Pathogenesis of ANCA-Associated Vasculitis: New Possibilities for Intervention

In Translation Pathogenesis of ANCA-Associated Vasculitis: New Possibilities for Intervention Cees G.M. Kallenberg, MD, PhD,1 Coen A. Stegeman, MD, PhD,2 Wayel H. Abdulahad, PhD,1 and Peter Heeringa, PhD3 The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) comprise granulomatosis with polyangiitis (GPA), primarily associated with antibodies to proteinase 3 (PR3-ANCA); microscopic polyangiitis (MPA); and eosinophilic granulomatosis with polyangiitis (EGPA), both principally associated with antibodies to myeloperoxidase (MPO-ANCA). Genetic and environmental factors are involved in their etiopathogenesis, with a possible role for silica exposure in AAVs and Staphylococcus aureus infection in GPA. The distinct associations of PR3-ANCA and MPO-ANCA with different HLA class II antigens, which are stronger than those with the associated diseases, suggest a pathogenic role for those ANCAs and indicate that GPA and MPA are different diseases. Both in vitro and in vivo experimental data have shown that MPO-ANCA can induce necrotizing small-vessel vasculitis and glomerulonephritis. The additional role of the alternative pathway of complement activation has been demonstrated in human and experimental pathology. Also, T cells seem to be involved in lesion development, particularly in the kidney. Granuloma formation, as seen in PR3-ANCA–associated GPA, is not well explained by the presence of autoantibodies in experimental models. Here, T cells seem crucial. Recently obtained insights into the pathogenesis of AAVs have led to more targeted treatment of these life-threatening diseases. Am J Kidney Dis. xx(x):xxx. © 2013 by the National Kidney Foundation, Inc. INDEX WORDS: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; antibodies to proteinase 3 (PR3-ANCA); antibodies to myeloperoxidase (MPO-ANCA); granulomatosis with polyangiitis (GPA); microscopic polyangiitis (MPA); pathogenesis.

BACKGROUND The antineutrophil cytoplasmic antibody (ANCA)associated vasculitides (AAVs) comprise granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA; formerly Churg-Strauss syndrome).1 When limited to the kidney, the condition is designated as isolated pauci-immune necrotizing crescentic glomerulonephritis (GN) or renal-limited vasculitis. These diseases are characterized by pauciimmune necrotizing small-vessel vasculitis and GN, combined with granulomatous inflammation, particularly in the airways, in GPA and EGPA. EGPA also shows blood hypereosinophilia and eosinophilic infiltration in affected tissues. Definitions of GPA, MPA, and EGPA have been proposed by the 2012 Chapel Hill Consensus Conference,1 but reliable and validated diagnostic criteria are still not available, although a large prospective international study aimed at establishing these criteria (Diagnosis and Classification of Vasculitis Study [DCVAS]) is underway.2 A hallmark of the AAVs is the presence of autoantibodies directed at neutrophil cytoplasmic constituents (ie, ANCAs). The target antigens of ANCA in the AAVs are proteinase 3 (PR3) and myeloperoxidase (MPO). GPA is associated primarily with PR3-ANCA, whereas MPA and EGPA are associated principally with MPOANCA (Table 1).3,4 In contrast to patients with GPA and MPA, a significant proportion of patients with Am J Kidney Dis. 2013;xx(x):xxx

EGPA have undetectable ANCA. ANCA status leads to a dichotomy in the clinical expression of EGPA: ANCA-positive patients largely show necrotizing small-vessel vasculitis, whereas ANCA-negative patients more frequently have a clinical phenotype dominated by tissue infiltration with eosinophils.5

CASE VIGNETTE A 47-year-old man without relevant medical history presented in mid 2005 with progressive nasal obstruction and crusting with epistaxis, loss of smell, migrating arthralgias, involuntary weight loss of 5 kg over the prior 2 months, and intermittent fever with night sweats for the last month. For presumed airway infection, he had received 2 courses of antibiotics, doxycycline, 100 mg, daily for 10 days followed by amoxicillin-clavulanic acid, 500/125 mg, thrice daily for 7 days, without effect. On referral, body temperature was 37.8°C, blood pressure was 150/90 mm Hg, and respiratory rate was 20 breaths/min. Body weight was 83 kg, and he was

From the Departments of 1Rheumatology & Clinical Immunology, 2Nephrology, and 3Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. Received December 12, 2012. Accepted in revised form May 14, 2013. Address correspondence to Cees G.M. Kallenberg, MD, PhD, Department of Rheumatology & Clinical Immunology, AA21, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. E-mail: [email protected] © 2013 by the National Kidney Foundation, Inc. 0272-6386/$36.00 http://dx.doi.org/10.1053/j.ajkd.2013.05.009 1

Kallenberg et al Table 1. ANCA Specificities in ANCA-Associated Vasculitides ANCA Specificity No. of Diagnosis Patients

GPA MPA NCGN EGPA

PR3-ANCA

MPO-ANCA

None

ANCA Positive

324 16 4 0

25 67 47 23

16a,b 2c 3c 13

96% 98% 94% 64%

364 85 54 36

Note: ANCA as present in 539 consecutive patients with ANCA-associated vasculitis seen at the University Medical Center Groningen, the Netherlands, in 1998-2008. Abbreviations: ANCA, antineutrophil cytoplasmic autoantibodies; EGPA, eosinophilic granulomatosis with polyangiitis; GPA, granulomatosis with polyangiitis; MPA, microscopic polyangiitis; MPO, myeloperoxidase; NCGN, necrotizing crescentic glomerulonephritis; PR3, proteinase 3. a Four were positive for elastase-ANCA. b Eleven had ear, nose, and throat–limited GPA. c One was positive for elastase-ANCA.

