Endothelium-Neutrophil Interactions in ANCA-Associated Diseases

BRIEF REVIEW www.jasn.org

† Lise Halbwachs* and Philippe Lesavre*

*Institut National de la Santé et de la Recherche Medicale INSERM U845, Université Paris Descartes, Sorbonne Paris Cité, France; and †Assistance Publique-Hôpitaux de Paris, Hôpital Necker, Paris, France

ABSTRACT The two salient features of ANCA-associated vasculitis (AAV) are the restricted without activation that could result from microvessel localization and the mechanism of inflammatory damage, independent neutrophil-neutrophil interactions, of vascular immune deposits. The microvessel localization of the disease is due to contact with endothelium, or distortion; the ANCA antigen accessibility, which is restricted to the membrane of neutrophils and neutrophil adhesion to inflamed en- engaged in b2-integrin–mediated adhesion, while these antigens are cytoplasmic dothelium and diapedesis through the and inaccessible in resting neutrophils. The inflammatory vascular damage is the vessel wall should occur without release consequence of maximal proinflammatory responses of neutrophils, which face cu- of toxic oxidants or proteases, which mulative stimulations by TNF-a, b2-integrin engagement, C5a, and ANCA by the should be delayed until cells reach the FcgRII receptor. This results in the premature intravascular explosive release by inflammatory focus. This review exam- adherent neutrophils of all of their available weapons, normally designed to kill ines current concepts of the ways ANCA IgG-opsonized bacteria after migration in infected tissues. disrupts these sophisticated regulatory mechanisms, leading to unwanted pre- J Am Soc Nephrol 23: 1449–1461, 2012. doi: 10.1681/ASN.2012020119 mature and improperly located neutrophil activation, almost exclusively in microvessels. The initial description of small-vessel with the endothelium in ANCA-associated – vasculitides dates back to 1950, when vasculitis (AAV).6 8 NEUTROPHILS IN THE BLOOD Wainwright and Davson, by ocular ex- Endothelium-neutrophil interactions FLOW: PHYSIOLOGIC CONTROL amination of kidneys conserved in the are essential to allow neutrophils to move AND ACTIVATION BY ANCA Pathologic Museum of Manchester, were toward inflammatory sites and regulate able to distinguish forms of vasculitis spatially and temporally neutrophil re- Physiologic Control of TNF-Primed fl with enlarged and in ammatory glomer- cruitment. Neutrophils contain intracel- Neutrophils uli involving mostly vessels smaller than lular pools of toxic proteins aimed to kill The central role of TNF-a in AAV is 1 fi — arteries. Their de nition of a micro- microbes and digest tissues. To perform demonstrated in vitro9,10 and in vivo by — scopic form of periarteritis nodosa innate immune responses to infections, the striking effect of anti-TNF anti- fi was speci ed in 1994 by an international neutrophils must adhere and migrate bodies in experimental anti-MPO– 2 consensus committee, who changed the toward the site of infection (Figure 1), induced GN11,12 and in human AAV.13–15 name to microscopic polyangiitis be- while avoiding collateral damage caused Circulating TNF-a results in neutro- fl cause of the localization of in ammatory by premature release of oxidants and phil priming, leading to weak degranu- lesions in arterioles, venules, but not in proteolytic enzymes. This implies highly lation, oxidative response, or adhesion medium-sized or small arteries.3 The regulated neutrophil-endothelial cell in- original observation that small-vessel teractions complying with the following vasculitis, including granulomatous demands: neutrophils must remain Published online ahead of print. Publication date polyangiitis (GPA) and microscopic nonadhesive in the arterial and arteriolar available at www.jasn.org. polyangiitis, was associated with anti- circulation, independently of their re- Correspondence: Dr. Philippe Lesavre, Néphrol- 4 proteinase 3(PR3) or antimyeloperoxidase cruitment in postcapillary venules of ogie Adultes, Hôpital Necker, 149 rue de Sèvres, (MPO)5 ANCA, produced numerous inflammatory organs; the 10-mmdiam- 75015 Paris. Email: [email protected] studies that demonstrated the central eter neutrophil must squeeze through Copyright © 2012 by the American Society of role of neutrophils and their interaction capillaries, smaller in diameter (7 mm), Nephrology

