ANCA)-associated vasculitides.
Wegener’s granulomatosis (WG), microscopic polyangiitis(MPA), and renal limited vasculitis (RLV) are inflammatory autoimmuneconditions that commonly are associated with circulating autoantibodies.These antibodies, known as anti-neutrophil cytoplasm antibodies(ANCA), are directed against proteinase 3 (PR3) or myeloperoxidase(MPO) that is present in the primary granules of neutrophilsand monocytes. MPA and WG are systemic diseases that often affectseveral organ systems; are associated with nonspecific inflammatoryfeatures such as fever, malaise, weight loss, and anemia; andare seen most commonly in the elderly, with a peak age of onsetbetween 65 and 74 yr (1). These conditions are commonly groupedtogether as the ANCA-associated systemic vasculitides (ASV);therefore, they show common histologic features in the kidneyand ANCA are thought to contribute to pathogenesis.
Detection of ANCA has aided diagnosis, as there is a high sensitivityand specificity of these antibodies for disease. As well asbeing associated with MPA and WG, less commonly ANCA may bepresent during infection or associated with drug-induced vasculitiswhen their significance is unclear, as they are often directedagainst other neutrophil antigens.
The renal features of ASV include oliguria, microscopic hematuria,and proteinuria. This may be associated with an impaired GFR,although renal function as measured by serum creatinine maybe normal. The gold standard test for the presence of glomerulonephritisremains renal biopsy. The histopathologic features have beenreviewed in two recent studies (2,3), but the central featureis a pauci-immune focal segmental necrotizing glomerulonephritisthat may become crescentic with the accumulation of macrophagesand epithelial cells in Bowman’s space. This review concentrateson recent developments in the diagnosis, pathogenesis, and managementof glomerulonephritis associated with WG, MPA, and RLV.
Environmental and Genetic Factors in Pathogenesis of ASV
Because of the low incidence of ASV, it has been difficult toconduct good quality population-based studies to investigateenvironmental or genetic factors that may contribute to thedevelopment or clinical course of ASV. A number of studies havesuggested that a variety of environmental factors, such as farming,solvent exposure, earthquake, or a protective effect of smoking,may be linked to development of ASV, although some of thesehave been contradictory (4–6). The most consistently reportedenvironmental factor is silica exposure, although the mechanismby which this could lead to ASV is unclear (4,7,8). Drug exposurealso may precipitate ANCA-associated vasculitis, including propylthiouracil,minocycline, and penicillamine. In those with high titers ofanti-MPO ANCA, exposure to these drugs should be considered(9). However, in a prospective study of 248 patients who weretreated with penicillamine, sulfasalazine, or minocycline, nopatients seroconverted to a positive ANCA (10).
PR3 is produced by neutrophils and can be expressed on the cellsurface, where levels may be genetically determined. Membraneexpression of PR3 (mPR3) can be measured by flow cytometry,and in most studies, there is a bimodal distribution of mPR3with both high- and low-mPR3-expressing neutrophils in a givenindividual (11). Although the total amount of mPR3 can be increasedby cytokines, the distribution of high- and low-mPR3-expressingneutrophils has been shown to be stable for an individual overtime (12,13). There is evidence that individuals with an increasedpercentage of high-mPR3-expressing neutrophils are at increasedrisk for developing WG, whereas patients who already have WGare at increased risk for relapse (14,15). Neutrophils withhigh mPR3 produce more superoxide in response to PR3-ANCA thanneutrophils with low mPR3 (16). Evidence that the proportionof high and low mPR3 is genetically determined is derived fromtwin studies in which a high degree of concordance is seen inmonozygotic but not in dizygotic twins (13).