182 cm tall. Examination was notable for purpura of the skin of his lower legs without edema, blistering, or defects and left foot drop in combination with sensory loss and paresthesias on touch in both feet and lateral and frontal portions of the lower legs. In addition, nasal obstruction with bloody crusting and postnasal drip was noted. Laboratory results showed the following values: white blood cell count, 14.3 ⫻ 109/L; hemoglobin, 9.2 g/dL; platelets, 612 ⫻ 109/L; sodium, 141 mEq/L; potassium, 5.4 mEq/L; serum urea nitrogen, 49.9 mg/dL; creatinine, 2.9 mg/dL (estimated glo-

prednisolone (mg/day) 60 40

80 60

-

merular filtration rate [eGFR], 25 mL/min/1.73 m2 by the 4-variable MDRD [Modification of Diet in Renal Disease] Study equation); erythrocyte sedimentation rate, 98 mm/h; and C-reactive protein (CRP), 235 mg/L. Serum lactate dehydrogenase level was slightly elevated at 311 (reference ⬍235) U/L, but all other liver function test results were unremarkable. Urine sediment evaluation showed numerous acanthocytes and occasional erythrocyte casts. Proteinuria was present with a spot urine protein-creatinine ratio of 1.9 g/g. Indirect immunofluorescence testing with serum of the patient on ethanol-fixed neutrophils showed ANCAs with a cytoplasmic granular pattern (C-ANCA) at a titer ⱖ1:640. Enzymelinked immunosorbent assay confirmed the presence of antibodies directed against PR3 (PR3-ANCA). Chest radiograph showed some ill-defined peripheral infiltrates without nodules or cavitation, compatible with alveolar bleeding in both lungs. A diagnosis of PR3-AAV with the clinical picture of GPA with involvement of the ear, nose, and throat; lungs; kidneys; and peripheral nervous system was made. Treatment with prednisolone, 1 mg/kg/d (80 mg), in combination with oral cyclophosphamide, 2 mg/kg/d (150 mg), was started. After 3 weeks of therapy, the patient’s clinical condition was much improved. The purpura had nearly disappeared without new lesions; arthralgias, fever, and night sweats had resolved. Nasal crusting had improved with some return of smell and absence of epistaxis. The foot drop on the left was still present. Laboratory results showed normalization of CRP level (⬍3 mg/L) and a persistently elevated C-ANCA titer at ⬎1:640 (Fig 1). Urinalysis still showed numerous erythrocytes with acanthocytes, but no erythrocyte casts on microscopy. Proteinuria was still present (protein excretion, 3.2 g/24 h). Serum creatinine level had increased slowly to 4.3 mg/dL (eGFR, 16 mL/min/ 1.73 m2). Kidney biopsy was performed: 19 glomeruli were present, of which 3 were globally sclerosed, 5 were normal, and

30

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20

cyclophosphamide 150 mg/d

5

250

-

40

-

30

-

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-

10

-

0

-

200

4

serum creatinine (mg/dl)

50

renal biopsy

150

3 100 2 50

1

C-reactive protein (mg/l)

eGFR (ml/min/1.73m2)

plasmapheresis

0

0

1

2

3

4

5

6

7

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12

13

14

15

16

follow up (weeks since presentation) C-ANCA titer (dilution) >640 >640 >640 320

80

20

40

40

40

20

40

Figure 1. Course of serum creatinine level [●], estimated glomerular filtration rate (eGFR) [〫] (left vertical axis), C-reactive protein level [Œ] (right vertical axis), antineutrophil cytoplasmic antibodies with a cytoplasmic pattern (C-ANCA) titer (dilution titer below the horizontal axis), and treatment (horizontal bars and open triangles at the top of the figure) in a 47-year-old man presenting with active granulomatosis with polyangiitis. Conversion factor for creatinine in mg/dL to ␮mol/L, ⫻88.4. 2

