anca-associated vasculitis: pathogenesis, novel … · medica copyright ® dhaygude anca-associated...

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ANCA-associated vasculitis: pathogenesis, novelmarkers of the disease and emerging therapies

A. DHAYGUDE 1, U. ERDBRUEGGER 2, M. HAUBITZ 3, A. WOYWODT 1

1Renal UnitLancashire Teaching Hospitals

NHS Foundation TrustPreston, Lancashire, UK2Division of Nephrology

University of Virginia at CharlottesvilleCharlottesville, VA, USA

3Division of Nephrology, Department of MedicineHannover Medical School, Hannover, Germany

Much has been learned in recent years on thepathogenesis of ANCA-associated small-vesselvasculitis. The interaction of primed neutrophilswith ANCA and endothelial cells is crucial to thedisease. Next we gained a better understanding,from animal models, of the pathogenetic impor-tance of the ANCA antibody. Very recent evi-dence provides intriguing data regarding thelink between infection and vasculitis, LAMP-2antibodies as novel markers, and NETs as a novelpathogenetic mechanism. It remains to be seenwhether others are able to corroborate thesefindings and whether testing for LAMP-2 anti-bodies will become part of the clinical routinein vasculitis. Recent years also saw the emer-gence of various new markers of endothelialdamage and the disease itself, such as circulatingendothelial cells and endothelial microparti-cles. These novel markers correlate well withdisease activity; they may well complement tra-ditional diagnostic tools, such as ANCA testing.Preliminary evidence has provided some insightinto the balance between endothelial damageand repair. Exciting preliminary data also indi-cate that circulating endothelial cells may notonly be markers of disease activity but that the-se cells may have pathogenetic importance intheir own right. These findings may haveprofound implications for the pathogenesis ofvasculitis and vascular disease in general. Recentyears also saw the publication of a number ofseminal studies for the treatment of ANCA-asso-ciated vasculitis. We now have a much better

understanding of the role of pulse intravenouscyclophosphamide and plasma exchange thanten or even five years ago. Further studies mustnow show whether plasma exchange is alsobeneficial for less severely ill patients with AASV.Finally, as ever, it is hoped that further progressin understanding the disease pathogenesis willalso provide more tailored and less toxic the-rapies. Key words: Vasculitis, diagnosis – Vasculitis,pathology – Vasculitis, drug therapy.

Twenty years or so since the discovery ofanti-neutrophil cytoplasmic antibodies 1

(ANCA), much progress has been made inunderstanding, diagnosing, and treatingANCA-associated small-vessel vasculitis(AASV).2 We now know that ANCA, neu-trophils and micro-vascular endothelial cellsare key players during the pathogenesis ofthe disease. Our understanding of their inter-actions and the interplay with adhesion mol-ecules has improved as well. The discussion

Corresponding author: Dr. A. Woywodt, FASN, ConsultantRenal Physician/Honorary Senior Lecturer, Renal Unit,Lancashire Teaching Hospitals NHS Trust, Royal PrestonHospital, Sharoe Green Lane, Preston, PR2 9HT, UK. E-mail: [email protected]

Vol. 61, No. 4 MINERVA UROLOGICA E NEFROLOGICA 411

MINERVA UROL NEFROL 2009;61:411-38

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DHAYGUDE ANCA-ASSOCIATED VASCULITIS

on the pathogenicity of the ANCA themselvesis still ongoing, fuelled by recent and excitingevidence. ANCA are also a sensitive diagnos-tic marker of the disease at presentationalthough specificity is an issue. Very recently,exciting new evidence has emerged regardingpotential mechanisms of molecular mimicryand new pathogenetic mechanisms, linkinginfection to vasculitis. In diagnosis, ANCAtitres themselves remain less than ideal toguide treatment during follow up, to detectrelapse, and to distinguish between vasculiticdisease activity and infection. New markers ofthe disease have therefore long been awaited.During the last decade, novel markers ofendothelial damage, such as circulatingendothelial cells and endothelial microparti-cles have become available to assess theextent and/or acuity of vascular damage in aclinical setting. There is also the interestingquestion of how endothelial repair occursand how both damage and repair evolve dur-ing the course of the disease, and in relationto treatment. There is also the intriguing ques-tion as to what effects circulating endothelialdebris may have on other cell populations.Finally, progress in understanding the patho-genesis has been mirrored by considerableadvances in treating vasculitis. Multicentre tri-als have largely set a standard for inductionand maintenance of remission, respectively.One of the most interesting developments inrecent years is targeting B-cells and furthernew treatment options may be on the horizon.In this article, we review recent developmentsregarding pathogenesis, discuss markers ofendothelial damage and provide a briefoverview of existing and emerging treatment.

Pathogenesis of ANCA-associated smallvessel vasculitis

The exact etiology of AASV is not known.It is likely that genetic factors predispose to thedisease while environmental factors triggerthe onset of inflammation. Research over thelast two decades, however, has successfullydelineated pathogenetic mechanisms of thedisease. This well established concept 3 focus-es on the interaction of ANCA, neutrophils,

and endothelium. ANCA were first describedby Davies et al.1 He described a series of eightpatients with constitutional symptoms, focalnecrotising glomerulonephritis, weight lossand arthralgia. A circulating IgG factor stain-ing the cytoplasm of neutrophils was identi-fied in the serum of these patients. It wasn’tuntil 1985 when van der Woude establishedthe link between ANCA and vasculitis.4Wegener’s granulomatosis (WG), microscop-ic polyangitis (mP) and the Churg StraussSyndrome (CSS) are nowadays regarded asthe ANCA-associated vasculitides. The dis-cussion around the continued use of theeponym “Wegener’s granulomatosis” isbeyond the scope of this article;5, 6 hence wewill continue to use the eponym here. Ofnote, WG has small vasculitis as well as gran-ulomatous inflammation. The reason why WGshows such a distinct phenotype while oth-erwise showing such similarities to mPremains enigmatic. In the presence of ANCA,neutrophils and monocytes when fixed withethanol, demonstrate diffuse granular cyto-plasmic (c-ANCA), perinuclear (p-ANCA) oratypical staining pattern. Enzyme linkedimmunosorbent assay (ELISA) analysis revealsthat c-ANCA are usually anti-proteinase 3 (PR-3) and p-ANCA are most often antimyeloper-oxidase (MPO). PR-3 and MPO are twoenzymes stored in the azurophilic granulesof neutrophils and monocytes.7 Alpha -1 antit-rypsin is a physiological inhibitor of PR3.Deficiency of alpha-1 antitrypsin (PiZZ phe-notype) has been associated with PR3 positivevasculitis 8 but a highly increased risk couldnot be demonstrated. Myeloperoxidase inneutrophils facilitates the formation ofhypochloric acid, a strong reducing agent anddefence mechanism against microorganismsand is a weak inhibitor of PR3. c-ANCA ispredominant in WG (70%) and p-ANCA ispredominant in CSS (60%). They are aboutequally distributed in MP (40% c-ANCA; 50%p-ANCA).9 Five to 30% patients with WG, MP,and CSS are ANCA negative. It must there-fore be appreciated that no ANCA test is spe-cific for one of the diseases and that a tissuediagnosis is required. A meta-analysis of 15studies has revealed that a positive c-ANCA byindirect immunofluorescence (IIF) has a sen-

412 MINERVA UROLOGICA E NEFROLOGICA December 2009

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ANCA-ASSOCIATED VASCULITIS DHAYGUDE

itoring of ANCA and preemptive treatmentwith immunosuppression for rising titres wasfound to prevent a clinical relapse.18 Howe-ver, it is important to note that up to 30% ofpatients with limited WG, are ANCA nega-tive but tend to become seropositive withprogression to systemic vasculitis. Moreimportantly, ANCA titres are by no meansinfallible markers of AASV. In fact the positivepredictive value of ANCA in predicting a clin-ical relapse in AASV is highly variable.19, 20

This has led some authors to suggest thatANCA titres should not be used to guide treat-ment in the first place.21 Nearly all expertsnow agree that a rise in ANCA titres withoutsymptoms should increase awareness, butnot necessarily mandate immunosuppressivetreatment. New markers of the disease and itsactivity are therefore eagerly awaited.

