Intravascular immunity as a key to systemic vasculitis: a work in progress, gaining momentum

Abstract

Vascular inflammation contributes to the defence against invading microbes and to the repair of injured tissues. In most cases it resolves before becoming apparent. Vasculitis comprises heterogeneous clinical entities that are characterized by the persistence of vascular inflammation after it has served its homeostatic function. Most underlying mechanisms have so far remained elusive. Intravascular immunity refers to the surveillance of the vasculature by leucocytes that sense microbial or sterile threats to vessel integrity and initiate protective responses that entail most events that determine the clinical manifestations of vasculitis, such as end-organ ischaemia, neutrophil extracellular traps generation and thrombosis, leucocyte extravasation and degranulation. Understanding how the resolution of vascular inflammation goes awry in patients with systemic vasculitis will facilitate the identification of novel pharmacological targets and bring us a step closer in each patient to the selection of more effective and less toxic treatments.

Systemic vasculitides: an overview

Vasculitides include disorders characterized by the inflammatory involvement of the vessel wall. They depend upon established extravascular diseases, e.g. systemic autoimmune diseases, or occur as primary conditions and share clinicopathological features with other conditions characterized by a prevalent vessel inflammation such as atherosclerosis, systemic sclerosis (SSc) and thrombotic microangiopathies. Inflammation is, in most cases, self-sustaining with heterogeneous clinical manifestations, depending on the size of the vessels and the anatomical districts that are preferentially involved. The incomplete insight into the underlying pathogenetic events makes a satisfactory classification of systemic vasculitis difficult [1]. The Chapel Hill Consensus Conference has recently released an updated classification of primary systemic vasculitides [2]. Seven main vasculitis groups have been proposed: large vessel vasculitides, small vessel vasculitides, medium vessel vasculitides, variable vessel vasculitides, single organ vasculitides, vasculitides associated with systemic disease and vasculitides associated with probable aetiology (Table 1).

Table 1.

2012 Chapel Hill Consensus Conference on the nomenclature of vasculitides

• Large vessel vasculitides

◦ Takayasu arteritis (TA)

◦ Giant cell arteritis (GCA)

• Medium vessel vasculitides

◦ Polyarteritis nodosa (PAN)

◦ Kawasaki disease (KD)

• Small vessel vasculitides

◦ Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV)

• Microscopic polyangiitis (MPA)

• Granulomatosis with polyangiitis (Wegener's) (GPA)

• Eosinophilic granulomatosis with polyangiitis (Churg–Strauss, EGPA)

◦ Immune complex small vessel vasculitides

• Anti-glomerular basement membrane disease

• Cryoglobulinaemic vasculitis (CV)

• IgA vasculitis (Henoch–Schönlein) (IgAV)

• Hypocomplementaemic urticarial vasculitis (anti-C1q vasculitis)

• Variable vessel vasculitides

◦ Behçet's disease

◦ Cogan's syndrome

• Single organ vasculitides

◦ Cutaneous leucocytoclastic angiitis

◦ Cutaneous arteritis

◦ Primary central nervous system vasculitis

◦ Isolated aortitis

◦ Others

• Vasculitides associated with systemic disease

◦ Lupus vasculitis

◦ Rheumatoid vasculitis

◦ Sarcoid vasculitis

◦ Others

• Vasculitides associated with probable aetiology

◦ Hepatitis C virus-associated cryoglobulinaemic vasculitis

◦ Hepatitis B virus-associated vasculitis

◦ Syphilis-associated aortitis

◦ Drug-associated immune complex vasculitis

◦ Drug-associated ANCA-associated vasculitis

◦ Cancer-associated vasculitis

• Others

Adapted from [2]