Am J Kidney Dis. 2013;xx(x):xxx

Pathogenesis of ANCA-Associated Vasculitis the other 11 showed segmental or global fibrocellular or cellular crescents with fibrinoid depositions and necrosis in 2 glomeruli. There was mild tubulointerstitial lymphoplasmocellular infiltrate, which was restricted to areas of tubular atrophy (⬃20% of the area). Therapy with cyclophosphamide and prednisolone was continued, and plasmapheresis (45 mL/kg) with substitution of 5% albumin in sodium chloride solution, 0.9%, 3 times per week for 3 weeks (9 sessions) was added. Despite ongoing hematuria and proteinuria, serum creatinine level improved to 1.9 mg/dL (eGFR, 41 mL/min/1.73 m2) after 3 weeks of plasmapheresis treatment. C-ANCA titer decreased to 1:20. Oral prednisolone dosage was tapered from 6 weeks onward and stopped 7 months after the start of therapy. Oral cyclophosphamide was switched to daily oral azathioprine (2 mg/kg) 4.5 months after the start of therapy, continued for 12 months, and then tapered by 25 mg every 3 months. In late 2009 (53 months after diagnosis, 23 months after discontinuation of azathioprine treatment), the patient presented with relapsed disease activity. Serum creatinine level, which had been stable at 1.5 mg/dL (eGFR, 53 mL/min/1.73 m2), had increased to 2.1 mg/dL (eGFR, 36 mL/min/1.73 m2) with renewed appearance of glomerular hematuria. Proteinuria increased from protein excretion of 0.7 to 2.1 g/24 h, and CPR level, from normal to 74 mg/L. In addition, arthralgias and malaise were present for 3-4 weeks. No change in upper-airway symptoms or reappearance of purpura was noted. Seven months earlier, C-ANCA titer had increased from 1:40 to 1:320 and remained at this level thereafter (Fig 1). Reintroduction of treatment with oral cyclophosphamide and prednisolone was associated with a rapid clinical response, normalization of CRP level, and decrease in C-ANCA titer, but serum creatinine level decreased to only 1.7 mg/dL (eGFR, 45 mL/min/ 1.73 m2) with persistence of proteinuria at protein excretion of 1.2 g/24 h. Again, prednisolone dosage was tapered off, and cyclophosphamide was switched to azathioprine after 3 months of stable remission. Currently, the patient is doing well with no symptoms or signs of active disease. Medication includes azathioprine, 100 mg, daily and enalapril, 10 mg, daily. Serum creatinine level is stable at 1.9 mg/dL (eGFR, 40 mL/min/1.73 m2), and proteinuria, at protein excretion of 0.8 g/24 h. Serum CRP level is ⬍3 mg/L and C-ANCA titer is 1:40. Cumulative cyclophosphamide dose is 31.9 g.

PATHOGENESIS This case illustrates a number of issues not yet resolved in the clinical approach to patients with AAV: Who is at risk of relapse? What triggers a relapse? Why do some patients have disease refractory to treatment? How can smoldering disease be detected, particularly in the kidney? More insight into the pathogenic mechanisms underlying disease expression will give answers to these questions. Ultimately, this will lead to personalized treatment based on distinctive pathogenic factors in individual patients. Nevertheless, great advances in our understanding of the pathogenesis of AAV have been made in recent years, giving clues for more targeted treatment. This research has focused particularly on the role of the PR3- and MPO-directed autoimmune response in the pathogenesis of AAV. Am J Kidney Dis. 2013;xx(x):xxx

Clinical Observations Suggestive of a Pathogenic Role of ANCA As shown in Table 1, most of our patients with AAV have detectable ANCAs, and this has been confirmed by others.6 Nevertheless, some groups have reported ANCA-negative pauci-immune necrotizing crescentic GN in up to 30% of patients.7 These ANCA-negative patients tend to be younger and have less systemic involvement and more chronic kidney lesions. However, given the close association of AAVs with ANCA, a pathogenic role for ANCAs is suggestive. When ANCAs are pathogenic, one would expect an increase in ANCA levels preceding disease activity. In a large prospective study of 85 patients with GPA with frequent serum sampling, an increase in PR3-ANCA titer had sensitivity of 79% for an ensuing relapse with specificity of 68%.8 However, this has not been confirmed in all studies, in part due to differences in sampling frequency and methods for quantitation of ANCA. A recent meta-analysis of all studies showed a positive likelihood ratio of 2.84 (95% confidence interval [CI], 1.65-4.90) of an increase in ANCA titer as a predictor of relapse, with a negative likelihood ratio of 0.49 (95% CI, 0.27-0.87).9 Also, data from patients with AAVs treated with the B-cell–depleting agent rituximab support a pathogenic role for ANCAs. In a series of 53 patients successfully treated with rituximab, relapses occurred in all except one patient only when B cells were reconstituted and ANCA levels had increased.10 Relapses in AAVs occur far more frequently in patients with PR3-ANCA than those with MPO-ANCA irrespective of the associated disease.11,12 In addition, PR3-ANCA is associated with granulomatous inflammation, more extrarenal disease, and a faster decline in kidney function.11 The reason for these differences in clinical presentation in relation to the antigenic specificity of the autoantibodies is not yet clear, but should be explained in order to substantiate that ANCAs are the primary pathogenic factors in AAVs. In this respect, a recent study found not only that GPA and MPA are genetically distinct, but that the strongest genetic associations are with the antigenic specificity of ANCAs and not with the clinical syndrome.13 PR3ANCA was found to be associated with HLA-DP, as well as with genes encoding for PR3 (PRTN3) and ␣1-antitrypsin (SERPINA1), whereas MPO-ANCA was observed to be associated with HLA-DQ. These data suggest that the autoimmune response to PR3 and MPO is genetically determined and precedes the clinical expression of GPA and MPA. As such, PR3ANCA vasculitis and MPO-ANCA vasculitis may be considered in the future nomenclature of the systemic vasculitides. However, in contrast to lupus, no data are available yet from population-based studies dem3