Animal models of AASV

Earlier attempts of animal model of vas-culitis were based on exposure to mercurychloride in Brown Norway rats.22 This modeldemonstrated arthritis, caecal vasculitis, raisedIgE and production of multiple autoantibod-ies including anti MPO antibodies. It did not,however, reflect the disease pattern of AASVin humans. Subsequent attempts to inducevasculitis in rodents with anti-MPO antibodieswere unsuccessful. Neumann et al developedSCG/Kinjoh, a new strain of mouse whichdeveloped spontaneous vasculitis and cres-centic glomerulonephritis with ANCA but thismodel was associated with significant immunedeposits.23 More recently, Xiao et al. preparedMPO knock-out mice and immunised themagainst murine MPO. Transfer of splenocytesfrom this immunised mice in to RAG2-/- miceinduced vasculitis and pauci-immune cres-centic glomerulonephritis.24 This animal mod-el has been regarded as compelling evidencethat ANCA themselves are pathogenic.25 Inaddition, Little et al. developed a rat model,which develops pauci-immune crescentic GNand pulmonary haemorrhage when immu-nised with human MPO.26 Using a similarmethod to develop an animal model of PR3-associated vasculitis has shown different


sitivity of 34% to 92% in diagnosing of WGand a specificity of 88% to 100%.10 IIF whencombined with ELISA, increases specificity ofdiagnosis to nearly 100% when comparedwith healthy controls.11 A potential pitfall isthat atypical ANCA, i.e. those not directedagainst PR-3 or MPO, are often detected innon vasculitic conditions, such as autoim-mune liver disease, inflammatory bowel dis-ease, tuberculosis,12 endocarditis and cysticfibrosis. Less often, cANCA is also seen inthese non-vasculitic conditions. These anti-bodies are directed against a broad variety ofother antigens, such as bacterial permeabili-ty protein (BPI) or lactoferin. On occasion,atypical ANCA are also seen concurrently withPR-3 ANCA in vasculitis.13 Rarely c-ANCA hasbeen reported as an epiphenomenon. Anoverview is provided elsewhere.14

ANCA and pathogenicity: an ongoingdebate

The role of ANCA in the pathogenesis ofsystemic vasculitis has long been debated.Clinical experience dictates that some patientshave persistently high ANCA titres but noclinical disease activity. More recently, how-ever, several clinical observations as well asin vitro and in vivo experiments have fuelleda new discussion of the pathogenetic impor-tance of ANCA.

Anecdotal but interesting evidence for apathogenetic role of anti-MPO ANCA wasprovided by a case report of a newborn baby,who developed pulmonary renal sydromeafter delivery from a mother with activemicroscopic polyangiitis.15 It is believed thatthe transplacental transfer of IgG MPO ANCAwas responsible for the clinical disease inthe newborn. However this does not seem tobe the rule in that healthy children withoutvasculitis are also seen in women with activevasculitis during pregnancy despite transpla-centar transfer of MPO.16

A change in ANCA titres over time hasbeen described to correlate with disease activ-ity. In one prospective study, 92% patientsshowed rising ANCA titres before clinicalrelapse.17 In another study, prospective mon-

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results, demonstrating that multiple mecha-nisms are responsible for loss of tolerance tothese autoantigens.27

Compelling as all these laboratory datamay be, clinicians are still not entirely con-vinced and argue that ANCA alone may notbe pathogenic enough or else one would notsee patients with high ANCA titres and nodisease activity, nor healthy children withhigh-titre MPO-ANCA born to women withactive AASV.

Interactions between neutrophils andendothelium

The established model of AASV focuseson the interaction of ANCA, primed neu-trophils and endothelial cells.3, 28 It has beenproposed that ANCA interacts with their tar-get antigens on the surface of neutrophilsand stimulate respiratory burst, when primedwith inflammatory cytokines, like tumournecrosis factor α (TNFα), interleukin 1 beta(IL-1 β). This mechanism eventually leads torelease of reactive oxygen species,29 pro-teases and other mediators.

It is also postulated that primed neutrophilsbind to primed endothelial cells, starting acascade of inflammation, leading to tissuedestruction. The interaction between neu-trophils and the endothelial surface is com-plex.30 Neutrophils go through different stagesof attachment, rolling, arrest and transmigra-tion through the endothelium. TNFα and IL-1βinduce the expression of the adhesion mole-cule selectin on the endothelial surface and P-selectin glycoprotein ligand 1(PSGL-1) on theneutrophil surface. PSGL-1 binds with P-selectin which results in attachment of neu-trophils to the endothelium. Chemokines,which attract neutrophils, are also expressedon the endothelial surface. PR3 itself is knownto induce IL-8, a strong chemotactic and acti-vating factor for neutrophils.31 Further stud-ies have confirmed that PR3 increases expres-sion of monocyte chemoattractant protein-1(MCP-1) and ICAM-1 at protein and m-RNAlevel and incubation of human umbilical veinendothelial cells (HUVEC) with PR3 for 24hours significantly increases adhesion of neu-

trophils which can be blocked by monocloanalantibodies to ICAM-1 or CD18.32, 33

Subsequently ANCA binding induces a con-formational change in the integrin moleculeson the neutrophil surface. Integrin binds toits ligands expressed on the endothelial sur-face. PR3, but not MPO interacts with endothe-lial cells (EC). PR3 from degranulated neu-trophils is partly internalised by the EC. It hasbeen shown that PR3 is capable of inducing apro-apoptotic signalling pathway.34 This obser-vation underpins the complex interplaybetween PR3 ANCA and EC. Activation of neu-trophils is more efficient when they are actu-ally attached to endothelium. Adherence ofneutrophils to endothelial surface is facilitatedby expression and activation of adhesion mol-ecules on both neutrophils and endothelialcells. Increased expression of α1(CD29) andα2 integrins (CD18) has been shown on neu-trophils and monocytes isolated from patientswith AASV comPred with normal controls.35 IncomPrison, transmigration of neutrophilsthrough the endothelium is poorly understood.Both transmigration and adhesion are α2 inte-grin dependant but transmigration alone canbe reduced by blocking of chemokine recep-tors CXCR2.36 Recent evidence suggests thatjunctional adhesion molecules, CD31 andCD99 may be involved in this process.37

Interestingly, P-selectin is absent in the normalglomeruli, possibly as a protective mechanismagainst the developement of glomeru-lonephritis. Pankhurst et al suggests an alter-native mechanisms in the emigration of neu-trophils in the inflamed glomeruli, which isindependant of the usual cascade of rolling,adhesion, transmigration but probably facili-tated by direct capture using α 4 integrins.30.

Old and new mechanisms of tissuedamage

Traditionally it has been postulated thatthe neutrophil respiratory burst and release ofsuperoxide and other mediators at the EC ischiefly responsible for endothelial damage.However recent evidence also supports therole of serine proteases like PR3 themselvesto mediate EC damage. EC can actually down-


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acterizes a unique type of neutrophil-relatedcell death that is linked to innate immunity.Kessenbrock et al. were now able to demon-strate that NETs can trigger vasculitis andshowed that ANCA stimulated neutrophils torelease NETs, which contain MPO and PR3.40

NETs can activate plasmacytoid dendritic cellsand B cells. Interestingly, they also showedthat the neutrophils ability to form NETs wasenhanced by bacterial infection with S.Aureus, which has long been linked to vas-culitis.43 NETs in turn bind Staphylococcus.41

Their results also suggest that PR-3/MPO co-localize with NETs, which may in turn presentPR-3/MPO as antigens to the remainder ofthe immun system, thus fuelling a viciouscircle.44 The importance of these new resultsis twofold: First, they provide a whole newpathogenetic mechanism for vasculitis.Moreover, they provide a new link betweeninfection and vasculitis. Figure 1 illustratesthe mechanism of NET formation and spec-ulative mechanisms linking it to vasculitis.


regulate the superoxide response of neu-trophils by suppression of NADPH activity.38

Lu et al proposed that EC release adenosine,which inhibits the respiratory burst of ANCAactivated neutrophils and protects theendothelial surface from the potentially dele-terious effects of superoxide.39 This obser-vation lead the authors to conclude thatANCA induced superoxide release was anunlikely mechanism for the EC injury in vivo.

Recently Kessenbrock et al.40 providedexciting new insight into a hitherto unknownmechanism of endothelial damage in ANCA-associated vasculitis. It has been known forquite some time that neutrophils are capa-ble of releasing gummous DNA upon celldeath. This DNA in turn can trap bacteria.41

Hence the term neutrophil extracellular traps(NET) has been coined.41 This mechanism isstimulated by Toll-like receptor 4 (TLR4).42

Eventually the gummous DNA can stick tothe endothelium and causes tissue damageduring sepsis. The release of NET thus char-

ANCABacteria

NETs

Other immuno-competent cells

Tissuedamage

IL-8Other cytokines

LPSTLR-4

NADPH

MPO orPR-3A B C D

O2 O-

?

Figure 1.—NET formation and possible implications for the pathogenesis of ANCA-associated vasculitis (hypothetical). A)Activation of neutrophils leads to production of reactive oxygen species; B) Next, the nuclear membrane starts to disin-tegrate, leading to vesicle formation; C) The nuclear membrane continues to disintegrate and the nuclei lose their lobules.Nuclear material now fills most of the cells. D: The cells release NETs, which bind bacteria. ANCA target antigens can betrapped within extracellular NETs and may thus act as auto-antigens and this perpetuate the process of ANCA production.Partly redrawn after.225 IL-8 denotes interleukin-8. LPS denotes lipopolysaccharide. TLR-4 denotes Toll-like receptor 4.

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Endothelial cells and antiendothelialcell antibodies

Histological findings in AASV suggest thatdamaged EC undergo necrosis and detachfrom the basement membrane. Theseendothelial cells are detectable in peripher-al blood as discussed in a separate chapterbelow. Could direct antibody-mediated cytol-ysis have a role in this? Antiendothelial anti-bodies are indeed detectable in patients withAASV 45, 46 and other vasculitic conditions.47

Their existence may provide yet anothermechanism of endothelial injury in vasculitis.However, there are considerable technicalproblems with AECA as well as a lack of con-sensus for their detection. Furthermore, AECAare a heterogeneous group of antibodies andare not entirely specific to endothelial cells,but may react with fibroblasts as well.