The group of large vessel vasculitides comprises giant cell arteritis (GCA) and Takayasu's arteritis (TA). GCA has a significant epidemiological impact in the elderly Caucasian population. The involvement of large arteries in GCA is restricted mainly to the supradiaphragmatic space and is segmental. New-onset headache, jaw claudication and scalp tenderness are common symptoms and are often accompanied by systemic inflammation [fatigue, fever, elevated erythrocyte sedimentation rate (ESR)] and by polymyalgia rheumatica (PMR), a related inflammatory disease of unknown cause characterized by pain of the neck, shoulders and hips. Vision loss, aortic aneurysm formation and ischaemic stroke represent typical and frequent complications of GCA. TA affects younger patients, in particular women of childbearing age, with a higher incidence in Asia and Latin America [3]. Large vessel involvement is widespread in TA, as the pulmonary artery and the whole subdiaphragmatic arterial tree are frequently involved. A lower rate of progression of vessel wall remodelling and enhanced formation of collateral vessels differentiate TA from GCA [4]. Aneurysm formation is more frequent in TA than in GCA, possibly reflecting still uncharacterized features of vessel inflammation in TA [5]. Despite epidemiological and clinicopathological dissimilarities, a clinicopathogenic continuum might encompass TA, GCA and some forms of chronic peri-aortitis [6–8]. For example, in a recent retrospective study the risk of arterial, but apparently not of venous, thrombosis characterizes both TA and GCA [9,10], with 10% of TA patients experiencing strokes [11]. The molecular correlates of the embolic diathesis are still poorly understood, also considering the epidemiological features of TA patients who, in general, are relatively young and not expected to be at risk of atherosclerosis.

High-dose corticosteroids are a mainstay in the therapy of GCA [12], even if a substantial fraction of the patients relapse upon steroid tapering. Steroids are also often used in TA in association with immunosuppressive drugs such as methotrexate or azathioprine [13]. Anti-tumour necrosis factor (TNF) agents are also effective in TA, whereas the use of biological agents in GCA is still under investigation and appears less promising [14].

Kawasaki's disease (KD) and polyarteritis nodosa (PAN) are medium vessel vasculitides. KD is a vasculitis of the childhood, almost invariably affecting patients under 5 years of age, more common in Japanese and Afro-Caribbean ethnic cohorts. Despite its self-limiting course, the disease is severe and potentially life-threatening because of the frequent involvement of coronary arteries. Common clinical signs include fever, cervical (often unilateral) lymphadenopathy, non-exudative conjunctivitis, polymorphic diffused exanthema, reddening and fixuration of lips and tongue, non-pitting oedema of the dorsa of hands and desquamating exanthema of palms and soles.

After an early phase of predominant perivasculitis, inflammation of medium-sized artery in KD progresses towards the involvement of the medial and intimal layers [15]. Infiltrating macrophages, CD8⁺ T cells and IgA-secreting plasma cells dominate, with frequent disruption of the vessel architecture and formation of aneurysms. Centripetal activation of the endothelium is associated with frequent thrombosis and end-organ ischaemia. KD late stages include aneurysms and stenoses due to intimal thickening. Myocarditis is almost invariably detectable at autopsy and coronary artery aneurysms and thrombosis occur in 15–25% of untreated patients. KD is, in fact, the primary cause of heart disease in children. Current therapeutic strategies in KD are based on the administration of intravenous immunoglobulins (IVIG) and anti-platelet agents such as aspirin and abiciximab [16]. Despite treatment, children with KD still have a 5–10% risk of developing coronary artery lesions, indicating the need for more effective, pathophysiologically targeted therapies [17]. A fraction (10–15%) of patients with KD do not respond satisfactorily to IVIG. In these subjects, anti-TNF agents are effective [18]. Dendritic cells endowed with a tolerogenic function expand during subacute phases of KD. This event is not apparently influenced by TNF blockade, revealing a somewhat complex remodelling of the immune network that occurs in KD patients responding to therapy [19].

PAN is an extremely rare necrotizing vasculitis involving medium arteries, characterized by micro-aneurysmatic and stenosing lesions [20]. Fibrinoid necrosis and dense neutrophil and lymphocyte infiltrate are frequent findings [21]. The pathogenesis is poorly known: most studies are biased by the lack of differentiation from other vasculitides, in particular microscopic polyangiitis (MPA) and by the epidemiological shift towards non-hepatitis B virus (HBV)-associated PAN [22,23]. Current therapeutic regimens are those employed in small vessel vasculitides [12,16,23] (see below).