Kallenberg et al

onstrating that the development of the autoantibodies precedes disease expression. The somewhat lower prevalence of PR3-ANCA in localized GPA versus generalized GPA might suggest that ANCAs are a secondary phenomenon. Thus, although associations of PR3-ANCA and MPO-ANCA with GPA and MPA, respectively, are strong, data from clinical observations do not prove the pathogenic role of ANCAs. Nevertheless, pulmonary hemorrhage and kidney involvement were observed in a neonate with MPO-ANCA in cord blood from a mother with a history of MPO-ANCA vasculitis and at that time preeclampsia and (low) MPOANCA titer. This is a strong argument for a direct pathogenic role of MPO-ANCA.14 Otherwise, a healthy newborn was described despite transplacental transfer of MPO-ANCA.15 These latter observations could suggest that not all ANCAs are pathogenic, requiring further analysis of possibly pathogenic epitopes. Distinct epitopes of MPO-ANCA16 and PR3ANCA17 have been described, but specific pathogenic epitopes associated with active disease were not defined. Very recently, Roth et al,18 using a highly sensitive epitope excision/mass spectrometry method, described MPO-ANCA epitope specificities with in vitro pathogenic properties that were present during active disease in patients with AAV and decreased during remission. Other nonpathogenic epitope specificities were not specific for active disease or also occurred in healthy individuals.18 Furthermore, Espy et al19 recently demonstrated that the sialylation ratio of PR3-ANCA was significantly lower in patients with GPA with active disease than in those in remission. Furthermore, the in vitro PR3-ANCA–induced oxidative burst of neutrophils was found to be correlated inversely with their sialylation levels. Thus, besides their epitopes, changes in the constant regions of the autoantibodies also may influence their pathogenicity. Pathogenic Role of ANCA as Derived From Animal Models Rats immunized with human MPO develop antibodies to MPO that cross-react with rat MPO. Local perfusion into the renal artery of these rats with the products of activated neutrophils (lytic enzymes, MPO, and its substrate hydrogen peroxide) results in pauciimmune necrotizing crescentic GN, which does not develop in nonimmunized rats.20 MPO-immunized rats also develop severe necrotizing crescentic GN when injected with a subclinical dose of heterologous anti–glomerular basement membrane antibodies.21 Even more convincing evidence for a pathogenic role for MPO-ANCA has come from studies in which MPO-deficient mice were immunized with mouse 4

MPO. Transfer of their splenocytes into immunodeficient mice resulted in pauci-immune necrotizing crescentic GN and systemic necrotizing vasculitis.22 Passive transfer of anti-MPO containing immunoglobulin G (IgG) alone into wild-type mice also resulted in focal necrotizing crescentic GN with a paucity of immunoglobulin deposition. Lesions in this latter model could be aggravated by injection of lipopolysaccharide.23 Although the immunohistopathology of the kidney lesions shows absence or paucity of immunoglobulins, the complement system appears to be involved because recipient mice deficient for factor B of the alternative pathway of complement activation or for complement factor C5 do not develop lesions.24 Furthermore, pretreatment with a C5-inhibiting antibody in this model can prevent disease development, and treatment 1 day after disease induction results in a marked reduction in glomerular crescent formation.25 Also, studies of patients with AAVs suggest activation of the alternative pathway of complement activation26 and show deposition of factors B and P of the alternative pathway, as well as the membrane attack complex, in their kidney biopsy specimens.27 Studies in rats and mice have further demonstrated the potential of MPO-ANCA to augment leukocyte-microvascular interactions in vivo, resulting in microvascular injury manifested as focal necrotizing crescentic GN and pulmonary hemorrhage.28,29 Like autoantibodies, T cells seem to play a strong contributory role in experimental MPO-ANCA– associated necrotizing crescentic GN. Immunization of C57BL/6 mice with either murine or human MPO in adjuvant has been found to induce a cellular and humoral anti-MPO response, but this alone does not cause vasculitis.29 However, when followed by a challenge with a subnephritogenic dose of heterologous anti-glomerular basement membrane antibodies to localize neutrophils and MPO to the glomerular basement membrane, mice develop necrotizing crescentic GN. This model has been found to depend on T-cell–mediated immunity because mice lacking the ability to produce circulating antibody still develop disease, whereas CD4⫹ T-cell depletion inhibits disease development.30 Subsequent studies in this model have demonstrated an important role of T helper 17 (TH17) cells in lesion development31 and have identified an immunodominant MPO T-cell epitope that is essential for cell-mediated glomerular injury.32 Thus, these observations indicate that tissue injury in MPOANCA–associated vasculitis is a 2-step process that involves local release of MPO by activated neutrophils that can be recognized by MPO-specific CD4⫹ T cells, causing delayed-type hypersensitivity-like vascular injury.32 Am J Kidney Dis. 2013;xx(x):xxx