There has been interest in an association between the developmentof ASV and polymorphisms in the gene encoding -1-antitrypsin(AAT), the major inhibitor of PR3 activity. The allele thatmost commonly is associated with reduced AAT activity is designatedPiZ and is reported to be associated with an increased riskfor developing WG (17,18) or either PR3- or MPO-ANCA (19,20).Two studies have reported an increased risk for death for patientswho have ASV and carry the PiZ allele (18,20), although a lowserum level of AAT was associated with a reduced incidence ofrenal involvement in one study (20). The mechanism by whichcarriage of the PiZ allele increases the risk for death forpatients with ASV is not clear but, because it is associatedwith a reduced amount of circulating AAT/PR3 complexes, theremay be increased circulating PR3 activity, although this hasnot yet been demonstrated.
ANCA are detected using indirect immunofluorescence (IIF) ofpatient serum on ethanol-fixed neutrophils and by ELISA. Severalstudies have compared the sensitivity and the specificity ofboth tests used alone or in combination for detection of ANCA.A meta-analysis of 15 studies revealed a positive cytoplasmicANCA pattern (c-ANCA); IIF had a sensitivity for WG of 34 to92% and a specificity of 88 to 100%, although the pretest probabilityof WG was high in most of the groups tested (21). A multicenterstudy that used standardized IIF and ELISA investigated thespecificity and the sensitivity of combined IIF and ELISA inWG, MPA, and RLV. The sensitivity of the combined IIF and ELISAresults for newly diagnosed WG, MPA, and RLV are shown in Table 1.The specificity of the combined results was nearly 100% comparedwith healthy controls (22). However, when clinicians rely ondata that are derived from commercial kits, they should be awarethat such kits vary widely. One study compared the sensitivityof 11 commercial direct ELISA and found that sensitivity rangedfrom 26.7 to 86.7% and from 13.3 to 73.3% for MPO- and PR3-ANCA,respectively, compared with IIF. Sensitivity can be increasedby changing the cutoff point but leads to a reduction in specificityfor diagnosis of ASV (23). Results of ANCA testing also varybetween centers, as shown by one multicenter international studyin which the same sera were tested in different sites (24).
The presence of circulating ANCA is closely associated withASV and often, although by no means always, reflects diseaseactivity. There are, however, a number of patients who remainANCA negative despite having a diagnosis of either MPA or WG(25,26). That patients can have active vasculitis with WG orMPA in the absence of ANCA suggests that ANCA themselves maynot play a direct role, that ANCA are necessary but not sufficientfor disease induction, or that other mediators that can mimicthe effects of ANCA exist. Nevertheless, strong evidence ofa pathogenic role of ANCA in ASV is provided by animal studies.
A mouse model of MPO-ANCA ASV has been developed in which MPO–/– mice develop murine MPO-ANCA after immunizationwith murine MPO. Recombinant activating gene 2 deficient (Rag2–/–) and WTB6 mice that received murine MPO-ANCAeither by passive transfer or by infusion of splenocytes developedrenal vasculitis. Glomerular accumulation of neutrophils andmacrophages was seen in association with the development ofglomerular necrosis and crescents. Mice that underwent neutrophildepletion were protected from the development of renal lesions,supporting the hypothesis that neutrophils play a central rolein the pathogenesis of ASV (27).
An animal model of PR3-ANCA ASV has been described. PR3 andneutrophil elastase double-deficient mice were inoculated withmurine PR3. These animals developed PR3-ANCA that recognizedboth murine and human PR3. IgG-containing PR3-ANCA or controlIgG was passively transferred to wild-type mice. Intradermalinjection of TNF- in wild-type mice led to development of panniculitisthat was significantly worse in mice that received the IgG-containingPR3-ANCA. Passive transfer of antibody to wild-type mice thathad received bacterial LPS did not lead to the development ofsystemic, lung, or renal vasculitis (28). The absence of systemicvasculitis in this model has many possible explanations, notleast of which is that the investigators were unable to detectPR3 expressed on the surface of murine neutrophils that wereisolated from blood, unlike human neutrophils; PR3 was presenton murine neutrophils that were isolated from the peritonealcavity after TNF- injection. This suggests that ANCA would beable to interact with neutrophils only in tissue, causing localizedinflammation.