Small vessel vasculitides include immune-complex and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV). IgA vasculitis (Henoch–Schönlein purpura) is a prototypic immune-complex vasculitis, more frequent in children and young adults. Palpable purpura, mainly at lower extremities, arthritis, gastrointestinal involvement (ischaemia, enteric haemorrhage, intussusception) and renal disease with features undistinguishable from isolated IgA nephropathy (Berger's disease) are relatively common [20,24]. Long-term kidney involvement can evolve into renal failure, more frequently in adult patients, whereas other manifestations are usually self-limiting or controlled by non-steroid anti-inflammatory drugs or corticosteroids. Severe renal manifestations are usually treated with high-dose corticosteroids, either alone or combined with immunosuppressive agents, plasmapheresis or IVIG [16,25].

The incidence of cryoglobulinaemic vasculitis (essential cryoglobulinaemia, mixed cryoglobulinaemia, CV) peaks in middle age and in areas with a high incidence of hepatitis C virus (HCV) infection. Cryoglobulins are formed by a combination of mono- or oligoclonal immunoglobulins with rheumatoid factor in the context of humoral responses associated with chronic infection (mainly HCV), autoimmunity (e.g. Sjögren's syndrome) or B cell malignancies. Purpura, mainly in the lower limbs, and skin ulcers, possibly with arthralgias or arthritis, are common features at presentation. Peripheral nerve involvement is usually mild and glomerulonephritis less frequent [26]. Treatments for HCV-related CV are based on anti-viral agents such as peg-interferon (IFN) plus ribavirin [27], whereas in non-viral CV AAV-like regimens are recommended [12]. Anti-B cell agents such as rituximab are being added to anti-viral agents as the standard of care treatment of CV, given their apparent safety and efficacy [28].

Autoantibodies directed against antigens usually contained in the neutrophil primary granules characterize AAV. These antibodies, which could be involved directly in the pathogenesis of the disease [29–31], are referred to as ANCA because of their target intracellular distribution at immunofluorescence analysis of fixed samples. Anti-proteinase 3 antibodies (cANCA) and destructive granulomatous lesions of the upper and lower respiratory tract, of the eye and of the ear, necrotizing crescentic glomerulonephritis and systemic vasculitis are hallmarks of granulomatosis with polyangiitis (GPA, formerly Wegener's granulomatosis [32]). Systemic features such as fever and arthromyalgias are frequent, while skin and nervous system involvement are less common. Persistent exposure to respiratory infectious and irritant agents might be involved in the natural history of the disease [33].

Pulmonary and renal involvements occur in both GPA and MPA. An epidemiological complementation exists, with MPA being more rare and severe in Europe and GPA less frequent in Japan [34]. Anti-myeloperoxidase (MPO) antibodies (pANCA) characterize MPA, and granulomatous lesions and the involvement of upper respiratory tract are less frequent. Skin, peripheral nerve, gastrointestinal and lung involvements are, in fact, exquisitely vasculitic in MPA. Renal involvement in MPA is frequent and often severe.

Eosinophilic granulomatosis with polyangiitis (EGPA, formerly Churg–Strauss' syndrome) is part of a wider clinicopathological continuum that includes allergy, asthma and eosinophilic syndromes. Clinical features include hypereosinophilia, nasal polyposis, palpable purpura, peripheral neuropathy and a history of allergic asthma, usually improving just before the onset of vasculitis. Cardiac, pulmonary and gastrointestinal involvement with either vasculitic lesions or granulomatous and eosinophilic infiltration are also common [35]. Renal involvement is less frequent than in other AAV. pANCA, often recognizing MPO, are detectable in 40–50% of patients. ANCA-positive patients apparently have a higher incidence of vasculitic manifestations, renal and peripheral nervous system involvement and higher relapse rates. ANCA-negative patients are at increased risk of heart and pulmonary involvement [36].