Pathogenesis of ANCA-Associated Vasculitis

In contrast to MPO-ANCA vasculitis, PR3-ANCA– associated GPA does not yet have an adequate animal model. Pfister et al33 used an approach similar to that of the MPO-ANCA model described by Xiao et al.22 They immunized mice deficient for PR3 and neutrophil elastase with recombinant mouse PR3. Serum of these mice, which contained anti-mouse PR3 antibodies that reacted with mouse granulocytes, was infused into lipopolysaccharide-primed recipient mice. Vasculitis and necrotizing crescentic GN did not develop, but mice injected with anti-PR3 serum developed somewhat larger skin infiltrates than control serum– injected mice after local challenge with subcutaneous TNF-␣ (tumor necrosis factor ␣). Also, rats immunized with human-mouse chimeric PR3, resulting in the development of anti-rat PR3 antibodies, did not develop lesions.34 More recently, Little et al35 injected PR3-ANCA–containing human IgG into mice with a chimeric human-mouse immune system including human neutrophils. These mice developed GN (in a minority, pauci-immune crescentic) and in a few, pulmonary capillaritis, but granulomatous inflammation was not observed. Primo et al36 induced an immune response to mouse PR3 in NOD mice (a murine model of insulin-dependent diabetes mellitus) by immunizing with recombinant mouse PR3 in complete Freund adjuvant. Splenocytes of these mice were transfused into immunodeficient NOD mice, resulting in segmental and necrotizing GN, but here also, granulomatous inflammation did not develop. Thus, an adequate animal model mimicking human PR3-ANCA GPA is not available. Pathogenic Role of ANCA as Derived From In Vitro Studies Falk et al37 were the first to demonstrate that both PR3-ANCA and MPO-ANCA have the capacity to further activate primed neutrophils to release reactive oxygen species and lytic enzymes from their primary granules. Priming of neutrophils was induced by lowdose TNF-␣, but also can be accomplished with other proinflammatory molecules, such as interleukin 1 (IL-1), IL-18, and the complement degradation product C5a.38-40 Priming includes the surface expression of the target antigens of ANCA (ie, PR3 and MPO) on the neutrophil membrane, thus allowing interaction with ANCAs.41,42 In contrast to MPO, PR3 is constitutively expressed on the neutrophil membrane, but the level of expression differs between individuals.43 High constitutive membrane expression of PR3 has been reported to be associated with vasculitis and rheumatoid arthritis44 and with relapsing disease in PR3-ANCA–associated vasculitis.45 In vitro, a correlation was found between levels of membrane PR3 expression on unprimed neutrophils and the degree of Am J Kidney Dis. 2013;xx(x):xxx

early activation of these neutrophils by PR3-specific monoclonal antibody, possibly explaining the relation between high membrane PR3 expression and relapse in PR3-ANCA disease.46 More recent studies have shown that PR3 is coexpressed with CD177 (also known as NB1) on the membrane, but PR3-ANCA– induced neutrophil activation seems independent of CD177 expression.47 However, another study suggested that CD177 is complexed also with Mac-1 (which comprises the CD11b/CD18 chains), and that Mac-1 functions as an adapter for PR3-ANCA– induced neutrophil activation.48 In vitro studies of patients with PR3-ANCA– and MPO-ANCA–associated necrotizing crescentic GN demonstrated that IgG fractions from the former are more potent activators of the respiratory burst and degranulation of neutrophils than IgG fractions from MPO-ANCA–positive patients.49 In order to activate neutrophils, ANCAs have to not only bind to surface-expressed PR3 or MPO, but also interact with receptors for the Fc part of the IgG molecule (Fc␥R), which are present on the neutrophil. Fc␥RIIa seems to be involved because blocking nonstimulating antibodies to these receptors abrogate oxygen radical release in response to stimulation with ANCA.50,51 Fc␥RIIa has a high affinity for the IgG3 subclass, and the IgG3 subclass of PR3-ANCA is relatively overexpressed during active disease.52 Genotype differences in both Fc␥RIIa and Fc␥RIIIa seem to be associated with disease relapse in GPA.53 In addition, the NA1 (neutrophil antigen 1) allele of Fc␥RIIIb was shown to be a risk factor for kidney involvement in GPA.54,55 A schematic representation of the signal transduction routes involved in ANCA-mediated neutrophil activation is shown in Fig 2, and this topic has been reviewed extensively.56,57 Williams et al58 showed that diacylglycerol kinase is selectively activated, resulting in the generation of phosphatidic acid, which in turn promotes neutrophil adhesion, in part through integrin activation. In addition, both during priming and ANCA-induced neutrophil activation, P38 mitogen-activated protein kinase (P38MAPK) and extracellular regulated kinase (ERK) are involved. Furthermore, phosphatidylinositol 3 kinase (PI3K) seems to play a central role in different stages of ANCAinduced neutrophil activation,59 and genetic ablation of the gamma polypeptide of PI3K (PI3K␥) and treatment with a PI3K␥ inhibitor attenuate the development of necrotizing crescentic GN in the anti-MPO animal model.60 Attenuation of necrotizing crescentic GN in this model also occurs with an inhibitor of P38MAPK.61 Neutrophil activation should not occur in the circulation to prevent anaphylactoid reactions. Reumaux 5

Kallenberg et al

PR3

PR3

ANCA

PR3 ANCA

ANCA

NB1 1

MPO MPO

Mac-1

ANCA

PR3 Mac-1

p22

NB1 1

Gp91

TNFR1

Mac-1

PR3

Mac-1

TNF α

Cyt b558

PLC

PI3-K

Tyrosine

PKC

PKB

ERK

TRANSLOCATION

kinase

p38

PKC

Tyrosine

Tyrosine

kinase

kinase

p110

PI3-K

p85

PLD

PI3-K

PKC

PKB

?