AECA when incubated with human umbil-ical vein endothelial cells (HUVEC), showincreased expression of adhesion molecules,chemokines and cytokines and may promoteleukocyte recruitment.48 In vitro, AECA havealso been shown to mediate complementand cell- cytotoxicity against a cultured mono-layer of endothelial cells.49 Further studiessuggest that AECA promote thrombogenicevents with increased production of tissuefactor and von Willebrand factor.50, 51 As oftoday, there are conflicting reports about theprevalence of circulating AECA in AASV andthe reported prevalence ranges from 8% to100%.45, 46, 50 Recent experiments usingglomerular endothelial cells as a specific sub-strate comPred with HUVEC, showed a lowprevalance of AECA (14% in WG and none inMP) in AASV.52 Others describe that AECAhave considerable pathogenetic importancein AASV, suggesting a kinase-dependentmechanism.53 For now, it is probably fair tosay that the role of AECA remains contro-versial and ill-defined. A reasonable next stepcould be a multi-centre effort towards tech-nical consensus and standardisation of AECA.

T lymphocytes

For a long time, the majority of researchinto AASV had been focused on neutrophils

and ANCA whereas in limited disease withonly granulomatous lesions the pathogenicrole of T lymphocytes has always been moreevident. However more recently the role oflymphocytes is emerging. Immunohistologicalstudies as early as 1983 showed involvementof T cells in the perivascular lymphoid infil-trate in patients with WG.54 Clinical observa-tions of induction of remission with anti Tcell antibodies also support the pathogenicrole of T cells in AASV.55, 56 Isotype switchingbetween IgG1 and IgG4, the two predomi-nant forms of ANCA, is dependant on IL-4, aT cell cytokine. Furthermore, disease activi-ty of WG correlates well with concentrationof IL-2, which is a marker of T cell activa-tion.57 T cells from patients of AASV, whetheractive or in remission, show significant pro-liferation when cultured with ANCA antigens(PR3 and MPO).58 The cytokine secretionprofile of T cells from granulomatous lesionsin WG shows a Th1 pattern.59 Other studieshave also confirmed the increased productionof interferon by T cells in patients of WG,suggesting a Th1 pattern.60 Of note, T cellactivation requires help of co-stimulatorypathway. In WG patients, it has been shownthat expression of CD28 is significantly lowand expression of B7 is increased.61 CD28costimulation is associated with Th2 type ofresponse and in absence of this T cells defaultto mount a Th1 type of response.62 Evidencesuggests that T cells remain activated in WG,even in remission state.63 It is tempting tothink that this may be related to the responseto superantigens produced by Staphlococcusaureus,64 as infection with these bacteria haslong been linked to WG. In addition, a defectin the suppressor function of natural regula-tory T cells (TReg) has been proposed inpatients with AASV, leading to persistantautoreactivity of T cells.65 Finally, geneticpolymorphisms in Protein tyrosine phos-phatase, non-receptor type 22 (PTPN22) andCytotoxic T-Lymphocyte Antigen 4 (CTLA-4)are known to negatively regulate T cellresponsiveness and thus offer an explana-tion for T cell hyperreactivity in AASV.66

Recent reasearch has also focussed on IL-17producing CD4+ T cells, termed Th 17 cells,as a crucial cellular subset in the pathogen-


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of CCR5 during the signalling pathway in therecruitment of T cells and macrophages. Itis also known that CCR5 is expressed main-ly on Th1 type of cells.75 Presence ofmacrophages and T cells in granuloma ofWG and Th1 type of response in WG makesCCR5 important in the pathogenesis of WG.Zhou et al showed in an immunohistologicalstudy of lung biopsies that patients with WGhave increased expression of RANTES andan increased number of CCR5+ve cells ininflamed lung tissue compared to adjacentnormal lung tissue. Interestingly, numerousCCR5+ve cells were found tethering to pul-monary vascular endothelium. In compari-son, cells within the lumen were predomi-nantly CCR5-ve, suggesting CCR5 to beinvolved in transmigration of cells acrossendothelium. A 32 basepair deletion in CCR5gene (CCR5 ∆32) confers resistance to humanimmunodeficiency virus (HIV) as CCR5 isessential for membrane binding cell pene-tration by HIV.76 When compared with ANCAstatus, this deletion is significantly under-rep-resented in ANCA negative patients.77

Conversely, CCR5∆ 32 was associated withpersistent ANCA positivity. Lack of this dele-tion in ANCA negative patients suggest impor-tance of CCR5 in pathogenesis of WG and itcan be postulated that absence of functionalCCR5 pathway and negative ANCA will dra-matically reduce development of WG.77

What induces ANCA production?

The true cause of ANCA production is notknown to the present day. Environmentalstudies suggested a connection with silicaexposure 78 but these data remain conflictingat present. There is surely a genetic back-ground although clusters of AASV in a fami-ly are rare.79 In this regard, it is of particularinterest that membrane expression of PR-3is genetically determined.80 It is also knownthat having a large subset of neutrophilsexpressing membrane PR3 is a risk factor forvasculitis.81 NET release by neutrophils dur-ing infection may present ANCA antigens tothe remainder of the immune system andrender them auto-antigenic as discussed


esis of autoimmune diseases.67 It is alreadyknown that T cells are important in granulo-ma formation in WG and a relative increaseof PR3 specific Th17 cells has been seen inANCA positive WG patients, in comparisonwith ANCA negative WG and healthy con-trols.68 This suggests a crucial role of Th17cells in autoantibody production in WG.68

Finally, the role of CD4+ T cells is furthersupported by their appearance in the renal tis-sue and urinary sediments 69 in patients withactive WG.

B lymphocytes

B cells are direct precursors of antibodyproducing plasma cells that must eventuallysecrete the ANCA. In addition, B cells arelikely to contribute to pathogenesis of AASVby other mechanisms, such as co-stimulationand antigen presentation within granuloma.70

Some now believe that the granuloma in WGmay be the site of ANCA synthesis.71 PR-3positive cells in the vicinity of antigen-pre-senting cells are also observed in granulo-mas.72 This observation also suggests thegranuloma as the site of the continuedimmune response against PR-3. Single-cellanalysis of B lymphocytes seems to confirmthis hypothesis.70 B cells are also highly effec-tive in antigen presentation and cytokine pro-duction (IL6, IL10 and TNFα). It has beenshown that there is increased level of B cellactivating factor (BAFF/BLYS) in patients ofWG.73 The recent marked success of anti Bcell directed therapy with rituximab furtherstrengthens the pathogenic associationbetween B cells and AASV as discussed ingreater detail below.

CCR5

CCR5 is a member of the beta chemokinereceptors. Its ligand is CCL5, previouslyknown as RANTES (Regulated uponActivation, Normal T-cell Expressed, andSecreted). An important role of CCL5/CCR5has been described in several autoimmunediseases.74 Current concepts suggest a role

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above. Previous data have also implicatedbacterial superantigens provoking autoanti-body production.82 Pendergraft and co-work-ers serependitously discovered another nov-el mechanism that causes ANCA production:They describe an immune response against apeptide that is antisense or complementary tothe auto-antigen, which then induces anti-idiotypic antibodies (auto-antibodies) thatcross-react with the auto-antigen.83

Another tempting hypothesis includes amolecular mimicry between microbial anti-gens and host proteins. Recently Kain et al.proposed molecular mimicry between lyso-somal membrane protein 2 (LAMP2) and bac-terial adhesin FimH. In neutrophils it is inte-grated in to membranes of MPO and PR3containing vescicles, hence LAMP2 is anoth-er and previously recognised target antigenfor ANCA.84 Their new findings suggest thatexposure to FimH induces autoantibodies tohuman LAMP2, which initiate pauci-immuneglomerulonephritis in rats immunised withFimH. The authors also showed that preva-lence of these autoantibodies to humanLAMP2 was more than 90% in patients withactive pauci-immune glomerulonephritis. Asonset of vasculitis is often preceded by infec-tions with FimH expressing bacteria, theyconclude that FimH triggers the autoimmunityto human LAMP2, which is responsible for thetissue injury in vasculitis syndrome.85 Ofcourse, these findings need corroboration infurther studies. Also, a standard for the detec-tion of LAMP-2 antibodies needs to be estab-lished. Larger multi-centre studies shouldthen establish the prevalence of these anti-bodies in the vasculitides. It would also be ofinterest whether these antibodies are capableof inducing NET formation.44