An increased incidence of venous thrombo-embolism in GPA has been established in the Wegener's Clinical Occurrence of Thrombosis (WeCLOT) study [37]. Results were confirmed and extended to all AAV (but not to patients with PAN) in a large retrospective analysis [38]. AAV patients also are at higher risk of arterial thrombosis, with 14% of patients experiencing a cardiovascular event within 5 years of GPA or MPA diagnosis [39]. Apparently, antibodies recognizing MPO are associated with a higher cardiovascular risk than antibodies against proteinase 3 (PR3) [39]. Venous and arterial thrombosis often occur during active phases of vasculitis and are not apparently associated with conventional pro-thrombotic risk factors, rather suggesting a potential link with inflammation and defective regulation of the thrombogenic action of leucocytes [10,40]. Significant efforts are being employed in AAV genetics, pathophysiology and therapeutics [41,42]. Current treatment approaches are based on disease severity (i.e. extension to vital organs or refractoriness to therapies [12,43]). A combination of oral or intravenous cyclophosphamide with glucocorticoids is often used for induction of remission in patients with generalized or severe AAV or with a five-factor score ≥ 1 [44]) [12,45]. Encouraging results from the Rituximab for ANCA-associated Vasculitis (RAVE) and Rituximab in Vasculitis (RITUXVAS) trials [46–48] suggest that rituximab might represent an alternative to cyclophosphamide, and as such it might be introduced in the standard of care treatment [49,50]. High-dose glucocorticoids plus methotrexate could be employed for remission induction in non-organ or non-life-threatening AAV. Plasma exchange is recommended in addition to standard treatment of patients with severe renal involvement, although it does not apparently influence the overall survival [12]. Various agents, including azathioprine, methotexate and rituximab, are used to maintain remission [51].

Behçet's disease is currently included in a group of ‘variable-vessel’ vasculitides: it involves arterial and venous vessels of variable dimensions, a feature that is somewhat unique, and is often associated with the development of arterial aneurysms. Recurrent mucocutaneous manifestations (including oral and genital ulcers), uveitis and endothelial activation characterize the disease. Joints, gastrointestinal tract and central nervous system are also involved [52]. Thrombotic events in Behçet's disease occur in 10–30% of patients and include superficial thrombophlebitis and deep vein, cerebral venous sinus, pulmonary artery and intracardiac thrombosis. Budd–Chiari syndrome and thrombosis of the vena cava are responsible for at least part of the disease morbidity [40]. Thrombosis and vasculitis appear intermingled, with diffuse neutrophilic inflammation of the vascular wall often being found in association with tightly adherent thrombi and activation of circulating leucocytes [53–55]: venous occlusion and arterial wall aneurysmatic remodelling are common outcomes, while embolism is relatively rare [40]. Enhanced T cell responses, due possibly to facilitating effects of human leucocyte antigen (HLA)-B51 and to genetically determined imbalances in the interleukin (IL)-10/IL-23 network [56–58]), and co-existing autoinflammatory processes are implicated [59,60]. Recent studies suggest that variants in ERAP1, an aminopeptidase that trims peptides for proper loading onto major histocompatibility complex (MHC) class I antigens, are associated with Behçet's disease, sustaining the contention that peptide–MHC class I interactions contribute to the pathogenesis of the disease [61].

There is consensus on the use of azathioprine and corticosteroids for eye disease [52]. Cyclosporin A, infliximab or interferon (IFN)-α are used in severe cases, colchicine in Behçet's disease-related arthritis and erythema nodosum and corticosteroids for dural sinus thrombosis [62]. By contrast, the management of vascular disease as well as gastro-Behçet's disease or neuro-Behçet's disease is based largely on expert opinion and includes conventional immunosuppressants, TNF-antagonists and thalidomide [52]. Of interest, immunosuppressive agents appear to substantially decrease the risk of thrombosis relapses [63], while anti-coagulants do not appear effective [64], highlighting the importance of the deregulated immune response in driving cardiovascular events in Behçet's disease.

Priming events in vessel inflammation

The blood transports oxygen and nutrients throughout the body. Pathogens or toxins also spread through blood vessels. As a consequence, the circulatory system must be able to sense potential threats to the organism integrity and to react. Intravascular immunity refers to the interaction between humoral and cellular constituents of the immune system and microbes [65]. Successful microbes have evolved strategies that divert and confuse the immune response by interfering with the chemotactic recruitment and the various functions of effector leucocytes by adhering to endothelial cells and thus avoiding clearance in the spleen, or by escaping detection by interference with humoral innate immunity [65]. Conversely, an exquisitely sensitive and tightly regulated immune patrolling system has evolved in higher eukaryotes. Patrolling cells detect not only direct signs of microbial invasion (pathogen-associated molecular patterns, PAMPs), but also signs of vascular injury that might indirectly reveal infection (damage-associated molecular patterns, DAMPs). More than 60 trillion endothelial cells line vascular lumens, accounting in the human body for an approximate 4 km² surface [66]. Changes on the surface of endothelial cells, which in physiological conditions are confined to relatively small areas, initiate the key event in vascular inflammation, i.e. the recruitment of blood leucocytes to the vessel wall. These changes reflect: (i) the activation of endothelium innate immune receptors, which are differentially distributed in the vasculature [67] and are apparently responsible for the pathological outcomes of vessel inflammation (e.g. panarteritis versus perivasculitis) [68]; and (ii) the paracrine action of a family of sentinel cells, which are strategically located in the vessels to detect potential or actual threats to the vessel integrity.