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homotypic aggregation,22 which is favored by the presence of cell-bound C3 fragments interacting with CR1 and CR3 (Mac-1) receptors, for C3b and iC3b, re- spectively, on bystander neutrophils. Moreover, TNF priming triggers the switching of the aMb2-integrin (Mac-1) to an active conformation, allowing its binding to intracellular adhesion molecule 3 (ICAM-3) on adjacent neutrophils.23 It is thus reasonable to propose an initial step of low-grade activation of circulating neutrophils (Figure 2B) promoted by TNF-a and complement, Figure 1. Neutrophil-endothelial cell interactions. The classic view of neutrophil inter- allowing neutrophil-neutrophil interac- actions with activated endothelium is a three-step process, although the development of tions through b2-integrin binding of intravital imaging recently revealed intermediate steps, such as the slow rolling and the iC3b and/or ICAM-3. This would re- intravascular crawling. Step 1 entails tethering and rolling involving selectins. Proin- sult in a transient homotypic aggrega- flammatory cytokines (TNF-a, IL1-b) or LPS induce the expression of endothelial selectins, tion, similar to that observed in the able to interact with different ligands (PSGL-1, ESL-1, CD44) on neutrophils. Selectin en- Shwartzmann phenomenon24 and to gagement mediates rolling and, together with chemokines induced on the endothelial that recently described after in vivo injec- surface, initiates the “inside-out” activation of neutrophil b2-integrins. The interaction of tion of granulocyte-colony stimulating partly activated b2-integrins leads first to neutrophils slow rolling on endothelial ICAM-1 25 and E-selectin. During this slow rolling, leukocytes integrate signals from chemokines or factor. These neutrophil-neutrophil in- lipid mediators important for downstream events. Step 2 entails firm attachment via interactions might be stabilized by ANCA, tegrins. The level of intracellular calcium rises, leading to full b2-integrin activation and firm as the b2-integrin–induced firm adhe- arrest on ICAMs. Chemokines trigger the polarization of leukocytes, with the formation of sion.26 This implies that b2-integrin– leading and trailing edges. The activation of aMb2 (Mac-1) at the leading edge promotes mediated homotypic interactions would intravascular crawling on the luminal surface to the point of transmigration. Intravital im- promote low ANCA antigen expression aging allows for distinguishing two ways of transendothelial migration. As shown in step 3, on circulating neutrophils, accessible the paracellular migration through endothelial junctions involves homotypic PECAM-1 and to ANCA autoantibodies in serum, as junctional adhesion molecule-A (JAM) interactions, resulting in unzipping of the endo- has been observed during b2-integrin– thelial cell junctions. In the transcellular migration, high density of ICAM-1 and VCAM-1 on mediated adhesion.27 This would allow specialized docking structures (migratory cup) captures crawling neutrophils and facilitates ANCA to bind neutrophils and trigger a their way through the endothelial cells.123,124 massive activation otherwise normally expected at infected sites in tissues. and to hyperresponsiveness to subsequent Early Misleading Signals and stimuli such as chemoattractants or im- Homotypic Aggregation munecomplexes.16,17 TNF-inducedprim- In AAV, excessive amounts of fluid phase NEUTROPHIL ADHESION AND ing has normally limited consequences inflammatory stimuli in plasma may ANCA-INDUCED ACTIVATION within the blood flow of circulation due provide misguided information to neu- to a strict control of neutrophil activation trophils, suggesting an intravascular in- Neutrophil Adhesion in Capillaries by the plasma itself (Figure 2A). fection and leading to premature cell and Postcapillary Venules Under physiologic conditions, flow- activation. Levels of circulating TNF-a Contacts with the vascular wall along ing neutrophils do not interact with the and IL-8 are indeed increased in ANCA capillaries would be favored by the de- resting endothelium. They adhere only vasculitis.18,19 High concentrations of creased deformability of neutrophils in response to the local expression of ad- Fc-reactive ANCA could provide these treated with TNF-a and C5a, particu- hesion molecules, chemokines, and bio- misleading signals as suggested by the larly in the presence of ANCA28 and by active lipids on the endothelial surface glomerular neutrophil adhesion ob- neutrophil homotypic interactions. Cir- due to tissue inflammation and release served in naïve LPS mice upon the in- culating IL-8 also inhibits the emigration of cytokines along the basal side of jection of high doses of anti-MPO of neutrophils and promotes their se- endothelium. The recruitment of neu- antibodies.20 C5a may also play an im- questration in the microvasculature trophils is thus restricted locally and portant role because neutrophils activate due to downregulation of neutrophil cell activation is delayed until they complement and release C5a when stim- CXCR1/CXCR2 receptors internalized have migrated and reached the site of ulated by inflammatory cytokines.21 C5a upon IL-8 ligation. High levels of IL-8 inflammation. is a strong neutrophil agonist triggering intraglomerular synthesis and serum