ERK

? NADPH OXIDASE

DEGRANULATION OF SECRETORY VESICLES AND SPECIFIC GRANULES

PKB

? NADPH OXIDASE

NADPH OXIDASE

NADPH OXIDASE

OXIDATIVE BURST

Figure 2. Signal transduction pathways involved in neutrophil activation by antineutrophil cytoplasmic antibody (ANCA). Tumor necrosis factor ␣ (TNF␣) results in neutrophil activation and, also at low dose, induces translocation of proteinase 3 (PR3) to the neutrophil membrane, where it localizes in conjunction with Mac-1 and NB1 (CD177), allowing interaction with monomeric ANCA; activation by ANCA-containing immune complexes via Fc␥-receptors; and hypothetical mechanisms of neutrophil activation by monomeric ANCA involving “activation clusters” in which Fc␥-receptors, Mac-1, and the autoantigens participate. Abbreviations: CYTb558, cytochrome b558; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PI3-K, phosphatidylinositol-3-kinase; PKB, protein kinase B; PKC, protein kinase C; PLD, phospholipase D; PLC, phospholipase C. Unknown or hypothetical pathways are indicated with a question mark.

et al62 showed that engagement of ␤2 integrins is a requirement for ANCA-induced neutrophil activation; prevention of adherence of neutrophils to a surface does not result in activation. Savage et al63 demonstrated that ANCAs stimulate neutrophils toward endothelial damage in an in vitro system. This group further showed that ANCAs stabilize adhesion and promote migration of neutrophils on endothelial cells in an elegant in vitro flow system.64 The CD11/CD18 heterodimer (Mac-1) and the chemokine receptor CXCR2 are involved in this process. Hong et al65 described a more indirect effect of ANCAs on endothelial cells in which ANCAs first induce the release of microparticles from primed neutrophils followed by binding of these microparticles to endothelial cells. After binding, endothelial cells show increased expression of adhesion molecules, release of IL-6 and IL-8, and production of reactive oxygen species. Based on all these in vitro data, a model for ANCA-induced small-vessel vasculitis has been proposed (Fig 3). Finally, neutrophil activation can be linked to (autoantigen-specific) B-cell activation. Holden et al66 showed that cytokine-stimulated nonprimed neutrophils or PR3-ANCA–stimulated primed neutrophils contain increased levels of B lymphocyte stimulator 6

(BLyS) and promote the survival of a centroblast cell line. Furthermore, ANCA-stimulated primed neutrophils release neutrophil extracellular traps, which contain the autoantigens PR3 and MPO.67 In addition, myeloid dendritic cells uploaded with and activated by neutrophil extracellular trap components induce MPO-ANCA and anti–double-stranded DNA antibodies when injected into mice, in conjunction with signs of vasculitis in kidney and lungs.68 Thus, neutrophils seem to play a crucial role in ANCA induction as well. Is Cell-Mediated Immunity Involved in AAV? Granulomatous inflammation suggests involvement of cell-mediated immune responses. T-Cell activation in AAVs, in particular GPA, has already been suggested by studies showing elevated levels of the soluble IL-2 receptor during active disease.69,70 Furthermore, whereas activated B cells have been described to be present in the circulation during active disease only, T-cell activation is reported to persist during remission.71 A recent study showed expansion of CD4⫹CD28⫺ T cells in cytomegalovirus-seropositive patients with GPA, which was associated with increased mortality and risk of infection.72 Also, peripheral-blood T-cells were shown to react to PR3.73 Am J Kidney Dis. 2013;xx(x):xxx

Pathogenesis of ANCA-Associated Vasculitis

C5a

C5b

C5 C5 Convertase

+ C3b C3bBbP C3 Convertase

C3bBb ROS Proteolytic enzymes

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capillaries ANCA

C5aR

ANCA antigen

Cytokine receptor

C 11 CD11b

F receptor Fc t

ICAM-1

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Factor B

Figure 3. Schematic representation of the neutrophil responses that are putatively involved in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated small-vessel vasculitis. Proinflammatory cytokines and chemokines (eg, tumor necrosis factor) that are released as a result of local or systemic infection cause upregulation of endothelial adhesion molecules (eg, selectins, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1) and prime neutrophils. Neutrophil priming causes upregulation of the expression of neutrophil adhesion molecules (such as CD11b, a ␤2-integrin cell-surface adhesion molecule involved in neutrophil adherence to and migration through vascular endothelial cells) and translocation of the ANCA antigens from their lysosomal compartments to the cell surface. Engagement of dimers of the antigen-binding portion of ANCA with ANCA antigens on the cell surface and interaction of the Fc part of the antibody with Fc receptors activate neutrophils and cause increased adherence of neutrophils to vessel walls. ANCA-mediated neutrophil activation also triggers production of reactive oxygen radicals and causes neutrophil degranulation. The consequent release of proteolytic enzymes leads to vasculitis. The alternative pathway of complement activation plays a role as an amplification loop of the inflammatory response; factor B and properdin are released from activated neutrophils and together with C3 activate the complement system, resulting in the production of the strong neutrophil chemoattractant C5a. Abbreviations: ICAM-1, intercellular adhesion molecule 1; C5aR, C5a receptor.