ANCA negative crescenticglomerulonephritis

Although not part of the AASV spectrum ofdiseases, ANCA-negative pauci-immune cres-centic glomerulonephritis is still of consid-erable interest to the field, not least due to thesimilarity of the lesions in histological spec-imens. It is probably fair to say that the dis-

ease is poorly understood with a lack ofpathogenetic concepts as well as paucity oftherapeutic guidelines. Up to 20% patientswith crescentic glomerulonephritis have noother underlying disease and never have cir-culating ANCA.86 Mei Ding’s experimentoffers a novel hypothesis for an alternativemechanism in such ANCA negative patients.In their mouse model experiment, deletion ofthe Von-Hippel-Lindau gene (Vhlh) fromintrinsic glomerular cells from mice was suf-ficient to initiate crescentic GN.87 Vhlh is anegative regulator of Hypoxia inducible fac-tor (HIF) which is a master regulator of oxy-gen homeostasis, angiogenesis and vascularremodelling.88 Loss of Vhlh leads to stabili-sation of HIF and up-regulation of the hypox-ia response downstream. Furthermore, Dinget al. also found strikingly increased expres-sion of the chemokine receptor CXCR4 inpodocytes. CXCR4 binds to stromal derivedfactor 1 (Sdf1), which is a growth factorsecreted by mesangial cells. Increased expres-sion of Sdf1 was also noticed in glomeruli.Increased Sdf1 production may recruit inflam-matory cells and contribute to formation ofglomerular crescents.89 Interestingly treat-ment of mice with neutralising antibodies toCXCR4 results in delayed onset and reducedseverity of glomerulonephritis in these mice.Ding et al. add further evidence to these find-ings by demonstrating increased expressionof HIF1 target genes and CXCR4 protein inglomeruli from humans with pauciimmuneRPGN. This study provides an important roadmap for further evaluation to determine ifincreased HIF expression and its targets arecritical in pathogenesis of pauci-immune cres-centic glomerulonephritis. Finally, it will beinteresting to see whether blocking antibod-ies to CXCR4 or HIF1 may have a therapeu-tic role.

Circulating endothelial cells as markersof endothelial damage

Circulating endothelial cells (CEC) are nota new concept to measure vascular damage.They were in fact first described almost 40years ago 90 on the basis of light microscopy


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insight into the pathogenesis from pheno-typic analysis of these cells. Even in the firstcouple of patients we encountered very highnumbers of CEC in ANCA vasculitis. Giventhat damage to and death of endothelial cellsis the hallmark of the disease, we were notsurprised that cell numbers correlatedextremely well with disease activity.93

Phenotypic analysis, however, provedmore difficult than anticipated. We alsoattempted laser capture microdissection tofurther characterise the cell phenotype.However very few if any intact CEC remainedfor analysis and further characterisation ofthe cells was just not feasible. In retrospect,one of the crucial obstacles in this endeavourand in our attemps to characterise CEC byimmunocytochemistry was the fact that evenin acute vasculitis CEC are rare in peripher-al blood. Hence the absolute number of CECavailable for any given analysis is always piti-fully small.

It is quite clear from the concept of smallvessel vasculitis that CEC cannot be specificto ANCA vasculitis and elevated CEC numbershave since been demonstrated in other vas-culitides, such as aortoarteritis 100 andKawasaki disease.101 CEC are also elevatedin systemic lupus erythematosus.102 A veryrecent study demonstrated elevated numbersof CEC and soluble markers in rheumatoidarthritis 103 and thus raised questions aboutthe pathogenesis of the disease. In a broad-er sense, CEC are not even specific for vas-culitis per se. They are also markedly ele-vated in other, non-vasculitic, forms of wide-spread acute vasculopathy. We studied CECin thrombotic microangiopathy. Not surpris-ingly, CEC numbers were also markedly ele-vated and a decline in cell numbers indicat-ed successful plasma exchange.104 Thereforeone has to remember that elevated CEC arenot specific to vasculitis, let alone to its ANCA-associated variant. Hence we don’t envisionthem to replace traditional diagnostic tools,such as tissue biopsy and ANCA testing, butto complement them.

In this regard a notoriously difficult clinicalscenario is the patient with known AASVwho becomes unwell during immunosup-pressive treatment. Distinguishing between


in smears of peripheral blood. A more wide-spread use of CEC was only possible withthe discovery of the S-Endo 1 antigen 91 andimmuno-magnetic isolation.90 This approachwas originally devised for the enumeration ofrare cells in body fluids. CECs are, even in themost acute vascular disorder, rare cells inperipheral blood; hence immuno-magneticisolation is well suited for this purpose. Morerecently, fluorescence-activated cell sorting(FACS) has also been employed.92 Immuno-magnetic isolation uses morphological crite-ria although the technique is time-consumingand cumbersome.93,94 At first glance, multi-parametric FACS 95 appears user-friendlier.FACS can avoid false positive counting ofleukocytes (a typical problem of the immuno-magnetic approach). The technique alsoholds some potential to elucidate the phe-notype of CEC. In this regard, analysis of CECwith six-colour FACS and demonstration ofapoptosis has recently been described.96

However, the technique does not permitdirect observation of the cell morphology.92

There is ongoing debate as to which of thetwo may be preferable. A standard forimmunomagnetic isolation and enumerationof CECs has been proposed already 97 butthere is no uniformly accepted consensusacross the field.90 Despite the lack for con-sensus CEC have been found to be reliablemarkers that correlate with clinical diseaseactivity across a variety of vascular disor-ders.90 For obvious reasons, cardiovasculardisease has received much attention 98, 99

while current studies focus on phenotypicanalysis, concurrent enumeration of CEC andEPC, and the clinical utility of CEC.

Circulating endothelial cells in ANCA-associated vasculitis

It is clear from the pathogenesis of ANCA-associated small-vessel vasculitis that endothe-lial cells are damaged and eventually dis-lodged from the basement membrane. Wethus became interested in circulating endothe-lial cells (CEC) in vasculitis and hypothesizedthat cell count would reflect disease activity.Another hope was that we could glean further

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disease activity and infection can be difficultto say the least. The limitations of ANCA test-ing have been discussed above. We foundCEC very useful to monitor treatment and todistinguish between relapse and infection indifficult cases.105 We also studied patientswith limited granulomatous disease and withrelapse.106 Patients with vasculitic relapseshad markedly elevated numbers of circulat-ing endothelial cells and indeed similar cellnumbers when comPred to patients at theirinitial vasculitic presentation. Patients withlimited disease due to granulomatous dis-ease had only slightly elevated cell numbers,which were similar to those seen in remission.These findings gave us confidence in theclinical use of CEC in vasculitis 94, 105 althoughour findings need to be corroborated by oth-ers. Finally, it would be interesting to seeprospective monitoring of CEC in vasculitispatients.

Endothelial microparticles in ANCA-associated vasculitis

In general, microparticles (MP) are sub-micrometric fragments derived from plasmamembranes. Increased numbers occur inresponse to a variety of events, such as acti-vation, injury, or apoptosis. Loss of phos-pholipid asymmetry is a crucial event dur-ing this process.107, 108 On their surfacemicroparticles express antigens that reflecttheir cellular origin. These surface markerspermit their enumeration and characterisa-tion by flow cytometry. Microparticles haveattracted considerable interest in vascular dis-ease although a consensus definition of theseparticles and a uniformly accepted protocolfor their enumeration is still lacking.109 In thisregard the situation resembles that of CECwhereby several techniques compete andprogress is hampered by lack of standard-ised criteria for definition and enumeration.Furthermore, endothelial microparticles onlyrepresent a small subgroup of all MP foundin human plasma.110 Specific endothelialmicroparticles were first described in 1990by Hamilton and colleagues.111 We studiedendothelial microparticles (EMP) by FACS

analysis and found elevated EMP in activevasculitis.112 Similar results had previouslybeen obtained in a paediatric cohort of vas-culitis patients.107 Particle counts also corre-lated with disease activity.112 It is probablysafe to assume that CECs and microparticlesdo not reflect the same disease process. Inother words, CEC, EMP and also solublemarkers, such as thrombomodulin, eachreflect different facets of vascular activationand damage although some degree of over-lap may exist. Finally, EMP may also havepathogenetic importance in vasculitis. Theyare now regarded as crucial players at theinterface of atherosclerosis and inflamma-tion.113, 114 Leukocyte MP induce endothelialIL-6 and MCP-1 production.115 It has beendemonstrated that EMP are tissue-factor pos-itive 116 and very recent evidence suggeststhat they can also convert plasminogen intoplasmin.117 Finally, elegant studies in flowchambers have demonstrated that MPenhance leukocyte rolling.118 Taken together,current data suggest that EMP may not onlybe a surrogate marker of vasculitis but thatthey may contribute to the proinflammatoryand procoagulant status of the endothelium.

Endothelial progenitor cells in ANCA-associated vasculitis

The role of endothelial progenitor cells(EPC) 119 in vascular disease and their poten-tial role for therapy 120 have been reviewedrecently.121 Of note, the field of EPC is par-ticularly hampered by lack of standardisa-tion.122, 123 Our knowledge about the kineticsof CEC detachment and EPC mobilisation aswell as their interaction is equally limited.Very recently, the margins between endothe-lial progenitor cells and haematopoietic stemcells became somewhat blurred after proofthat endothelial cells can be hematopoietic inmice.124 We studied numbers of circulatingCD34+ progenitor cells and EPCs in vasculi-tis and demonstrated that these cells increasedsignificantly after the institution of immuno-suppressive therapy and with disease remis-sion.125 Others have previously described anincrease in EPCs in inflammatory vascular


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dard as to their definition and enumeration iseagerly awaited. Figure 2 provides anoverview of mechanisms of endothelial repair.