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levels are reported in AAV with a negative correlation between IL-8 serum levels and CXCR1/CXCR2 neutrophil expression.29,30 In these conditions of high systemic secretion of inﬂammatory stimuli, triggering b2-integrin activation, circulating neutrophils adhere to the endothelium while they squeeze into capillaries before reaching the usual site of neutrophil emigration, particularly postcapillary venules. For those neutrophils having passed through capillaries, they interact in the classic way along selectin-expressing postcapillary venules (Figure 1), but their rolling is converted by ANCA into ﬁrm adhesion even at minimal TNF-a concentration.26,31,32 Firm adhesion mainly involves neutrophil b2-integrins interacting with ICAM-1 with a likely participation of a4b1 on mouse neutrophils or a9b1on human neutrophils.33,34 Increased ICAM- 1andde novo vascular cell adhesion molecule 1 (VCAM-1) expression are indeed observed on glomerular endothelia from ANCA patients.35 Moreover, increased levels of b2andb1-integrins are described on circulating neutrophils from patients with ANCA+, active GPAthat dis- sipates with treatment.36

signals for the firm adhesion of neutrophils to the endothelium.31 (C) Explosive responses of neutrophils adherent to endothelial cells in the presence of ANCA. Neutrophils then adhere to ICAM-1– expressing endothelial cells. During this firm adhesion, TNF/b2-integrin joint signals result in degranulation and oxidative burst37,128 and a massive increase of ANCA Figure 2. Neutrophil activation promoted by TNF-a and amplified by ANCA. (A) Ho- antigen expression, particularly mPR3, on meostatic control of TNF-induced neutrophil activation in the blood flow. Plasma proteins the neutrophil surface.27 Neutrophil-bound and oxidants prevent untimely intravascular activation of neutrophils. In particular, serum ANCAs trigger the complement classic albumin prevents the shedding of leukosialin (CD43), a prerequisite for neutrophil adhe- pathway (CP), exclusively on adherent neu- sion and inhibits most neutrophil responses to low concentration of inflammatory stim- trophils and in synergy with the alternative uli.125 Neutrophil degranulation and oxidative responses are delayed by the endogenous pathway (AP) activated on TNF-stimulated ceramide, generated upon TNF-a priming.126 Finally, anti-PR3 ANCA do not react with cells.45 C5a fragments and C5b-9 soluble membrane PR3 in the presence of plasma127 and particularly of a1-antitrypsin.95 (B) Initial complexes, released close to endothelial step of low-grade activation of circulating neutrophils involving cell-cell homotypic inter- cells, are able to activate these cells and to actions. TNF-stimulated neutrophils activate the complement alternative pathway via the promote cell retraction and endothelium secretion of endogenous properdin.21 This releases the powerful agonist C5a, able to permeability.98,100,129 The synergy between promote homotypic aggregation of circulating neutrophils,22 via interactions of the TNF- signals promoted by TNF, b2-integrin en- activated aMb2 (Mac-1)-integrins with ICAM-3 or iC3b on bystander neutrophils.23 TNF/ gagement, and ANCA-bound FcgR leads to b2-integrin joint signals would promote sufficient ANCA antigen membrane expression to an intense degranulation and the release of overcome the plasma inhibitory effect27 and allow the access of ANCA Fab to their antigen, proteases and oxidants highly toxic for the whereas their Fc portion interacts with FcgRIIa receptors. By doing so, ANCA give the final endothelium.