A more detailed analysis showed that expansion of T cells in GPA occurs within the CD4⫹ effector memory population (characterized by being positive for CD45RO and negative for the lymphoid homing receptor CCR7).74 Surprisingly, these effector memory T cells, expanded during remission, decreased in numbers during relapsing disease. Presumably they had migrated into the involved tissues, and effector memory T cells were observed to appear in urine during active kidney disease in GPA.75 CD4⫹ effector T cells differ in function reflected by their cytokine pattern: TH1 cells express interferon ␥ and TNF and are involved in defense against intracellular pathogens; TH2 cells produce IL-4, IL-5, and IL-13 and contribute to humoral immunity and defense against worms; TH17 cells expressing IL-17 are involved in activation of endothelial and epithelial cells and attraction of neutrophils; and T follicular helper (TFH) cells express IL-21. Effector memory T cells in GPA have been observed to show an increase in TH17 and TFH cells.76 In vitro stimulation of peripheral-blood monoAm J Kidney Dis. 2013;xx(x):xxx

nuclear cells with PR3 has shown that most proliferating cells belong to the TH17 subset.77 Also, levels of IL-17 and IL-23, the inducing cytokine for TH17 cells, are increased during active disease.78 As mentioned,31 IL-17 proved essential in an experimental model of anti-MPO necrotizing crescentic GN. A schematic representation of the role of T cells in AAVs is shown in Fig 4. Furthermore, several studies have reported a defective function of regulatory T cells (Tregs) in AAV,79-81 although not confirmed by others.82 Also, the frequency of Tregs has been reported to be increased79 or decreased.80,81 However, plasticity of T cells seems changed in AAV/ GPA with a shift in balance between Tregs and T effector cells. Taken together, T cells play a major role in the pathogenesis of AAV. Due to the success of B-cell depletion therapy, there also is increased interest in the immunoregulatory role of B cells in AAV. Detrimental effects of B cells include the production of pathogenic autoantibodies, but also generation of proinflammatory cytokines 7

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S. aureus

8

1 Breg

Treg

?

PR3 PR3

IL-1β β

3

M

Pc

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Th

B

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IL-15 IL 15

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TEM

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TEM TEM

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TNFα MICA NKG2D TEM

ANCA

TLR2 LTA Pgn

TLR9

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7

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CpG

CD11b

Fc receptor

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Perforin

Figure 4. Innate and adaptive immune mechanisms putatively involved in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs). Step 1: infection, possibly by Staphylococcus aureus, triggers bronchial epithelium and macrophages (M) in a Toll-like receptor (TLR)-dependent manner to release proinflammatory cytokines. Step 2: cytokines prime/ activate neutrophils to release proteinase 3 (PR3) and endothelial cells to express adhesion molecules, thus recruiting inflammatory cells (monocytes/macrophages). Step 3: recruited monocytes/macrophages sense TLR ligands and secrete more proinflammatory cytokines, including interleukin 23 (IL-23), driving T cells toward a T helper cell (TH)17 phenotype. Step 4: secreted IL-17 can attract neutrophils and stimulate granuloma formation. Step 5: in this proinflammatory environment, neutrophils are primed and adhere to the endothelium. Step 6: release of the autoantigen PR3 in an environment with antigen-presenting cells (APCs), B cells, and T cells leads to the local production of ANCA. IL-21 from follicular helper T cells (TFH) and B-cell activating factor (BAFF) from activated neutrophils activate B cells to the production of PR3-ANCAs. Step 7: PR3-ANCAs fully activate primed neutrophils, resulting in degranulation and the production of reactive oxygen species (ROS), thereby causing damage to vascular endothelial cells (“leukocytoclastic vasculitis”). Step 8: PR3-ANCA formation is perpetuated by the lack of function and/or numbers of regulatory T cells (Treg) and possibly regulatory B cells (Breg). Step 9: effector memory T cells (TEM) are produced under stimulation with IL-15 and interact by the NKG2D (natural killer group 2 member D) expressed on their surface with MICA (MHC class I related chain A; expressed on endothelial cells), resulting in chronicity of inflammation of the vascular wall. Abbreviations: ICAM-1, intercellular adhesion molecule 1; LTA, lipoteichoic acid; NET, neutrophil extracellular trap; Pc, plasma cell; Pgn, peptidoglycan.

(TNF-␣ and IL-6) that may activate pathogenic T cells and other inflammatory cells. Otherwise, B cells may suppress the inflammatory response through the production of anti-inflammatory cytokines such as IL-10 and transforming growth factor ␤, stimulation of Tregs, and inhibition of effector T cells. Studies in other autoimmune disorders such as systemic lupus erythematosus and multiple sclerosis indicate that the interplay between pathogenic and protective B-cell functions in autoimmunity may be dysregulated, but to date, these properties of B cells have received little attention in AAV.83 What Triggers Relapse in AAV? Presently, no distinction is made between patients with higher or lower risk of relapse regarding mainte8

nance treatment. From a clinical point of view, the presence of PR3-ANCA and cardiovascular involvement has been associated with a higher relapse rate, whereas decreased kidney function has been associated with a lower relapse rate.12 This raises the question of what triggers AAV in general and relapses in particular. The answers to these questions are still elusive. Both genetic and environmental factors are involved in disease expression. Interestingly, PR3ANCAs are associated with HLA-DP, and MPOANCAs, with HLA-DQ, even more than the associated diseases.13 Furthermore, PR3-ANCAs are associated with genes encoding for ␣1-antitrypsin and PR3 itself, suggesting that genetic variance in PR3 and/or its inactivation plays a role in the development of PR3-ANCAs.13 As environmental factors, silica Am J Kidney Dis. 2013;xx(x):xxx