Are there other mechanisms that may lead to tissue injury invasculitis and that do not require the presence of ANCA? Otherautoantibodies, for example the anti-endothelial cell antibodies(AECA), discussed next, have been proposed as a pathogenic mechanism,whereas other mechanisms of tissue injury may include the productionof matrix metalloproteinases (MMP). A recent study describedan increased production of MMP from peripheral blood mononuclearcells and increased plasma levels in patients with active WGcompared with healthy control subjects. Although the MMP alsowere increased in patients who were in remission, this was accompaniedby an increase in tissue inhibitors of MMP that was not seenin patients with active disease. The authors of this study suggestthat the imbalance of MMP and tissue inhibitors of MMP in patientswith active WG may contribute to tissue injury (29).
For some years, the presence of circulating AECA has been reportedin patients with ASV as well as other inflammatory conditions.These antibodies have been shown to be reactive against constitutivelyexpressed endothelial cell antigens. Incubation of AECA-containingIgG from patients with cultured human umbilical vein endothelialcells induced increased expression of adhesion molecules, cytokines,and chemokines, suggesting that these autoantibodies may promoteleukocyte recruitment across endothelium (30). A more recentstudy using endothelial cells that were cultured from humannasal, kidney, liver sinusoidal, and lung microvascular endothelialcells showed increased binding of AECA to unstimulated kidneyendothelial cells and nasal endothelial cells and cytokine-treatedlung microvascular endothelial cells compared with human umbilicalvein endothelial cells and liver sinusoidal endothelial cells(31). This raises the possibility that AECA are specific forthe endothelial cells in the target organs of ASV and may promoteleukocyte recruitment to these organs via the production ofcytokines and increased expression of adhesion molecules, aswell as cause endothelial cell injury.
ANCA, Neutrophil, and Macrophage Involvement in ASV
Study of interactions between ANCA and neutrophils has providedinsights into potential pathogenic mechanisms of ASV. ANCA activationof neutrophils requires both antigen binding and Fc receptorengagement. Neutrophils from individuals who are deficient inMPO are unable to respond to MPO-ANCA; however, there is nocorrelation between neutrophil surface expression of MPO anddisease activity (32). Patients with ASV show increased transcriptionof PR3- and MPO-related genes (33,34). Despite ANCA antigenexpression on the neutrophil cell surface, the evidence forin vivo binding of PR3-ANCA to neutrophils has been controversial(35,36).
Cross-linking of MPO and PR3 with Fc receptors on the surfaceof cytokine-primed neutrophils by ANCA in vitro leads to intracellularsignal transduction, resulting in the production of superoxide,proinflammatory cytokines, and degranulation (37–39).Interaction of intact ANCA and ANCA F(ab')2 fragments with neutrophilsinitiates complementary intracellular signaling cascades, indicatingthat engagement of both surface antigen and Fc receptors contributesto activation of multiple signaling pathways (40–42) (Figure 1).p21ras, a molecule that is a branch point in multiple signalingcascades, controls a number of cellular processes in the neutrophil,including the respiratory burst. It was shown recently thatboth intact ANCA IgG and ANCA F(ab')2 fragments activate onlyone of the two isoforms of p21ras present in the neutrophil,Kirsten-ras, during initiation of a neutrophil respiratory burst(41). This potentially could permit specific inhibition of theANCA-activated signaling pathways without complete inhibitionof all p21ras functions.
Figure 1. Signaling pathways in neutrophil activated by anti-neutrophil cytoplasm antibodies (ANCA) F(ab')2 fragment and whole antibody.
ANCA activation of cytokine-primed neutrophils in vitro leadsto accelerated and dysregulated apoptosis that ultimately maylead to neutrophil death by secondary necrosis, thereby promotinginflammation (43). Apoptotic neutrophils also express on thecell surface MPO and PR3 antigens that are capable of bindingby ANCA, but these neutrophils cannot undergo cellular activation.The opsonization of apoptotic neutrophils by ANCA induces increaseduptake by macrophages and release of proinflammatory cytokines.In vivo, this may lead to a failure to resolve inflammationin the normal manner (44,45). In addition, ANCA may have a directeffect on monocytes inducing increased CD11b expression, sheddingof CD62L, and the production of superoxide (46).