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Proinflammatory Effects of Adhesion: Amplification by ANCA and Complement During neutrophil adhesion and spreading, b2-integrin outside signaling synergizes with TNF-induced signals to trigger neutrophil responses such as degranulation and oxidative burst (Figure 2C). As well described by Carl Nathan, “in contrast to results with cells in suspen- sion, the responses of TNF-activated adherent neutrophils last over an hour andleadtoaccumulationofnearlyas much antimicrobial product as can be ” elicited by bacteria or phorbol esters Figure 3. Clustering, in lipid rafts, of neutrophil functional receptors required for ANCA- (p 178).37 mediated responses. The ANCA-induced oxidative burst is promoted by joint signals These inflammatory responses of ad- triggered by TNF priming, b2-integrin engagement, and the clustering of Fcg- herent neutrophils are further enhanced receptors.9,16,39,42 TNF-a mobilizes intracellular pools of aMb2-integrin (Mac-1), nicotin- by the presence of ANCA and of com- amide adenine dinucleotide phosphate (NADPH) oxidase protein gp91phox, and PR3 from plement. Indeed, an important conse- secretory vesicles to the plasma membrane, resulting in a co-localization of gp91phox, b2- quence of adhesion is the appearance, on integrins, and FcgRII.16 TNF-a, together with the engagement of b2-integrins, triggers the 16 130 the neutrophil surface, of high levels of clustering of FcgRII receptors required for FcgR-induced signaling. The same signals b g antigens such as mPR3 that is accessible induce the association of 2-integrins, NADPH-oxidase components, and Fc RII with the cytoskeleton, the latter being increased in the presence of anti-PR3 or anti-MPO anti- to ANCA in plasma.27 ANCA binding to bodies.16,131 Upon TNF-a priming, PR3 forms a complex with NB1 and with CD11b/CD18, membrane antigens, through their Fab all three proteins being localized in cholesterol-rich domains (lipid rafts),60,132 together portion, and to FcgR receptors, through with signaling molecules, cross-linked FcgRII,133,134 and NADPH-oxidase membrane their Fc portion, results in a further syn- components.135 ergy between TNF-a, b2-integrins, and FcgR signaling (Figure 3), leading to a striking increase of ANCA antigen complement classic pathway exclusively Consequences of the Biphasic membrane expression27 and to an ex- on adherent neutrophils, a condition Expression of Membrane PR3 plosive oxidative burst.38,39 Both allowing access of ANCA to their anti- An important point in ANCA-induced FcgRIIa and FcgRIIIbseemtobein- gens in plasm45 and in synergy with the amplification of neutrophil activation is volved,39–41 although the latter is not es- alternative pathway activated on TNF- the heterogeneous membrane distribu- sential because polymorphonuclear stimulated cells.21 Mice treated with a tion of one of the main ANCA antigens, neutrophils from FcgRIII–deficient in- C5-inhibiting mAb46 or deficient in C5 PR3, due to its association with CD177 dividuals showed a normal oxidative or in the C5a-receptor,47,48 but not in (NB-1). PR3 and CD177 are coexpressed response to ANCA antibodies.42 Differen- the downstream component C6,49 are on the membrane of a neutrophil subset ces have been observed between ANCA protected against anti-MPO GN. This and the proportion of mPR3+/CD177+ IgG subclasses, with an IgG3 . IgG1 . underscores the important role of C5 neutrophils, variable in the normal pop- IgG4 order of efficiency to induce neu- fragments, C5a, which are released on ulation, is increased in AAV, a high pro- trophil static adhesion.43 IgG glycosyla- the neutrophil surface and enhance cell portion being a risk factor for relapses.52 tion, critical for the interaction with responses such as degranulation and –56 Positive correlations are also reported FcgRs, is important in ANCA effects oxidative burst, creating an inflamma- between a high percentage of mPR3+ and treatment with endoglycosidase S tory amplifying loop.21 One should neutrophils and abnormal renal func- significantly decreased ANCA’s ability to point out that C5a significantly enhan- tion parameters in patients with GPA.57 activate neutrophils without interfering ces the oxidative burst promoted by Priming with TNF-a increases the 2 with their antigen-binding capacity.44 FcgRIIa cross-linking, which implies mPR3 expression in both CD177 and The synergy between TNF-a, b2-integrins, the existence of crosstalk between CD177+ subpopulations55 and the low 2 and FcgR was confirmed in vivo in ex- C5aR and FcgRIIa receptors, possibly level of mPR3 then present on CD177 perimental anti-MPO–induced AAV.6 important in ANCA-mediated neutro- neutrophils appears to be sufficient for Complement further amplifies the phil activation.50 Interestingly, analo- some of the effects promoted by anti- proinflammatory responses of adherent gous mechanisms have been elegantly PR3 antibodies, such as actin polymeri- neutrophils in the presence of ANCA. dissected in experimental rheumatoid zation and oxidative bursts.55,58 More Neutrophil-bound ANCAs trigger the arthritis.51 recently, anti-PR3 ANCA were shown