Circulating endothelial cells aspotential mediators of disease

Several lines of evidence have long sug-gested that CEC themselves could be pro-inflammatory.132 Damaged eukaryotic cellshave been shown to release a variety of pro-inflammatory factors, to initiate a Toll-like-receptor-2/NFκB-dependent reaction inmonocytes and fibroblasts.133 Evidence hasalso emerged that ANCA accelerate apopto-sis in neutrophils and imPired clearance ofapoptotic neutrophils has been described 134.Kirsch et al. showed in a recent study thatapoptotic and necrotic endothelial cells andtheir fragments are rapidly internalized byhealthy endothelium (Figure 3). Support forthese findings came from other studiesdemonstrating the phagocytic capability ofendothelial cells.135 Kirsch et al. could alsoshow that endothelial cells exposed to apop-totic and necrotic cells exhibit enhancedadhesion properties for leukocytes and that


diseases: Avouac et al., for instance, describedincreased EPC numbers in scleroderma.126 Incontrast to de Groot et al.,125 other studiespostulate an imbalance between CECs andEPCs in patients with vasculitis.127, 128 Whatmake these studies so difficult to comPre is,again, the lack of standardisation and the useof different assays and surface markers.Therefore, these studies provide interestingfood for thought but require independentconfirmation. What stimulates EPCs in reac-tion to ischemia or other forms of insult? Asa nephrologist, it is worthwhile to remem-ber that erythropoietin (EPO) regulatesEPCs.129 Hence EPO treatment must alwaysbe corrected for when EPCs are measuredin renal patients. Statins also influence EPCnumbers.130 Other factors that have beenimplicated as regulators of EPCs include vas-cular endothelial growth factor (VEGF), theangiopoetins, and platelet-derived growthfactor CC (PDGF-CC). EPCs are capable ofhoming in to sites of vascular damage.Mechanisms include CD18/ICAM-1 and sdf-1/CXCR4. Endothelial commitment requireshistone deacylase (HDAC) activity anddepends on the expression of the homoeo-box transcription factor HoxA9.131 It is prob-ably fair to say that EPCs will receive furtherscientific attention in vasculitis while a stan-

Figure 2.—Mechanisms of endothelial repair. BM, base-ment membrane; EPC, endothelial progenitor cell. ANG:angiopoetin; EPO: erythropoetin; PDGF: platelet-derivedgrowth factor; VEGF: vascular endothelial growth factor;SDF: stromal-cell derived factor; CXCR: chemokine motifreceptor; HDAC: histone deacetylase activity; HoxA9:homoeobox transcription factor A9; D: cluster of differen-tiation 18; ICAM: intercellular adhesion molecule. FromWoywodt A, Erdbruegger U, Haubitz M,119 with permission.

Figure 3.—Engulfment of apoptotic endothelial cells (green)by untreated human umbilical vein endothelial cells(HUVEC). From Woywodt A, Kirsch T, Haubitz M,226 withpermission.

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isolated CECs from patients with vasculitiseven aggravated these effects.136 These effectson binding properties could be explained,at least in part, by release of the proinflam-matory chemo-attractants IL-8 and MCP1which serve as chemo-attractants. Interestin-gly, apoptotic and necrotic cells induced dif-ferent patterns of effects in healthy endothe-lium. Enhanced IL-8 and MCP1 levels inserum have been detected in patients withactive vasculitis.137 Endothelial synthesis ofthese mediators triggered by ANCA 138 andcirculating endothelial cells 136 may contributeto the pro-inflammatory state associated withvasculitis. Kirsch et al. have recently investi-gated this topic further and became interest-ed in thrombospondin (TSP-1). This multi-domain, multi-functional glycoprotein mod-ulates cell adhesion and proliferation.139

Kirsch et al. were able to show that apop-totic cells induce enhanced expression of

TSP-1 in human endothelial cells and demon-strated that TSP-1 facilitates engulfment ofapoptotic cells by phagocytes.140 It is tempt-ing to speculate that endothelial-derived ele-vated TSP-1 may serve as a signal for phago-cytes promoting enhanced clearance of apop-totic cells. Figure 4 summarizes proposedinteractions of circulating endothelial cellswith healthy endothelium.

Therapy of ANCA-associated vasculitis

General principles

A common feature of the ANCA-associat-ed vasculitides is marked heterogeneityregarding disease manifestation, severity andprognosis, which affects treatment strategies.Two different classifications, namely theChapel Hill system and the American College


Figure 4.—Interactions of damaged circulating endothelial cells with healthy endothelium. IL-8 denotes interleukin-8.MCP-1 denotes Monocyte chemotactic protein-1. HMGB denotes High-mobility group box 1. TRAIL denotes tumornecrosis factor-related apoptosis-inducing ligand. Note that some interactions, for example those with other cell sub-sets in peripheral blood, are as yet unproven 226.

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vigilance regarding malignancy, and urothe-lial neoplasms in particular, is advised in allpatients who have been treated withcyclophosphamide. However prospectivestudies on screening strategies in these highrisk patients are lacking.

Immunosuppression also increases the riskof infertility, whereby the risk is particularlyhigh with cyclophosphamide. In the NIHstudy, cyclophosphamide therapy in womenresulted in a 57% incidence of amenorrhealasting more than one year or inability tobecome pregnant.151 The risk relates to cumu-lative dose and regimen and pulse cyclophos-phamide is probably less detrimental to fer-tility.152 These days counselling should bemandatory, particularly in younger patients.Data in men treated with cyclophosphamideare not nearly as good in vasculitis but stud-ies using cyclophosphamide to treat tumorsshow there is no reason to believe their fer-tility should be spared by alkylating agentssuch as cyclophosphamide in vasculitis.Cryopreservation of sperm should thereforebe considered if appropriate.

Inflammatory vascular disease also con-fers an increased risk of cardiovascular dis-ease 153 although the evidence is stronger forlupus 154 than for ANCA-associated vasculi-tis.155 It is not quite clear whether theincreased risk relates to the disease, its treat-ment, or both. Anti-hypertensive therapy iscrucial, as is treatment of risk factors such ashyperlipidemia.


of Rheumatology criteria, are in use concur-rently. A unified classification has been pro-posed but is not yet widely used.141 TheEuropean Vasculitis Study group (EUVAS)has previously proposed definitions of diseaseactivity and stages to guide therapy and clin-ical studies (Table I).142 Briefly, non-organ ornon-life-threatening disease, which can belocalized or early systemic, is distinguishedfrom organ-threatening disease, which canbe generalized, severe or refractory. Otherinstruments are also in use to assess diseaseactivity. Merkel et al. correlated these differ-ent tools and found them highly correlatedand reliable.143 A second generation of suchtools for the assessment of disease activity iscurrently under development.144

Treatment strategies have evolved sincethe introduction of glucocorticoids in 1948.Methotrexate, azathioprine and cyclophos-phamide were introduced in the 1960’s, andplasma exchange in 1976. It is worthwhileto remember that long-term survival andremission only became feasible with the intro-duction of Cyclophosphamide by Fauci.145

Before the introduction of cyclophos-phamide-based therapeutic regimens, mor-tality of Wegener’s granulomatosis at 1 yearwas 80%.146 Since then, much has beenlearned through numerous studies, includ-ing multi-centre, international and prospec-tive trials over the last 20 years. It is throughthis body of evidence that ANCA-associatedvasculitis has now become a chronic andtreatable disease. 5-year survival rates nowapproach 80% 147 although relapses are notuncommon.148 Finally, it is noteworthy thatelderly patients still have a poor prognosis.149

The cytotoxic drug regimens currently usedhave a significant toxicity, which can con-tribute to morbidity and mortality. A consid-erable share of the current mortality is indeeddue to infections. Malignancy is of similarconcern and cyclophosphamide is usuallyimplicated whereby the cumulative doseseems to be of greatest importance. A largestudy from Scandinavia reported a twofoldincreased risk of cancer when compared tothe general population.150 Cancer of theurothelium and skin as well as lymphomaand leukemia are most common.150 Life-long

TABLE I.—Definitions of disease stages of ANCA-asso-ciated vasculitis.156

Category Definition

Localised Upper and/or lower respiratory tractdisease without any other systemic invol-vement or constitutional symptoms

Early systemic Any, without organ or life-threateningdisease

Generalized Renal or other organ threatening disease,serum creatinine <500 µmol/litre (5.6mg/dl)

Severe Renal or other vital organ failure, serumcreatinine >500 µmol/litre (5.6 mg/dl)

Refractory Progressive disease unresponsive to glu-cocorticoids and cyclophosphamide

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Given this considerable toxicity of treat-ment, more recent studies have attempted toestablish treatment strategies with reducedtoxicity that are more tailored to the individ-ual patient’s characteristics. Therapeutic con-cepts are also changing from permanent cyto-toxic treatment to sequential treatment strate-gies using less toxic drugs after remission hasbeen achieved. This approach has surelyreduced mortality and morbidity and must beused as a reference to test the efficacy andsafety of newer drugs. A recent paper from theEuropean Vasculitis Study Group has sum-marised current recommendations for themanagement of ANCA-associated small andmedium vessel vasculitis.156 Nevertheless itremains sobering that some forty years afterthe introduction of cyclophosphamide thistoxic drugs still remains the mainstay of treat-ment in severe disease. Finally the issue ofprophylaxis of treatment toxicity, for examplewith trimethoprim/sulphamethoxazol to pre-vent pneumocystis pneumonia or with mes-na during cyclosphosphamide treatment, isas important as the therapy itself but beyondthe scope of this article.