Role of Cytokines
Circulating neutrophils from patients with ASV are in a moreactive or "primed" state than those from healthy control subjectswith increased expression of activation markers and increasedbasal superoxide production (45,47). This may be due to circulatingproinflammatory cytokines such as TNF- (48). TNF- has severaleffects on neutrophils in vitro that are vital for neutrophilsto interact with ANCA and endothelial cells and produce proinflammatoryand cytotoxic molecules. TNF- priming of neutrophils causesthe mobilization of PR3 and MPO from granules to the cell surface,as well as transcription of PR3 mRNA and de novo synthesis ofPR3 (38,49). Cytokine priming of neutrophils leads to increasedexpression of 2-integrin adhesion molecules, which are necessaryfor their interaction with endothelium (50). Cytokine primingis necessary for the ANCA-stimulated production of superoxideby neutrophils. This priming of the respiratory burst involvesthe phosphorylation and activation of intracellular signalingmolecules and the mobilization of the components of the NADPHoxidase complex (51–53). Priming also enhances cytokineproduction (54,55). Cytokines other than TNF- also have a primingeffect on neutrophils, e.g., GM-CSF. Recent work from our institutionhas demonstrated that IL-18 primes the ANCA-stimulated neutrophilrespiratory burst response via the phosphorylation of p38 mitogen-activatedprotein kinase without increasing the surface expression ofPR3 or MPO (56).
The importance of cytokines in this disease is underlined bythe animal model using MPO-ANCA described above. Mice that weregiven LPS and MPO-ANCA developed exaggerated renal injury, associatedwith a rise in circulating TNF-. Treatment with anti-TNF- attenuatedthe LPS-mediated aggravation of the renal lesion, highlightingthe importance of proinflammatory cytokines (57).
Endothelial injury occurs early in ASV, and there is compellingevidence that ANCA mediates the interactions between cytokine-primedneutrophils and activated endothelium. A large study of 65 renalbiopsies from patients with ASV found that neutrophils and macrophageswere the predominant leukocytes in glomerular lesions in ASV(58), whereas another study suggested that macrophages weremore commonly present than lymphocytes (59). This partly maybe because the production of IL-8 and monocyte chemoattractantprotein-1 induces recruitment of neutrophils and macrophagesacross the glomerular capillary endothelium (39,60). Furthermore,ANCA has been shown in vitro to cause changes in the actin cytoskeletonof neutrophils, which could lead to their retention in glomerularcapillaries (61).
Interactions among ANCA, neutrophils, and endothelial cellshave been studied using in vitro flow models and intravitalmicroscopy. Flow models involve perfusing neutrophils acrossa monolayer of endothelial cells that have been preactivatedwith a cytokine to increase the expression of endothelial celladhesion molecules. After treatment with low concentrationsof TNF-, endothelial cells capture neutrophils from flow andsupport rolling adhesion via selectin molecules. Perfusion ofANCA over rolling neutrophils induces stationary adhesion ofneutrophils to endothelial cells and causes increased transmigration.Both adhesion and transmigration are 2-integrin dependent; transmigrationbut not adhesion is reduced by the blockade of the chemokinereceptor CXCR2 (62–64). ANCA do not alter the level ofexpression of 2-integrins on neutrophils but do cause a conformationalchange in CD11b, revealing its activation epitope (63).
Similar effects were seen in an animal model of MPO-ANCA ASVin the WKY rat. ANCA promoted neutrophil attachment to and transmigrationthrough endothelium into mesentery in response to GRO (a rathomologue of IL-8; Figure 2). Rats that were immunized withhuman MPO or received MPO-ANCA by passive transfer developedevidence of microvascular injury with areas of mesenteric hemorrhage(Figure 3) (65).