1452 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 1449–1461, 2012 www.jasn.org BRIEF REVIEW to specifically trigger degranulation and terms of vessel size (15–50 mm) and rhe- microvessels (Figure 5A) may explain the extracellular release of superoxide from ology. Usual intravital models analyze ANCA-associated accumulation of the CD177+ neutrophil subset by a such events by videomicroscopy acute neutrophils in the glomerulus6;it mechanism involving b2-integrins.59,60 preparations of mesenteric or cremas- does not involve endothelial adhesion The requirement of cytochalasin b in teric microvessels. Those models are molecules and is reminiscent of selectin- these observations implies a reorganiza- partially relevant to AAV where renal independent neutrophil sequestrations tion of the actin cytoskeleton similar to and lung capillaries are primarily in- observed in pulmonary capillaries.72 that induced by neutrophil adhesion and volved. New protocols of intravital im- Participation of platelets in neutrophil spreading. It is thus tempting to specu- aging65 delineate neutrophil recruitment adhesion to the endothelium has been late that anti-PR3 ANCA preferentially in capillaries in brain, lung, liver,66,67 observed in experimental anti-GBM promotes the release of oxidants and en- and kidney, where unilateral ureteric li- GN (Figure 5B). This model, however, zymes by mPR3+/CD177+ neutrophils gation permits visualization of glomeruli cannot be extrapolated to the ANCA sit- adherent through b2-integrins to endo- in the hydronephrotic kidney.68 uation, because the primary event is the thelial cells. endothelial lesions in the anti-GBM The potential pathogenicityof mPR3+ Renal Capillaries model and the neutrophil activation in /CD177+ neutrophils is, however, not AAV preferentially involves capillaries, AAV; a peculiar mechanism of glomeru- only related to the effect of anti-PR3 venules, and arterioles (Figure 4). Two lar leukocyte adhesion, mediated by ANCA, because a high proportion of distinct types of renal lesions are seen: the a4b1-integrin, has been observed in mPR3+/CD177+ neutrophils is observed glomerular necrosis versus peritubular LPS-free mice injected with high doses in AAV regardless of the ANCA anti- inflammatory capillaritis. Inflammatory of anti-MPO (Figure 5C) and is reminis- PR3 or anti-MPO specificity and is tubulointerstitial lesions are more pro- cent of chronic vasculitis induced by also observed in lupus patients.53,55 nounced in AAV, where AKI is largely Mycobacterium butyricum in rats73 The major reported difference between reversible, than in anti-glomerular and anti-MPO injection to LPS-treated 2 2 mPR3+/CD177+ and mPR3 /CD177 basement membrane (GBM) GN, mice results in a glomerular b2-integrin– cells is the higher transendothelial mi- where AKI is mainly due to irreversible dependent leukocyte adhesion (Figure gration of mPR3/CD177 positive neu- glomerular lesions. Interestingly, peri- 5D). These collected data indicate that trophils.59,61 This difference could be tubular inflammatory capillaritis may ANCA can induce leukocyte adhesion related to the ability of CD177 to bind be observed in AAV with few and even in glomerular capillaries through multi- CD31 (PECAM-1) on endothelial cells sometimes without any glomerular ple pathways and raise the possibility and to trigger PECAM-1–induced sig- lesions.69 than antiadhesion molecule antibodies nals, which modify the cell junctional Thus, by analogy with the threshold might represent potential therapies integrity and facilitate neutrophil trans- model proposed in transfusion-related in AAV. migration.61,62 acute lung injury,70 we suggest that AAV In conclusion, ANCA antibodies lower capillary lesions evolve with increasing Interstitial Postcapillary Venules and the threshold of neutrophil responses to levels of neutrophil priming and activa- Peritubular Capillaries inflammatory cytokines and result in a tion (TNF-a, b2-integrins, chemokines, The ability of ANCA to promote conver- vigorous response in the vascular bed and ANCA levels). We hypothesize that sion from rolling to adhesion in flow before any diapedesis and migration. the threshold is higher for glomerular chambers in vitro,26,32 and to enhance The cumulative effect of ANCA and che- sequestration than for peritubular in- chemokine-induced adhesion and trans- mokines has been shown in GPA, where flammatory capillaritis. migration both in mesenteric74 and cre- heterozygous CCR5 deletion is never ob- masteric6 microvessels in vivo, suggests served in ANCA-negative patients.63 To Nonconventional Pathways of the canonic three-step neutrophil adhe- note, CCR5 is present on TNF-activated Leukocyte Recruitment in Glomeruli sion model (Figure 1) is operating in neutrophils.64 Leukocyte adhesion is likely to be differ- AAV in postcapillary venules (Figure 4). ent in glomerular and lung capillaries Peritubular capillaries are potentially compared with postcapillary venules. recruited because they exhibit postcapil- SPECIFICITIES OF GLOMERULAR Because of the unique structure of glo- lary venule-like transformation that en- VERSUS PERITUBULAR merular capillaries, including the absence hances the influx of inflammatory cells MICROVESSELS of P-selectin,71 leukocyte recruitment in after kidney allograft rejection,75,76 after glomeruli bypasses the requirement for renal ischemia-reperfusion,77,78 and in In most tissues, leukocyte-endothelial initial rolling interactions. Both physical inflammatory and crescentic GN.79,80 interactions associated with inflamma- trapping and immediate arrest involving The importance of neutrophil recruit- tory responses do not occur in capillaries, adhesion molecules have been demon- ment and transmigration into the kid- butarepredominantinpostcapillary strated in different models (Figure 5). In ney interstitium has been recognized in venules, which are more favorable in particular, the trapping of neutrophils in human AAV.81