These sentinels include constitutively rolling/crawling neutrophils and monocytes and innate lymphocyte populations, invariant natural killer (NK) T cells in particular, and even mast cells [69]. Immobilized populations of phagocytes that reside in specialized vascular districts also play a role. Küpffer cells in liver sinusoids are the best-characterized vessel guardians, specialized in purging the circulating blood from opsonized particulate substrates. Platelets also express innate pattern recognition receptors and interact with leucocytes in the blood and at sites of vessel injury [70]. Platelets respond to lower amounts of selected PAMPs, such as bacterial endotoxin, than most leucocytes, releasing a wide array of activatory signals. As such leucocyte sensitivity to potentially harmful agents is much higher in the presence of platelets [71,72]. The interaction might have dangerous outcomes [73], even considering the ability of platelet-derived microparticles, which extend the reach of platelets even to quite distant districts, to elicit, amplify and maintain vascular inflammation (see also below) [74].

Sentinel cells react to PAMPs and DAMPs by generating various mediators that act on endothelia, including cytokines, histamine and cysteinyl–leukotrienes [65,71] and even functional microRNA [75]. Endothelial cells react by up-regulating the expression of P-selectin, which is stored in Weibel–Palade bodies, and of E-selectin, which is synthesized de novo. Selectins engage the leucocyte PSGL1 leucocyte receptor, enforcing the tethering of flowing leucocytes to the endothelium and initiating their subsequent rolling along the vessels in the direction of the blood flow. Long tethers are formed under shear at the rear of the rolling neutrophils that ‘catapult’ to the front of the cell [71]. PSGL1 activation determines a conformational change of leucocyte integrins, which interact with higher affinity with their counterparts on the endothelium. As a consequence, leucocytes progressively slow down, adhere firmly to endothelial cells and eventually arrest. The endothelium surface is enriched in negatively charged residues, such as heparan sulphates, that anchor positively charged chemokines. Chemoattractant signals, specifically including formylated moieties derived from mitochondria, organelles with a putative endosymbiotic origin [76,77–79], appear to play a key role in directing neutrophils towards transmigration sites in inflamed vessels.

PSGL1 activation also results in the redistribution of the primary granules content to the neutrophil plasma membrane, including MPO and PR3 that are targeted by ANCA (see above). The redistribution of oxidizing and proteolytic activities at the membrane may facilitate transendothelial migration and the further movement of leucocytes away from the chemokines on the endothelial cells and within the extracellular matrix, even if this contention has not been demonstrated formally in in-vivo systems.

Reactive oxygen species (ROS) generation is a hallmark of immune cell activation. ROS are required to cope with invading microorganisms in the blood and in the peripheral tissues. In zebrafish the wound response entails a localized rise in hydrogen peroxide concentration at the margins of the wound, which is necessary for the swift recruitment of leucocytes at the site of injury [80], where they are likely to be in charge both of restricting microbe proliferation and of delivering appropriate signals to stem/progenitor cells which, in turn, reconstitute damaged tissues [81].

Subendothelial vessel wall cells/constituents contribute to vessel homeostasis under inflammatory conditions. For example, leucocytes that have extravasated from post-capillary venules crawl along processes of pericytes, mural cells of blood vessels, in a β2 integrin-dependent manner. This event enables them to reach gaps between adjacent pericytes [82]. Traffic through the subendothelial matrix in inflamed tissues is facilitated by enlargement of these gaps [82] and by the remodelling of the cytoskeleton of pericytes interacting with the leucocytes [83]. Once extravasated, leucocytes receive migratory and survival signals from pericytes associated with nearby capillaries and arterioles. Pericytes broadcast the news of ongoing vessel injury and attract extravasated neutrophils and monocytes at the site of vascular inflammation [84,85]. So far, the actual implications of these events for maintained small vessel inflammation in AAV and other systemic vasculitis have not been investigated.