Induction of remission in generalizedvasculitis

Oral cyclophosphamide (Cyc) and gluco-corticoids represented the first treatment to

permit long-term survival in generalized andorgan-threatening disease (including renalfailure with serum creatinine >500 µmol/L).151,157 However oral cyclophosphamide is asso-ciated with considerable toxicity from treat-ment. In the seminal NIH study haemorrhag-ic cystitis occurred in 50%, bladder cancer in5.6%, myelodysplasia in 2%, and lymphomain 0.7%.151 Subsequent studies were per-formed to determine whether intravenouspulse cyclophosphamide was equally effectivefor the induction of remission.158 Three ran-domized-controlled trials showed that bothregimens have similar efficacy in achievingremission when compared to oral cyclophos-phamide (Table II).159-161 However, intra-venous cyclophosphamide resulted in lowercumulative doses (over 50% reduction) andless side effects such as infection and leu-copenia.161 A meta-analysis concluded thatintravenous cyclophosphamide was equallyeffective but associated with a higher relapserate as compared to oral cyclophosphamide.157A very recent randomized trial, published thisyear, showed that intravenous pulsecyclophosphamide was equally effective ininducing remission when compared to oralcyclophosphamide.162 These data give us fur-ther confidence to adopt intravenous pulsecyclophosphamide as the new standard forinduction of remission with less drug toxici-ty. However this issue remains debated andlonger follow-up remains desirable. Several


TABLE II.—Treatment of ANCA-associated vasculitis, modified after.156

Category Induction Maintenance

Localized

Early systemic

Generalized

Severe

Refractory

Methotrexate and steroids

Steroids and either methotrexate orcyclophosphamide

Cyclophosphamide (preferably intrave-nous for 3-6 months; oral 2mg/kg bodyweight/day) and steroids (initially intra-venous boli, followed by 1mg/kg/dayfor 1 month)

Cyclophosphamide (intravenous or oral)and steroids plus plasma exchange

Rituximab, steroid pulses, infliximab,immunoglobulins, Deoxyspergualin,anti-thymocyte globulin

Azathioprine or methotrexate plus lowdose steroids

—

Steroids and either Azathioprine,Methotrexate (in pa-tients with normalrenal function) or alternativelyLeflunomide

MMF in patients with intolerance toAzathioprine

No consensus

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considerable experience in these patients.Smaller centres may also struggle with thelogistics, particularly out of hours. Thus, itremains to be seen whether plasma exchangeis also advantageous in less severe cases(serum creatinine <500 µmol/L) and furtherstudies, such as PEXIVAS, are awaited. Finally,in MEPEX the mortality of severe ANCA-asso-ciated with renal failure vasculitis remainedhigh, i.e. around 25%, regardless of whethersteroid pulse or plasma exchange was used.168This may be attributed to the use of highsteroid dosis and oral cyclophosphamide inan old patient population.

Induction of remission in non-organ-threatening and granulomatous disease

Early disease of the upper respiratory tractcan be managed with steroids and oraltrimethoprim/sulphamethoxazole althoughthis treatment option has become somewhatless popular in recent years. Topical treat-ment is also crucial especially if severe rhini-tis is present.168 Nasal oil and saline nosespray are useful as well as topical Mupirocinif Staphylococcus is present.

Induction of remission in non-organ-threat-ening disease without renal imPirment can beachieved with low-dose once weeklymethotrexate (MTX), which is somewhat lesstoxic than Cyclophosphamide although notentirely without problems. The NORAM studyconfirmed its efficacy in early systemic diseasebut also showed a high rate of relapse.169

Furthermore, the drug is contra-indicated inrenal failure. There is also a need for propercounselling in once weekly oral treatment isbegun: Fatal leukopenia may ensue if patientstake the drug daily. Pulmonary and hepatictoxicity is another matter of concern.Additionally, one small, randomized trial ofmoderate renal vasculitis found similar induc-tion rates with mycophenolate mofetil com-pared to cyclophosphamide 170 althoughthese data require confirmation in larger stud-ies. For now, methotrexate remains an alter-native to cyclophosphamide in mild diseasewithout renal involvement.

A feared manifestation of WG is bronchial/


variants of intravenous pulse cyclo-phos-phamide are in use and doses are around 0.5to 1.0 g/m2 body surface area. Many clini-cians use 750 mg/m2 as standard dose. Someadjust the dose according to the leukocytenadir at 14 days; newer regimens also adjustthe dose for age and renal impairment.163 Theduration of intravenous cyclophosphamidetreatment is also unclear. Traditionally mostclinicians would treat for one year and thenswitch to Azathioprine but now the tendencyis towards shorter duration, for example threeto six months. There is currently no firm evi-dence in this regard and most clinicians willmake an individual decision in each case,based on the extent and severity of diseaseand the risk of immunosuppression. Of note,we have learned from CYCAZAREM 164 andWEGET 165 that most patients achieve remis-sion within six months after diagnosis.

Plasma exchange has long been used insevere cases of ANCA-associated small-vesselvasculitis, particularly those with pulmo-renalsyndrome and diffuse alveolar haemorrhage.An earlier small retrospective study in 20patients with alveolar hemorrhage syndromeshowed resolution of hemorrhage in allpatients using plasma exchange.166 More con-vincing evidence has now emerged. In therecent MEPEX study, plasma exchange hasbeen shown to improve renal recovery forpatients with ANCA-associated vasculitis pre-senting with renal failure (serum creatinine>500 µmol/L): this trial showed a 69% renalrecovery rate in patients with AAV with serumcreatinine >500 µmol/L treated with plasmaexchange comPred to 49% with methylpred-nisolone.167 All patients received continuouscyclophosphamide and oral prednisolone aswell. On that basis, the role of plasmaexchange in severe ANCA-associated small-vessel vasculitis seems to be well established.However, plasma exchange is not a risk-freetreatment. Increased heparin exposure andvolume overload may prove detrimental inpatients who are dialysis-dependent alreadyand especially those with pulmonary haem-orrhage. Whether the issue of heparin expo-sure can be circumvented in by citrate anti-coagulation remains unproven. Use and tim-ing of plasma exhange will for now require

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laryngeal stenosis.171, 172 These lesions candevelop largely independent of all other dis-ease activity and prove be very refractory totreatment. These cases require a multi-disci-plinary approach involving interventionalistsfrom other fields. Intra-lesional steroids havebeen used with good success in active diseaseand repeated dilation of chronic stenoses isfeasible in experienced hands.173

Maintenance of remission

It is well known that remission can bemaintained with oral cyclophosphamide andtapering doses of prednisolone, albeit at thecost of substantial drug toxicity and sideeffects. Newer drug regimens using sequen-tial drug therapy have aimed to maintainremission with less toxicity. In the CYCAZ-EREM trial in 2003,164 144 AAV-patients(WG/MP or renal limited variant) with gen-eralized disease (Crea <500 µmol/L) receivedoral cyclophosphamide plus glucocorticoidsand were randomized to either continue oralcyclophosphamide or to switch to azathio-prine (Aza) after induction of remission. Nodifference in relapse rates, renal function andprogression to end-stage renal disease wasseen after 18 months (13.3% vs. 15%) . Thesedata suggested that a switch to azathioprineafter remission is effective and safe. Are therealternatives to azathioprine? In the WEGENTtrial, published in 2008, 126 patients (WG ormP) were randomized to receive either MTXor Aza for 12 months after successful induc-tion of remission with intravenous cyclophos-phamide.174 There was no difference inrelapse free survival and toxicity betweentheses two drugs, demonstrating thatmethotrexate can also be used as an alter-native maintenance drug.174 Thereforemethotrexate may be regarded as an alter-native in patients who cannot tolerate aza-thioprine or who are younger, as one wouldlike to avoid the effects on skin and devel-opment of secondary malignancy. Howeverone must bear in mind that somewhat high-er relapse rates have previously been report-ed with the drug.175 Are there further alter-natives? Mycophenolate mofetil (MMF) can

also be used as an alternative, but data fromuncontrolled prospective and retrospectivestudies also show a higher relapse rate.176, 177

Preliminary data from a EUVAS study(IMPROVE) showed earlier relapses in patienttreated with MMF than in the azathioprinegroup. Others have used a novel drug,Leflunomide, an immuno-modulatory druginhibiting dihydroorotate dehydrogenase, tomaintain remission.178 They subsequentlydemonstrated that leflunomide was moreeffective than methotrexate 175 although thedrug has frequent side effects. Neuropathy isof particular concern.179

To maximize the effectiveness of mainte-nance regimens, the treatment strategy mayhave to be individualized according to theclinical context. For example, maintenanceAzathioprine has been associated with sig-nificantly higher relapse rates in patients thathad positive ANCA levels at the time ofswitching.180 Also, patients with WG have ahigher tendency to relapse than patients withMP. Earlier studies have linked relapse in WGto nasal carriage of staphylococcus aureus.64

Interestingly, relapse rates in patients withrespiratory tract involvement could bereduced by adding sulfamethoxazole/trime-thoprim to the maintenance regimen.181 Morerecently, this strategy has been somewhatforgotten and no recent studies haveaddressed this therapeutic approach.