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edema that is observed in AAV.81 After these dissociations of cell-cell junctions, endothelial apoptosis and detachment are promoted by neutrophil-derived proteases and oxidants.84–90 Neutrophil secretion of these toxic oxidants and enzymes is greatly enhanced by ANCA9 and large numbers of circulating endothelial cells have been observed in patients, their levels decreasing with remission.91 Endo- thelial cell detachment results in denuda- tion of the subendothelial matrix leading to fibrin deposition as seen by electron mi- croscopy in AAV glomeruli,92 which implies platelet recruitment and thrombosis. Plasma protease inhibitors, such as a1 antitrypsin (a1AT), regulate the toxicity of serine proteases released during neutrophil degranulation. This could, in part, explain the predisposition to ANCA-positive GPA reported for a1AT deficiency alleles Z and S.93,94 Moreover, a1AT prevents the anti-PR3–mediated neutrophil activation95 and release of IL-8 by monocytes.96 Finally, a1AT was recently shown to control neutrophil activation independently of ANCA by binding IL-8 and preventing its reaction with its receptor CXCR1.97 All of these Figure 4. Renal small vessels. AAV results in necrotic and crescentic GN and lung hem- activities imply that a1AT deficiency fa- orrhage and is characterized by microvascular inflammation and necrosis in a variety of vors neutrophil-induced inflammation, organs. The two organs that are most extensively injured in this manner are the kidney, particularly in the presence of ANCA. through the development of pauci-immune crescentic GN, and the lung, with consequent alveolar hemorrhage. In the kidney, AAV preferentially involves glomerular and peritubular Role of Complement capillaries and venules, and arterioles. AAV does not affect interlobar and arcuate arteries and rarely interlobular arteries. Thus, the vascular distributions of AAV and of medium-sized- Although endothelial cells themselves vessel and large-vessel vasculitides minimally overlap each other. The predilection of AAV do not trigger the complement cascade, for glomerular capillaries (necrotic capillaritis in A) and peritubular capillaries and venules their close contact with adherent neu- (peritubular inflammatory capillaritis in B) is so exclusive that it is likely to be related the trophils activates surface complement unique structure of renal capillaries. Interestingly, peritubular inflammatory capillaritis may and may result in complement-mediated be observed in AAV with few, and even without any, glomerular necrosis,69 suggesting that endothelial injury (Figure 2C). Indeed, distinct mechanism of neutrophil-endothelial cell interactions are involved in AAV in glo- C5a receptors are present on endothelial meruli and in peritubular capillaries. cells and C5a induces rapid P-selectin membrane expression,98 cell retraction and increased endothelial permeabil- UNWANTED NEUTROPHIL/ endothelium through b2-integrins/ ity,99,100 and the expression of different ENDOTHELIUM INTERACTIONS ICAM-1 interactions, leading to a rise genes encoding for adhesion molecules, LEAD TO ENDOTHELIAL LESIONS ofcytosolicfreeCa++ and cytoskeletal cytokines, chemokines, and related re- changes required for neutrophil diapede- ceptors.101 Neutrophil-Mediated Endothelial sis.82 This signaling produces a micro- Cell Lesions vascular permeability, which is further Role of Platelets One of the hallmarks of AAV is massive enhanced by proteases and oxidants re- Neutrophil-platelet interactions play an endothelial injury, mainly produced by leased at the immediate proximity of important role during inflammation102 neutrophils and resulting in necrotiz- endothelial cells during TNF-induced ad- and provide a link between thrombosis ing vasculitis (Figure 6A). Neutrophil hesion in the presence of ANCA.83 Vascu- and inflammation103 and between in- firm adhesion transmits signals to the lar permeability leads to subendothelial flammation and immune response.104