In-vivo microscopy studies have also indicated that the density of the interstitial collagen network controls its ability to provide physical guidance to extravasated neutrophils. Neutrophil migration through the interstitial matrix depends upon the integrity of the actin-based cytoskeleton and on metalloproteinase (MMP)-sensitive adhesion/signalling molecules on neutrophils. In contrast, it does not apparently require pericellular degradation of the collagen network [86].

Mechanisms that enable inflamed vessels to cope with microbial and non-microbial threats and to heal

The mechanisms involved in the defence against microbes and in the reconstitution of vessel integrity largely overlap: this possibly reflects an evolutionary process initiated at the level of an ancestral unspecialized haemolymphatic system [87]. Aberrant deployment and/or maintenance of responses, that have probably been selected evolutionarily because they are advantageous, constitute a priming event of vasculitis leading to vessel wall disruption (with subsequent haemorrhage) and thrombosis, on one hand, and on deregulated vessel wall remodelling on the other hand. After detection of a potential threat to vessel integrity, counter-regulatory responses contrast the injurious activity and the diffusion of pathogens or toxins in the acute phase and at later times promote the reversal of vessel damage through neoangiogenesis and repair responses.

Thrombosis

Arterial and venous thrombosis can also occur as processes that are triggered by the active participation of immune cells, which are dispensable for haemostasis or vessel repair. Immune-mediated thrombosis, or immunothrombosis [88], might have a homeostatic role in protection against microbial threats in the vasculature. Thrombi might contribute to the capture of blood microbes, preventing their spreading into tissues and entrapping them in a microenvironment that is enriched in microbicidal activities.

Neutrophil extracellular traps (NETs), threads of deconsensed chromatin released by activated neutrophils and decorated with microbicidal signals (Fig. 1), represent crucial structural constituents of immune-elicited thrombi [89]. Neutrophils generate NETs as a response to stimuli that cannot be removed easily by phagocytosis. NETs are known: (i) to be implicated in AAV [90]; and (ii) to contribute to the organization of venous thrombi [91,92]. As such, they represent intriguing candidates to link paroxysmal neutrophil activation and thrombosis in AAV. Neutrophils of patients with active AAV appear to express high amounts of tissue factor (TF), a critical signal for the activation of the coagulation cascade. Moreover, they generate TF-expressing NETs and microparticles which can be detected in the blood, the bronchoalveolar lavage and the patient's kidney [93]. NET generation is possibly associated with the direct effect of ANCA on primed neutrophils [93]. However, direct clinical evidence of NET involvement in atherothrombosis is lacking, and the limitations associated with the available animal models of vasculitis for the study of thrombotic events [94] make experimental proof of a cause–effect relationship difficult.

Fig. 1.

Neutrophils degranulate and generate neutrophils extracellular traps (NETs) in response to sterile stimuli. Neutrophils freshly purified from a healthy donor were either left untreated (a) or challenged with purified human P-selectin (b). The expression of pentraxin (PTX)3 (red) and myeloperoxidase (MPO) was analysed by confocal microscopy. (c,d) The generation of NETs upon challenge of adherent neutrophils with the N-formylated formyl-methionyl-leucyl-phenylalanine (fMLP) peptide, which mimics microbial of mitochondrial protein degradation products or interleukin (IL)-8. Hoechst (blue) was used for counterstaining nuclei and extracellular DNA.

NETs promote thrombosis by activating factor XII, by neutralizing endogenous anti-coagulants (e.g. tissue factor pathway inhibitor: TFPI) and by promoting platelet recruitment and activation through captured von-Willebrand factor and histone proteins [88]. In turn, activated platelets commit neutrophils to NET generation with unusual efficacy [95]. During sepsis, NETs play a critical role in the capture of circulating bacteria, thus preventing their dissemination to distant sites. This represents a formal proof of the protective role of intravascular immunity [96]. These pathways might also be involved in the response to sterile vessel injury.