Figure 2. Neutrophil firm adhesion and transmigration in the mesentery of a WKY rat after induction of ANCA by immunization with myeloperoxidase (MPO). A topical inflammatory stimulus was provided by mesenteric superfusion with CXCL-1 (3 x 10–9 M). This process may be quantified in real time using intravital microscopy. Magnification, x40, hematoxylin and eosin.
Figure 3. Mesenteric microvascular hemorrhage in a WKY rat after infusion of anti-MPO antibodies and superfusion with CXCL-1 (3 x 10–9 M). Magnification, x20, hematoxylin and eosin.
The mediators of the endothelial damage that is associated withvasculitis have been widely assumed to comprise both reactiveoxygen species and serine protease released from neutrophils.However, a recent study found that serine proteases, ratherthan reactive oxygen species, were responsible for injury (66),which equates with the ability of PR3 and elastase to induceapoptosis after uptake into endothelial cells (67).
Circulating T cells in patients with ASV show persistent evidenceof activation during active disease and remission, with a Th1-typecytokine profile (68–73). There has been particular interestin T cells that do not express the co-stimulatory molecule CD28,with expanded populations of both CD4+ CD28– and CD8+CD28– cells in the peripheral blood of patients with WG;the increase in the CD28– population may correlate withmore severe disease manifestations (74). There also is evidenceof an increase in circulating peripheral blood memory T cellsthat express the inducible chemokine receptors CCR3 and CCR5,which may be recruited along chemotactic gradients into sitesof inflammation (75,76).
Within tissues, a Th1 cytokine profile has been reported also,particularly in T cells from granulomatous tissue of patientswith WG (74). CD4+ CD28– cells, present in granulomas,seem to contribute to production of IFN- and TNF- (77,78). Thesecells show evidence of an effector-memory phenotype, oligoclonality,and replicative senescence, suggesting that they are the productof antigen exposure but not necessarily MPO or PR3 (78,79) (reviewedin [80]).
Given the important role that ANCA probably plays in the pathogenesisof vasculitis, it is surprising that we understand so littleabout the factors that initiate and control its production.ANCA is largely an IgG class-switched antibody, which suggeststhat T cell help is involved in its production by B cells andplasma cells.
Both MPO and PR3 are normal human proteins, so regulatory processesthat govern the development of T and B cells should preventthe development of autoreactive cells that are capable of causingdisease. That ANCA are found in patients with ASV suggests thatthere has been a failure in these normal regulatory processes.
B cell function is disturbed in patients with active ASV. Bcell activation is related to disease activity, and B cellsthat are capable of producing ANCA seemingly without T cellhelp have been identified in the circulation of patients withactive ASV. The suggested explanation for this is that B cellsin these patients have already been maximally stimulated invivo and therefore may have escaped normal control (72,81).
MPO and PR3 reactive T cells have been demonstrated inconsistentlyin patients with vasculitis and also are found in normal individuals,calling into question whether MPO and PR3 indeed are the antigensthat drive the aberrant T cell populations. Potential otherantigens include proteins that are present in Staphylococcusaureus, which show homology to human PR3 (82). In one study,peripheral blood mononuclear cells from patients with activeWG showed significantly more proliferation to coagulase-positivestaphylococci than controls, whereas some T cell lines thatwere derived from peripheral blood mononuclear cells showedweak cross-reactivity with PR3 (83).
An alternative mechanism for the production of PR3-ANCA wasproposed recently. Complementary PR3 (cPR3), a protein thatis encoded by the antisense strand of the PR3 encoding gene,may be expressed. Antibodies develop against cPR3 and, in turn,induce anti-idiotype antibodies that cross-react with PR3 (PR3-ANCA)and lead to the development of ASV (84).