The strongest predictor of relapse is with-drawal of immunosuppression.162 There ispresently no consensus on the duration ofmaintainance therapy, and recommendationsrange from 18 months to 5 years in patientswith persistent ANCA positivity. This issue isaddressed in another ongoing trial (REMAIN).This interesting study will evaluate whetherprolonged maintenance therapy with low-doseprednisolone and azathioprine reduces long-term morbidity in systemic vasculitis, by reduc-ing the frequency of relapse, when comparedwith cessation of therapy in the second year.

Emerging therapies

Empiric cytotoxic treatment has improvedthe survival of patients with AAV significant-


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Some have reported that granulomatoushead and neck manifestations of WG are alsoamenable to rituximab.185 Others describefailure of rituximab treatment in granuloma-tous disease.186 It is probably fair to say thatthese findings highlight the need for a betterunderstanding of the interplay between gran-ulomatous lesions and B cells. Furthermore,the exact mechanism of action of rituximabin ANCA vasculitis is incompletely under-stood, since CD20 is not expressed on plas-ma cells themselves, but ANCA titres decreasefollowing rituximab treatment. Another unre-solved problem is the timing of re-treatment.A recent multi-centre survey, while confirm-ing the efficacy of rituximab, showed thatANCA or B cell levels are not ideal to guidere-treatment.183 Potentially, the effect can beexplained by the interference of rituximabwith B-cell proliferation in the setting of highplasma-cell turnover,187, 188 or being located inanatomical niches. Despite our limited under-standing regarding its mechanism of actionand concerns about long-term toxicity, rit-uximab has probably been the most signifi-cant addition to our therapeutic armamen-tarium in the last five years. Two large con-trolled studies (RITUXVAS and RAVE) gavefirst very promising results regarding its useas a first line therapy with the chance toreduce (RITUXVAS) or avoid (RAVE)cyclophosphamide.

Newer B-cell depleting therapies such asocrelizumab (humanized anti CD20), ofatu-mumab (human anti-CD20) and epratuzum-ab (humanized anti-CD22) have been devel-oped. Some of these agents have alreadybeen used in systemic lupus erythematosusand rheumatoid arthritis while no data haveyet been reported in vasculitis. Anotherpotential target of therapy is the B lymphocytestimulator (BAFF/BLYS), a member of theTNF superfamily that is critical to B-cell devel-opment.189 The crucial role of BAFF/BLYS inlupus is only just emerging.190 TNF canincrease the release of BAFF/BLYS from neu-trophils 191 and BAFF/BLYS serum levels havebeen found to be increased in patients withvasculitis.73 Therapeutic manipulation isalready available through Belimumab (huma-nized anti-BAFF/BLYS). It remains to be seen


ly and newer regimens have reduced treat-ment-related toxicity and morbidity. Howeverby today’s standards many of these drugs,such as cyclophosphamide, seem crude andnot targeted on the pathogenetic mechanismsof vasculitis. In addition, relapsing and refrac-tory disease remain challenging to treat.Newer treatment strategies are thereforeeagerly awaited. It must be assumed that pre-venting the initiation of the inflammatory cas-cade is the most effective treatment in vas-culitis. If this is impossible, the goal is toattenuate subsequent immune responses bytargeting crucial pathogenetic mechanismsor inflammatory mediators. Directing thera-pies at these sequential targets will go beyondnon–specific immunosuppression and hope-fully expose patients to less toxicity. Most ofthe newer drugs are used either as addition-al or adjunctive treatment, only some of themare investigated as an alternative to the stan-dard of care.

Therapy directed at B cells

One of the most exciting new develop-ments in the treatment of ANCA-associatedvasculitis is the emergence of a new target fortreatment, the B lymphocyte. B-cells play animportant role during the pathogenesis ofthe disease as they differentiate to plasmacells that must eventually produce the ANCAantibodies. These ANCA-secreting B cells arebelieved to mature and reside within granu-lomatous lesions in WG.70 Rituximab, an anti-CD20 chimeric monocloncal antibody, hasallowed partial or complete remissions inrelapsing or refractory AAV in 70-100% insmall case series or case reports.182 The largestretrospective multicenter study of 65 patientsin the UK showed at least partial response in99% of all cases.183 Clinical relapses occurredfrequently, approximately 30% within thefirst 2 years. Most relapses occurred afterreconstitution of peripheral B cells. Leucope-nia and infectious complications such aspotential reactivation of hepatitis B virus 182

and cytomegalovirus is a matter of concernalthough clinically the drug is remarkablywell tolerated. Progressive multifocal leukoen-cephalopathy has also been reported.184

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whether BAFF/BLYS could become a thera-peutic target in vasculitis. Figure 5 providesan overview of current and future therapeu-tic interventions directed at B cells.

Therapy directed at T cells

The production of ANCA is dependenton T cells. T cells are highly activated withincreased expression of HLA-DR and CD25and predominantly show a Th1 cytokinephenotype. They are found in affected tis-sues, in granulomas and vasculitic lesions.192

Effector memory T cells are increased andpossibly not adequately controlled by reg-ulatory T cells, leading to chronic inflam-

mation in AASV.65 Many of the establisheddrug treatments, such as azathioprine ormycophenolate mofetil (MMF), affect T lym-phocytes albeit in a non-specific manner.15-deoxyspergualin (gusperimus) is a nov-el T cell directed drug.193 It inhibits the inter-leukin-2-stimulated maturation of T cellsand the polarization of T cells into IFN-gamma-secreting Th1 effector T cells result-ing in growth of activated naive CD4 T cells.Deoxyspergualin also inhibits cytokine andmonocyte activation. It has been effective ina small group of patients resistant or intol-erant to cyclophosphamide.194 Leukopeniais a significant side effect, but seems to be


Figure 5: B cells as current and future targets for therapeutic intervention. BAFF denotes the BAFF/BLYS factor. BAFFRdenotes BAFF/BLYS receptor. CTLA4 denotes Cytotoxic T-Lymphocyte Antigen 4 and B7 its receptor. APRIL denotes aproliferation-inducing ligand. TACI denotes transmembrane activator and CAML interactor, the receptor for BAFF/BLYSSand APRIL. BCMA denotes B cell maturation antigen. TCR denotes T cell receptor. CD40 denotes cluster of differenti-ation 40 and CD40L its ligand. CD 20 denotes cluster of differentiation 20. Note that BAFF/BLYS and APRIL promote sur-vival of B lymphocytes. BAFF/BLYS and APRIL work via TACI whereas APRIL can also activate via BCMA. Therapeuticmanipulation of BCMA and TACI is not yet available. Anti-CD40 is also available (Dacetuzumab).

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Therapy directed at individual cytokines

In vitro and in vivo evidence demonstratesthat TNF-α plays a central role during thepathogenesis of ANCA-associated vasculitis.TNF-α mediates granuloma formation,endothelial activation and neutrophil primingin ANCA-mediated vascular damage. TNF-αis also increased in serum of patients withactive vasculitis.202 In two animal models anti-bodies against TNF-α prevented or attenu-ated ANCA induced crescentic glomeru-lonephritis.203, 204 Pilot studies showed a ben-efit of treatment with Etanercept, is a recom-binant, soluble, human, TNF receptor fusionprotein.205, 206 A larger randomized, placebo-controlled trial (WGET) evaluated the effica-cy of Etanercept in inducing and maintainingremission in 180 WG patients as an adjunct tostandard therapy. No additional effect ofEtanercept was found and there were moreadverse-events.164 The increased incidenceof malignancy is of particular concern.207

Infliximab, a chimeric anti TNF monoclonalantibody has been studied in open-label tri-als including 51 patients with AASV.205, 208-210

In this heterogenous group Infliximabinduced remission in 70-88% of patients withrefractory vasculitis. However an increasedrate of serious infections has been seen withthe drug,211 most notably tuberculosis.212 Asof today, no randomized trial of infliximab inpatients with AASV exists. Infliximab maypossibly also help in resistant granulomatousdisease, as efficacy against granulomatousinflammation has been shown in patientswith Crohn’s disease.213

Interleukin-5 (IL-5) can also be targetedby an antibody (mepolizumab) whichreduced dosage of glucocorticoids in thehypereosinophilic syndrome 214 and is cur-rently investigated in patients with ChurgStrauss Syndrome. IL-5 is intimately linkedto the eosinophil inflammatory response. Itsutility may therefore be limited to the ChurgStrauss syndrome, in which eosinophils playa crucial role.