Neutrophils that are involved in immune-mediated thrombosis are likely to be activated, even if the extent of the activation is not sufficient to licence them for migration to extravascular districts. The expression of ANCA antigens, such as PR3 and MPO, on the surface of neutrophils is a genetically determined event, possibly enhanced further by inflammatory stimuli [41,97]. Membrane PR3 and MPO interact with ANCA which, in turn, activate the alternative complement pathway. As a consequence, neutrophils undergo degranulation and respiratory burst and release NETs [24,90,98,99]. ANCA-induced NET generation increases the autoantigen burden due to the exposure of PR3 and MPO along NETs. Recent studies have indicated that antigen-presenting dendritic cells challenged with NET components reproduce ANCA and autoimmunity when injected into healthy experimental animals, implicating the cross-presentation of NET-associated antigens in the in-vivo maintenance of AAV [100].

Vessel remodelling

Neointimal and medial thickening are part of a stereotyped response to the injury of large vessels [101–103]. Enhanced recruitment of inflammatory cells, disruption of the vessel wall architecture due to proteolytic activities and deregulated activation of the repair programme of the vessel wall contribute to the vessel wall remodelling. The adventitial layer has a privileged role in the natural history of large vessel vasculitis [4]. The current pathogenic model implicates a biased activation of polarized T helper type 1 (Th1)/Th17 lymphocytes by adventitial dendritic cells within the vessel wall, release of cytokines and of IFN-γ in particular from activated T cells, with ensuing macrophage recruitment and activation. In turn, macrophages infiltrate the adventitial and medial layers and secrete cytokines such as IL-1β and IL-6 and growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) [101,104,105]. Late events include medial smooth muscle cells proliferation, intima invasion and deposition of poorly structurally organized extracellular matrix, on one hand, and neoangiogenesis on the other hand.

The generation and release of growth factors, including VEGF and PDGF, from platelets and circulating or infiltrating immune cells is critical for the remodelling of the vessel wall in medium and large vessel vasculitides [106–109]. Levels of VEGF are high in patients with large vessel vasculitides [107], particularly in GCA patients with recent optic nerve ischaemia [108]. Vessel-associated macrophages are a possible source of the signal [104]. PDGF co-operates with VEGF in sustaining neoangiogenesis [110], with pericyte and smooth muscle cell recruitment and myofibroblast proliferation [110].

The balance between angiopoietins 1 (Ang1) and 2 (Ang2) is critical to regulate angiogenesis and tissue remodelling [111,112]. Ang2 expression is restricted to inflammatory [113,114] or to physiological and non-physiological pro-angiogenic conditions [115,116]. Persistent hypoxia or high levels of growth factors are associated with Ang1 expression which, in turn, participates in a vasoprotective response. In contrast, upon release from Weibel–Palade bodies, Ang2 initiates the neoangiogenic process by promoting endothelial destabilization, an event which is critical for vessel sprouting, possibly via interference with the activation of the Tie2 receptor [117]. Little is known about the pathogenic role of angiopoietins in systemic vasculitides: higher levels of Ang1 and Ang2 have been detected in Behçet's disease and AAVs, without a clear correlation with disease activity [114,118]. A recent study [17] reports an association between single nucleotide polymorphisms (SNPs) in the VEGFA and ANGPT1 gene and KD susceptibility as well as between SNPs in VEGFR2 and ANGPT2 and coronary artery disease. Ang1 was significantly lower in the serum of patients with active disease and scarcely expressed in tissue samples of affected arteries, while Ang2 was detected only at sites of post-thrombotic neovascularization, suggesting that Ang1/Ang2 deregulation was involved in KD subendothelial oedema, gap formation, fenestration of endothelial cells and eventual inflammatory cells infiltration [17,119].

The hypoxia-associated response is a target to limit the progression of aneurysmal lesions associated to systemic vasculitides [114,115,120,121]. Ang2 blockade has yielded promising results in cancer preclinical models when combined with anti-neoplastic and anti-VEGF agents [122]. This approach could have additional advantages in an Ang1/TGF-β-rich anti-angiogenic/profibrotic environment [123,124]. Selective Ang2 blockade and Ang1/Ang2 dual blockers such as trebananib proved clinically efficacious in ovarian cancer in a Phase II study [125], and several trials are ongoing for other solid (NCT01664182, NCT01538095, NCT01553188, NCT01666977) or haematological malignancies (NCT01555268). A dual anti-VEGF–Ang2 CrossMab is currently being tested at a preclinical stage [122].