Lymphocytes and the Development of Glomerular Crescents
Crescent formation, with little in the way of immune complexdeposition, is the hallmark lesion in patients with ANCA-associatedvasculitis. A number of animal models of glomerulonephritishave demonstrated a role for T cells in the development of theselesions (see associated article by Tipping and Holdsworth inthis issue of JASN85). However these models are dependent ona response against an implanted antigen or hapten in the kidney,which has not been reported in ASV, and the glomerular lesionis notable for its marked T cell component, which is not commonlyreported in human ASV (58,59). The relationship between thedevelopment of crescents in ASV and the initial focal segmentalnecrotizing lesions, which frequently contain neutrophils andare pauci-immune in nature, is not clearly understood.
Current treatment strategies for patients with ANCA-associatedglomerulonephritides are based on broad immunosuppression usingcorticosteroids, purine antimetabolites, and alkylating agents.Other approaches are suggested for ASV without renal involvement(86). The introduction of cyclophosphamide and corticosteroidshas changed the natural history of the disease from an almostinvariably fatal condition to a relapsing and remitting one,with greatly increased survival. However, the use of corticosteroidsand cyclophosphamide is associated with a high degree of toxicityand treatment-related morbidity and mortality. The initial managementof the disease is induction of remission with aggressive immunosuppressionto control inflammation and prevent further organ damage. Thesecond phase of treatment is the maintenance of remission withless intensive immunosuppression to limit the adverse effectsof the therapeutic agents while retaining control over the disease.Currently, there is debate as to the intensity of initial immunosuppressionand the duration of induction regimens using cyclophosphamide;in addition, it is unclear exactly how long patients shouldremain on immunosuppression and at what point, if any, immunosuppressioncan be discontinued safely. As our understanding of the pathogenesisof ASV improves, so should our ability to generate safer, moretargeted therapies.
Several recent studies have aimed to optimize the use of corticosteroidsand cyclophosphamide in inducing disease remission in ASV, manyconducted by the European Vasculitis Study Group (EUVAS; http://www.vasculitis.org).A meta-analysis suggested that pulsed cyclophosphamide may besuperior to continuous cyclophosphamide at inducing diseaseremission, associated with fewer treatment-associated adverseevents, although potentially associated with a higher relapserate (87). Preliminary results of the EUVAS CYCLOPS study, alarge, prospective, randomized, controlled, international trialthat compared pulsed and continuous cyclophosphamide, has suggestedthat for non-life- or -organ-threatening disease, pulsed cyclophosphamideis as efficacious as continuous daily oral cyclophosphamidewith a lower cumulative dose (88). There was no difference inrelapse rates.
More aggressive immunosuppression is required in patients withlife- or organ-threatening disease. A number of case seriesand retrospective studies have suggested benefit of additionalplasma exchange for patients with dialysis-dependent renal failureor pulmonary hemorrhage with ASV. A prospective open-label trialof cyclophosphamide and corticosteroids either with or withoutplasmapheresis for patients with ASV found that plasmapheresiswas of additional benefit only to patients who were dialysisdependent at the time of presentation (89). Preliminary resultsof the EUVAS MEPEX trial have suggested that plasmapheresisis superior to methylprednisolone for recovery of renal functionin patients with a creatinine >5.6 mg/100 ml (90).
Cyclophosphamide is extremely effective at inducing and maintaininglong-term remission in patients with WG. The regimen used atthe National Institutes of Health is cyclophosphamide 2 mg/kgdaily until patients have been in full remission for 1 yr. Cyclophosphamidewas given in conjunction with a tapering dose of oral corticosteroids(91); although effective in maintaining disease remission, thisregimen is associated with adverse effects, including bone marrowsuppression, gonadal suppression, and hemorrhagic cystitis.The incidence of bladder cancer in patients with WG that weretreated with cyclophosphamide, was reported as 5% at 10 yr,representing a 31-fold increase compared with the general population(92). This has led to studies that have examined whether WGor MPA can be treated safely with shorter courses of cyclophosphamideto reduce the risk of treatment-related morbidity. The EUVASCYCAZAREM trial, which compared 12 mo of cyclophosphamide treatmentwith early cessation of cyclophosphamide and substitution ofazathioprine as maintenance therapy, showed equivalence in bothgroups for patient survival, relapse rate, disease activity,and renal function; there was a nonsignificant reduction ofadverse events in those who were randomly assigned to receiveazathioprine after disease remission. This supports an earlychange to less toxic azathioprine when the patient has enteredremission (93). A caveat to this is that in a study of disease-freesurvival in patients with PR3-ANCA ASV, patients who had a positivePR3-ANCA test at the moment of switching from cyclophosphamideto azathioprine had a relative risk for relapse of 2.6 comparedwith those who were ANCA negative. Patients who were ANCA negativeat switching to azathioprine had the same risk for relapse aspatients who received cyclophosphamide only (94). Several EUVAStrials are ongoing, investigating the most effective drugs andduration of treatment for maintenance of disease remission.