IL-6 is another pro-inflammatory cytokine.Its role in other inflammatory diseases, suchas inflammatory bowel disease, is well char-acterised although its importance in vasculi-


manageable.195 Another issue is that thedrug is administered by subcutaneous self-injection, which may not be feasible in eachand every patient. Unfortunately, diseaseflares were frequent after the drug was with-drawn.

Another unspecific T cell directed drug isantithymocyte globulin, which represents acollection of polycloncal antibodies direct-ed against the surface antigens of activated Tcells. In a small study in 15 patients with WGresistant to cyclophosphamide it has beenshown to be effective.196 The monoclonalanti-CD52 antibody alemtuzumab 197 is amore selective T cell drug. Alemtuzumab hasbeen used earlier to treat patients with refrac-tory or relapsing AASV.56 In the largest avail-able study conducted in 71 patients, 65%went into clinical remission and 20% had asignificant improvement. Disease relapse andinfectious complications, however, were quitecommon.198

T cell activation is thought to be due to a“two-signal model” in CD4+ T cells. Activationof CD4-positive T cells occurs throughengagement of the T cell receptor and co-stimulation of CD28 on the T cell surface bythe major histocompatibility complex pep-tide and B7 family members on the antigenpresenting cell. Cytotoxic-T-lymphocyte-asso-ciated antigen 4 (CTLA 4) can prevent “deliv-ery” of the second co-stimulatory signalrequired for complete activation of T-cells.The gene encoding CTLA-4 has multiple poly-morphisms and a positive associationbetween WG and longer alleles of (AT)n inthe CTLA-4 gene has been demonstrated.199

In addition, CTLA4-IG has prevented diseaseprogression in an animal model of crescen-tic glomerulonephritis.200 Unfortunately, a tri-al investigating CTLA-4 IG as an adjunctive toMTX to induce and maintain remission inearly systemic ANCA-associated vasculitiswas terminated, apparently due to lowrecruitment and supply issues with the spon-soring manufacturer. Finally, it has to be saidthat the catastrophic experience with the anti-CD28 Monoclonal Antibody TGN1412 201 hasdampened the initial enthusiasm regardingtherapeutic manipulation of co-stimulatorysignals.

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tis is not quite clear. IL-6 can be targeted bytocilizumab, a fully humanized monoclonalanti-IL-6 antibody. Tocilizumab did showbenefit in patients with rheumatoid arthritis216. Animal data and studies in systemic lupuserythematosus show some favorable effecton extrarenal manifestations 216 but its efficacyin vasculitis remains unclear.

Therapy directed at other targets

Intravenous immunoglobulin (IVIG) is animmune-modulatory drug with multipleeffects. It is thought to interact with theinhibitory Fc receptor. A few open-label stud-ies have shown that IVIG is effective in refrac-tory ANCA-associated vasculitis and in earlyactive disease.217 IVIG is a potential treat-ment alternative if standard care is not feasi-ble, e.g. in the context of active infection orpregnancy.

Furthermore, recent evidence also suggestsa crucial role for the alternative pathway ofcomplement activation.218 Traditionally, theANCA-associated vasculitides have been clas-sified as pauci-immune, suggesting that com-plement plays no major role in the diseaseprocess. More recently, however, it hasemerged that complement may be moreimportant than initially believed. Accordingly,monoclonal antibodies against the comple-ment component C5 can significantly atten-uate MPO-ANCA crescentic glomeru-lonephritis in mice.203 Also, C5a and its recep-tor have beed described as an amplificationloop for ANCA-mediated neutrophil activa-tion.219 Eculizumab, a humanized monoclonalantibody can inhibit C5 cleavage. The drughas been used in hemolytic uremic sydnrome220 but no data are currently available in vas-culitis.

A common theme in recent years has beenan increased understanding of the pathwaysof neutrophil signaling in vasculitis. Moreand more of these pathways are thereforebecoming potential targets for treatment.Imatinib mesylate, a tyrosine kinase inhibitorused in malignancies as chronic myeloidleukemia, has been used in vitro on cellsfrom patients with active vasculitis. Theseisolated cells showed less expression of

CD25, and decreased conversion of T cells tomemory T cells in vitro. Its use in experi-mental models of rheumatoid arthritis 221 alsosuggest a possible benefit in other autoim-mune diseases, although data in vasculitisare lacking. Another crucial pathway seemsto be that of an isoform of the protein kinasePI3K, which suppresses ANCA-mediated neu-trophil chemotaxis and activation. It mightbe another treatment target.222 Inhibition ofthis kinase can decrease the inflammation inSLE and RA.223, 225 P21ras is also crucial tosuperoxide production and vascular necrosis,two salient features of ANCa-associated vas-culitis. Farnesylthiosalicylic acid is a selec-tive inhibitor of mediator p21ras. The inhibi-tion of this downstream mediator p21rascould diminish superoxide production andvascular necrosis. Nevertheless, the use ofkinase inhibitors as an antiproliferative agenthas still to be proven in clinical studies.

Conclusions

Much has been learned in recent years onthe pathogenesis of ANCA-associated small-vessel vasculitis. We already knew that inter-action of primed neutrophils with ANCA andendothelial cells is crucial to the disease. Nextwe gained a better understanding, from ani-mal models, of the pathogenetic importanceof the ANCA antibody although cliniciansremain sceptical, given the fact that somepatients have high titres but no disease. Veryrecent evidence provides intriguing dataregarding the link between infection and vas-culitis, LAMP-2 antibodies 85 as novel mark-ers, and NETs as a novel pathogenetic mech-anism. It remains to be seen whether othersare able to corroborate these findings andwhether testing for LAMP-2 antibodies willbecome part of the clinical routine in vas-culitis. Recent years also saw the emergenceof various new markers of endothelial dam-age and the disease itself, such as circulatingendothelial cells and endothelial microparti-cles. Available data suggest that these novelmarkers correlate well with disease activityand that they may well complement tradi-tional diagnostic tools, such as ANCA test-


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ultimi anni è stato osservato un incremento nellepubblicazioni di studi in merito al trattamento dellavasculiti ANCA-positive. Rispetto a cinque o diecianni fa, conosciamo meglio il ruolo della ciclofosfa-mide somministrata per via endoveosa e dei plasmaexchange. Sono tuttavia necessari ulteriori studi alfine di valutare gli effetti dei plasma exchange nelleforme di vasculiti meno gravi. Infine si spera che inun futuro prossimo si possano capire meglio i mec-canismi patogenetici della malattia al fine di potergarantire terapie più specifiche e meno tossiche.

Parole chiave: Vasculiti, diagnosi – Vasculiti, anatomiapatologica – Vasculiti, terapia farmacologica.

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ing. Preliminary evidence has provided someinsight into the balance between endothelialdamage and repair. Exciting preliminary dataalso indicate that circulating endothelial cellsmay not only be markers of disease activitybut that these cells may have pathogeneticimportance in their own right. These find-ings may have profound implications for thepathogenesis of vasculitis and vascular dis-ease in general. Recent years also saw thepublication of a number of seminal studies forthe treatment of ANCA-associated vasculitis.We now have a much better understanding ofthe role of pulse intravenous cyclophos-phamide and plasma exchange than ten oreven five years ago. Further studies mustnow show whether plasma exchange is alsobeneficial for less severely ill patients withAASV. Finally, as ever, it is hoped that furtherprogress in understanding the disease patho-genesis will also provide more tailored andless toxic therapies.

Riassunto

Vasculiti da ANCA-positive: patogenesi, nuovi markerdi malattia e terapie emergenti

Negli ultimi anni la patogenesi delle vasculiti deipiccoli vasi ANCA-positive è stata oggetto di moltistudi. L’interazione dei neutrofili con gli ANCA e le cel-lule endoteliali è cruciale nella patogenesi della malat-tia. Successivamente studi su modelli animali hannopermesso di capire l’importanza patogenetica deglianticorpi ANCA. Evidenze recenti hanno mostratonuovi modelli patogenetici quali la relazione fra leinfezioni e le vasculiti, gli anticorpi LAMP-2 e NET.Rimane solo da stabilire se altri studi siano in gradodi corroborare questi risultati e se il dosaggio deglianticorpi LAMP-2 possa essere utilizzato come inda-gine di routine nelle pratica clinica. Negli ultimi annisono stati introdotti nuovi marker di danno endote-liale della malattia, come cellule endoteliali circolan-ti e microparticelle endoteliali. Questi nuovi markersono correlate con la attività della malattia e posso-no essere utilizzati come mezzi diagnostici oltre aquelli tradizionali come i test per gli ANCA. Alcuni stu-di preliminari hanno osservato una correlazione fra ildanno e la ripazione endoteliale. Sono stati riporatirisultati promettenti che indicano che le cellule endo-teliali circolanti non sono marker della attività dellamalattia, ma presentano comunque un ruolo fonda-mentale nella patogenesi della malattia stessa. Questirisultati hanno implicanze nella patogenesi dellevasculiti e delle malattia vascolari in generale. Negli

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