Pathogenesis of ANCA-Associated Vasculitis

exposure and Staphylococcus aureus carriage have been suggested. Silica exposure, with reported odds ratios of 4.4 (95% CI, 1.36-13.4) and 1.9 (95% CI, 1.0-3.5), has been associated with AAV.84,85 Silica could act as an adjuvant and also is associated with other autoimmune diseases. Whether S aureus carriage precedes the development of GPA is not clear, but chronic carriage has been documented from onset in 63% of patients with GPA.86 Several mechanisms have been proposed to explain the relationship between S aureus carriage and GPA.87 S aureus contains various superantigens that stimulate T cells expressing particular T-cell receptor V-beta chains. Analysis of the superantigens expressed by S aureus strains from patients with GPA who were chronic carriers showed that carriage of S aureus strains expressing the toxic shock syndrome toxin 1 was associated with a strongly increased risk of relapse (relative risk, 13.3; 95% CI, 4.2-42.6).88 Otherwise, microbial products may interact with Toll-like receptors (TLRs) on and in immune cells, activating these cells. Tadema et al89 demonstrated increased expression of TLRs by monocytes and natural killer cells of patients with GPA. In particular, monocytes from nasal carriers of S aureus were observed to express increased levels of intracellular TLR9. Bacterial DNA is a ligand for TLR9 and contains many CpG dinucleotide motifs, which are known to be immunostimulatory. CpG together with IL-2 has been reported to be able to trigger autoreactive B cells to the in vitro production of PR3ANCAs.90,91 Furthermore, a complementary protein to PR3 (cPR3) was demonstrated to induce antibodies to cPR3 that were suggested to result, by idiotype–antiidiotype interaction, in antibodies to PR3, and this complementary PR3 showed homology to staphylococcal proteins.92 However, antibodies to complementary PR3 were not increased in another cohort of patients with GPA.93 Molecular mimicry between microbial peptides and autoantigens, as a mechanism for the induction of autoimmunity, also has been described for antibodies to the human lysosomal membrane glycoprotein 2 (hLAMP-2), present in neutrophils and other cells. Autoantibodies to hLAMP-2 were described in 1995 as sensitive and specific markers for pauci-immune necrotizing crescentic GN.94 More recently, an immunodominant epitope of hLAMP-2 was shown to exhibit 100% homology with the bacterial adhesin FimH of Gram-negative bacteria.95 Furthermore, immunization of rats with FimH was shown to result in antibodies that react with rat and hLAMP-2 and pauciimmune focal necrotizing GN.95 Anti–hLAMP-2 autoantibodies were shown to be sensitive for AAV in Am J Kidney Dis. 2013;xx(x):xxx

3 European cohorts,96 but this was not confirmed in another cohort from the United States.97

RECENT ADVANCES Following the detection of ANCAs, renewed interest in the AAVs has led to a number of large randomized controlled trials that have refined immunosuppressive treatment and reduced the amount of cyclophosphamide needed to control disease activity.98,99 Given the immediate and long-term side effects of cyclophosphamide, this has been a major step forward in the treatment of AAV. Furthermore, insight into the role of ANCAs in these disorders has led to the additional use of plasma exchange, which has been shown to be beneficial in patients presenting with life-threatening disease.100 Studies of the efficacy of plasma exchange in less severe disease are underway. A major breakthrough in the treatment of AAV is based on experimental studies showing the role of B cells in these diseases. B-Cell depletion with rituximab has been shown to be equivalent to cyclophosphamide and even superior in patients with relapsing disease.101,102 Intermittent treatment with rituximab may prevent relapses,103 and its role in maintaining patients in remission is being tested in a large randomized controlled trial. Results of in vitro and in vivo experimental studies in AAV are directly translated in patient care with respect to the role of the complement system.25-27 Presently being tested in a controlled clinical trial is the hypothesis that blocking the C5a receptor, being an essential factor in initial lesion development, could reduce the need for high-dose corticosteroids (www.ClinicalTrials.gov; NCT01363388). More trials based on new insights into the pathogenesis of AAV are underway.

SUMMARY Tremendous progress has been made in understanding the pathogenesis of the AAVs. Besides genetic and environmental factors involved in the cause of these diseases, pathogenic pathways, related to both humoral and cellular immune responses, have been elucidated. The case vignette reported here shows that targeting autoantibodies with the addition of plasma exchange when immunosuppressive treatment is not fully effective could improve kidney function. More targeted treatment, in particular B-cell depletion with rituximab, has been proved efficacious. Nevertheless, relapsing disease is still a major issue. Further understanding of the etiopathogenesis, in particular further insight into the mechanisms leading to disease relapse, will allow a personalized therapeutic approach to patients presenting with AAV. 9

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ACKNOWLEDGEMENTS Support: This work was supported by the European Union Seventh Framework Programme (FP7/2007-2013) grant 261382. Financial Disclosure: The authors declare that they have no relevant financial interests.

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