Newer therapies that are based on improved understanding ofthe pathogenesis of vasculitis also have been reported. Theproinflammatory role of TNF- in ASV led to several reports andpilot studies that used anti-TNF- therapies. An open-label pilotstudy of 20 patients who had WG and were given etanercept (TNFreceptor fusion protein) in addition to their existing immunosuppressionsuggested that the drug was safe and probably effective in controllingWG disease activity (95). An open-label pilot study of 32 patientswho had WG and MPA and received infliximab (anti-TNF mAb) inaddition to standard therapy for new, relapsed, or resistantdisease suggested that infliximab may be effective in controllingASV, although there were a number of relapses during or soonafter infliximab therapy, as well as a number of significantinfectious episodes (96).
As a result of these pilot studies, a randomized, controlledtrial of etanercept compared with placebo, added to standardimmunosuppression, for the induction and maintenance of remissionof WG was conducted in 180 patients. The trial failed to showany benefit in adding etanercept to standard immunosuppression(97). The relapse rates in both groups were high, and >55%of patients in both groups had at least one severe or life-threateningadverse event. Six patients died, although none as a resultof active WG. Significantly more patients developed solid-organcancers in the etanercept group than in the control group. Allof the patients who developed cancer had received cyclophosphamideduring the trial, and several had received previous prolongedcourses of cyclophosphamide, which may have increased theirrisk for developing cancer. One possible criticism of the designof this clinical trial is that, although the patients were stratifiedby disease severity before randomization, the trial was notpowered to detect any differences in outcome for subgroups ofpatients with more or less severe disease, limited or generalizeddisease, or newly diagnosed or relapsed disease. The etanerceptgroup was less likely to achieve sustained remission, but thisgroup contained significantly more patients who had pre-existingdisease and may have been less likely to achieve a sustainedremission, although the disease duration was not significantlydifferent between the two groups.
Differences in the efficacy of the various anti-TNF therapieshave been seen in the treatment of other granulomatous diseases,such as Crohn’s disease and sarcoidosis, in which infliximabbut not etanercept is of benefit (98,99). Etanercept predominantlybinds to soluble TNF-, whereas infliximab also binds to membrane-boundTNF-, and this may lead to infliximab-specific effects suchas lysis or apoptosis of membrane TNF--expressing cells (100,101).Although etanercept has not been shown to be of benefit in WG,large-scale trials using anti-TNF- antibodies would be justifiedas its different biologic actions may prove to be therapeuticallyuseful in either WG or MPA.