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During rolling and crawling steps, activated endothelium induces neutrophil polarization. Active b2-integrins, clustered at their leading edge, capture red cells and platelets resulting in cell aggregates.102 In addition, platelets bind exposed collagen during vascular injury and express very large amounts of P-selectin that avidly recruits neutrophils.104 Thus, additional platelets may accumulate in the glomerular microvasculature through neutrophil-platelet interactions. The involvement of platelets in AAVis suggested by the increased frequency of venous thromboembolic events in GPA. In the Wegener’s Granulomatosis Eta- nercept Trial, including 180 patients, 7.0 venous thromboembolic events per 100 patient-years were reported with thrombosis occurring during a period of active disease.105 Plasma levels of platelet-derived soluble CD40L and P-selectin positively associated with the activity of disease in GPA, underscoring the participation of platelet activation in AAV.106

Neutrophil-Platelet Microparticles Membrane microparticles, released in vitro by fMLP or C5a-stimulated neutrophils, carry a large set of cell adhesion molecules and proteases such as PR3 or elastase107 that activate com- Figure 5. Nonconventional pathways of leukocyte recruitment in glomeruli. (A) In- plement.21 These interactions suggest travascular physical trapping independently of endothelial adhesion molecules. Neutrophil a dissemination of neutrophils’ toxic sequestration in capillaries results from ANCA-induced actin polymerization (green line) effects, although they were initially re- 28 and increased cell rigidity and homotypic aggregation, mediated by TNF-activated b2- ported to have anti-inflammatory activ- integrins interacting with ICAM-3 on bystander neutrophils.23 (B) Glomerular leukocyte ities.108,109 adhesion induced by anti-GBM in mice and involving platelets. After anti-GBM antibody68 or immune complex136 deposition, platelets adhere to the glomerular endothelial lesions Microparticles released by TNF- via platelet glycoprotein VI, fibrinogen being a potential ligand. Activated platelet express primed neutrophils in the presence of P-selectin, which recruits neutrophils by binding PSGL-1. The resulting neutrophil ANCA bind endothelial cells and trigger adhesion, which also involves b2-integrin/ICAM-1 interactions but bypasses the initial expression of adhesion receptors and pro- requirement for rolling, is restricted to glomerular capillaries and not observed in post- duction of oxidants and inflammatory capillary venules. (C) Glomerular leukocyte adhesion induced by high doses of anti-MPO in cytokines that generate thrombin in mice. Infusion of high doses of anti-MPO antibody to LPS-free mice resulted in a glomerular plasma.110 Microparticles released by ac- leukocyte adhesion, which was b2-integrin independent, but instead required neutrophil tivated or apoptotic neutrophils circulate 20 a4-integrin and an unknown ligand distinct from VCAM-1. Human neutrophils do not in the plasma of patients with AAV and 137 express a4b1, except in extreme septic conditions, but they express the a9b1-integrin, their level is strikingly increased during which has similar specificities.33,34 (D) Anti-MPO–induced glomerular leukocyte adhesion acute vasculitis.111 Most of these neutro- in LPS-treated mice. In this AAV experimental model, the synergistic effect of LPS with anti- MPO antibodies is directly related to both LPS-induced TNF synthesis11 and to increased phil microparticles form aggregates with expressions of CXCL1, CXCL2 (homologs of human IL-8), which participate in neutrophil platelet-derived microparticles and ex- glomerular recruitment.138 TNF-primed neutrophils, bearing anti-MPO antibodies, adhere press neutrophil and platelet-adhesion to the endothelium of glomerular capillaries, via the b2-integrin LFA-1 interacting with receptors with potential prothrombotic endothelial ICAM-1.20 and proinflammatory activities.

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are important in the promotion of gran- uloma formation113 and autoimmunity in AAV. A surprising number of patients with anti-GBM disease are known to express ANCA. The recent description of anti-PR3 and anti-MPO antibodies, long before the onset of anti-GBM injury, suggests that ANCA plays an antecedent role in glomerular pathophysiology.114 ANCA may induce low-grade intravascular activation, capillary lesions, disrup- tion of the basement membrane with exposure of cryptic antigens, and possibly recruitment of immune cells involved in autoimmunization and epitope spreading. This epitope spreading driven by ANCA long before the onset of anti- GBM disease may be involved in AAV.

NETosis and Endothelial Damage Upon activation, neutrophils release webs of DNA and histones, called neutrophil extracellular traps(NETs) (Figure 6C) that are designed to trap bacteria.115 Premature formation of NETs within Figure 6. Consequences of intravascular activation of adherent neutrophils by ANCA. (A) capillaries induces endothelial cell in- Endothelial cell (EC) lesions. During neutrophil adhesion to endothelium, b2-integrins jury116,117 and thrombosis by stimulat- induce the clustering of ICAM-1 molecules, which results in intraendothelial signaling and ing platelets.118 microvascular hyperpermeability,139,140 by altering adherens junctions141,142 and by The ANCA-mediated activation of increasing the caveolae-mediated transcytosis.143 Vascular permeability is enhanced TNF-primed neutrophils induces NETs by neutrophil proteases and oxidants83 and leukotrienes, such as LTB4, which play an formation, as shown in vitro and in kid- 144 important role, in particular via the release of neutrophil azurocidin (HBP). Neutrophil- ney biopsies of patients with AAV, where secreted proteases (elastase, PR3) or oxidants also induce the apoptosis and the de- typical components of NETs, DNA, his- tachment of endothelial cells.84–90 Hyperpermeability results in plasma leakage and tones, and proteins from neutrophil edema, whereas cell detachment results in the exposure of the prothrombotic subendothelial matrix. (B) Innate/adaptive immunity. Activated neutrophils participate in the granules are found in affected glomeruli 119 recruitment, activation, and programming of antigen-presenting cells. Cathepsin G acti- and in the interstitium. This presence vates prochemerin to produce chemerin, one of the few cytokines that attracts both im- of NETs in blood vessels, in the absence mature dendritic cells and plasmacytoid dendritic cells.37 Moreover, neutrophils secrete of microbial infection, further conﬁrms TNF-related ligand B-lymphocyte stimulator (BLyS), which helps to drive proliferation and that neutrophils integrate the conjunction maturation of B cell differentiation,145 and IFN-g, which helps to drive differentiation of T of signals from TNF, ANCA (Fcg), and cells and activation of macrophages. (C) NETosis. ANCA binding to TNF-primed neu- b2-integrins as alarm signals similar to trophils results in the release of extracellular traps NETs, similar to those observed after that sent by infectious microorganisms. a microbial challenge. This neutrophil cell death mechanism, involving the nicotinamide In this review, we favor a functional adenine dinucleotide phosphate oxidase and the production of reactive oxygen species paradigm for the ANCA antigen: a neu- (ROS), produces webs of DNA covered with antimicrobial molecules, including the auto- trophil intracellular antigen, whose mem- antigens PR3 and MPO and LL37. LL37 is able to convert the self DNA in an activator of plasmacytoid dendritic cells and may play a role in the transfer to adaptive immunity.146 braneexpressionisinducedinanexplosive Finally, ANCA from AAV patients binds to MPO and PR3 on NETs, indicating that epitopes manner by the autoantibody itself fol- of target autoantigens are present,119 which suggests that NETs might participate in the lowed by four signaling pathways are autoimmune response by providing high concentrations of extracellular autoantigens. recruited simultaneously: neutrophil pre- activation by TNF-a; b2-integrin engagement during homotypic aggregation and Transfer from Innate to Adaptive crucial decision to set up an adaptive adhesion to the microvascular endo- Immune Response immune response.112 As shown in Figure thelium; activation of the complement Neutrophils are a key component of 6B, neutrophils recruit dendritic cells, alternative pathway, releasing C5a, and; innate immunity and contribute to the monocytes, and lymphocytes, which FcgRII receptor recruitment by ANCA

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