Other recently reported new treatments that show promise forASV include the anti-B cell therapy rituximab (anti-CD20). Severalcase and small series reports of its safe, effective use inresistant or difficult-to-manage ASV have now been reported(99,102), although two recent, prospective, open-label studieshave reported both the successful and the unsuccessful use ofrituximab in patients with ASV (103,104). Despite the fact thatCD20 is not expressed on plasma cells, the use of rituximabis associated with a reduction in ANCA titer as well as B celldepletion, without evidence of a generalized immunoparesis (105).It has been suggested that the reduction in ANCA that is seenafter administration of rituximab is due to the concurrent administrationof corticosteroids (106) and that rituximab may owe any therapeuticefficacy to interference with other B cell functions; however,there also are reports of the reduction in ANCA titers in theabsence of steroids (103) or with an unchanged corticosteroiddose (105). If the ability of rituximab to reduce ANCA titersis confirmed, then a better understanding of the way that thisantibody seems to induce selective deletion of ANCA-producingcells would greatly improve our understanding of the mechanismsthat control ANCA production. Either way, the role of rituximabboth as a potential therapeutic intervention for patients withASV and as a tool to help define the role that B cells playin ASV merit further investigation. Currently, the Immune ToleranceNetwork is recruiting patients to a prospective, randomized,controlled, double-blind trial of rituximab in patients withASV (http://www.immunetolerance.org/RAVE/).
Anti-T cell therapies using anti-thymocyte globulin or anti-CD4or anti-CD52 antibodies have been reported in small series.Remission has been achieved using these agents in patients withresistant vasculitis, albeit with high relapse rates and a highrate of infectious complications (107–109).
Treatment should be tailored to disease severity. The currentevidence-based treatments are shown in Table 2 according toEUVAS disease severity classification. Other possible futuretherapeutic strategies might include new anticytokine therapiessuch as IL-18-binding protein to reduce neutrophil priming andendothelial activation, intervention in the neutrophil-endotheliumadhesion cascade, or the neutrophil intracellular signalingpathways that are activated by ANCA.
Disease mortality has significantly improved to approximately80% survival at 5 yr with introduction of current therapeuticregimens. Poor prognostic markers that are consistently associatedwith ANCA-associated vasculitis include older age and more severerenal impairment (110–112). Renal biopsy gives furtherinformation regarding outcome. Chronic lesions, particularlyinterstitial fibrosis, are associated with poor renal outcomeand correlate with renal function at 18 mo (86,113). It is ofinterest that there are no specific differences between WG andMPA in renal pathology; however, patients with MPA are likelyto present with more chronic lesions.
Treatment-related morbidity is common; 25% of patients experienceadverse effects of treatment within the first year, with infectionbeing the most common cause of early death. Identification ofthose who are at greatest risk for morbidity is important whenweighing benefits of potent immunosuppressive treatments. However,we have no good markers to identify those who will respond uniformlypoorly to treatment.
Laboratory and clinical research has improved our understandingof the complex interplay of the components of the immune systemand the roles that they play in the pathogenesis of ASV. Asour understanding of the pathogenesis has improved, particularlyof the role of ANCA, this has improved our understanding ofwhy empirically derived broad immunosuppression has been effectivein controlling this autoimmune disease. Our current understandinghas led to the development of newer therapies for ASV, targetedat specific immune system components, that will potentiallyprovide better disease control with less treatment-related morbidityand mortality.
Acknowledgments
We thank Dr. M.A. Little for permission to use the images inFigures 2 and 3.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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-1-antitrypsin (AAT), the major inhibitor of PR3 activity. The allele that most commonly is associated with reduced AAT activity is designated PiZ and is reported to be associated with an increased risk for developing WG (17,18) or either PR3- or MPO-ANCA (19,20). Two studies have reported an increased risk for death for patients who have ASV and carry the PiZ allele (18,20), although a low serum level of AAT was associated with a reduced incidence of renal involvement in one study (20). The mechanism by which carriage of the PiZ allele increases the risk for death for patients with ASV is not clear but, because it is associated with a reduced amount of circulating AAT/PR3 complexes, there may be increased circulating PR3 activity, although this has not yet been demonstrated. 
2-integrin adhesion molecules, which are necessary for their interaction with endothelium (50). Cytokine priming is necessary for the ANCA-stimulated production of superoxide by neutrophils. This priming of the respiratory burst involves the phosphorylation and activation of intracellular signaling molecules and the mobilization of the components of the NADPH oxidase complex (51–53). Priming also enhances cytokine production (54,55). Cytokines other than TNF-
and TNF-
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Abstract
Introduction