Innate immunity in vascular inflammation

Humoral innate immunity

The long pentraxin PTX3 is a prototypic innate pattern recognition receptor, highly conserved through evolution [126,127]. In contrast to short pentraxins such as C-reactive protein, that are usually produced in the liver, PTX3 is mainly generated and released at sites of inflammation. Neutrophils store a large and non-renewable reservoir of preformed PTX3 in the specific granules (Fig. 1). As such, neutrophils are responsible for the prompt release of PTX3 in response to acute vessel and tissue injury [128,129]. In this context, PTX3 represents a regulator of the inflammatory response: it regulates neutrophil access to inflamed vessels negatively by interfering with the P-selectin/PSGL1 system [130], binds to activated platelets and quenches their inflammatory activities [129]. Indeed, activated platelets in the presence of PTX3 interact less effectively with leucocytes, aggregate less and bind with lower efficacy to fibrinogen [129]. Vessel cells, including endothelial cells and macrophages, produce PTX3, which possibly has a role in the persisting vessel remodelling and inflammation associated with systemic vasculitis [108,131,132].

PTX3 interacts with cell remnants, regulating their clearance [133] and controlling the immunogenicity of antigens associated with dying cells [134], events that could be implicated in the persistence of cell remnants at sites of inflammation, a hallmark of AAV [131,132,135,136]. Interestingly, the action of PTX3 on the clearance of cell debris depends upon its physical interaction with other inflammatory molecules such as ficolins [137,138], highlighting the role of PTX3 as a critical regulator of apoptotic cell phagocytosis in inflammatory conditions.

PTX3 concentration is associated with cardiovascular risk factors but apparently not with subclinical atherosclerosis [139], and the molecule accumulates at sites of active vessel remodelling in patients with GCA [108] and TA [140]. PTX3 physical association with various matrix components modifies the biological characteristics of the matrix. PTX3-assisted reorganization is required for the cumulus matrix organization [141] and for effective smooth muscle cell migration. Moreover, PTX3 acts as a selective inhibitor of neovascularization triggered by fibroblast growth factor (FGF)2, without influencing physiological angiogenesis [142]. Other inflammatory molecules, such as TNF-stimulated gene 6 protein (TSG-6), are able to compete with PTX3 for the binding and thus to revert the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis, suggesting that the relative levels of interacting inflammatory molecules generated at inflammatory sites are crucial to fine-tune the angiogenic outcome of the process [143]. Further studies in patients with systemic vasculitis are necessary to verify whether or not PTX3-assisted organization of the matrix influences the functions of leucocytes that have extravasated and the outcome of the vessel wall remodelling [101,109].

Exogenous administration of soluble pattern recognition receptors has been proposed to have therapeutic potential [144–146]. A single injection of a viral vector carrying the PTX3 gene inhibited intimal thickening after balloon injury in rat carotid arteries [147,148], suggesting that PTX3 delivery might prove valuable in large vessel vasculitides and especially TA, in which angioplasty is commonly used.

The high mobility group box 1 (HMGB1) protein has well-characterized functions in the nucleus and in the cytosol of living cells. It is released as a consequence of cell and tissue necrosis and further secreted actively by activated immune cells, broadcasting the news that active inflammatory responses are needed to prompt long-term repair and defence programmes [149,150]. HMGB1 enhances and accelerates the action of multiple stressors, endogenous inflammatory signals (e.g. DNA and chromatin components), microbial constituents and cytokines [150,151]. HMGB1 blood concentration increases as a consequence of localized ischaemia-associated tissue injury. This is the case of acute myocardial infarction, where circulating levels of HMGB1 predict the extension of ischaemia and the residual contractile function of the myocardium [152] and of diseases characterized by the extensive involvement of the microcirculation and systemic peripheral ischaemia. In patients with SSc, platelet-derived, bioactive HMGB1 release correlates with platelet activation [153] and serum HMGB1 concentrations are associated with disease activity [154]. Elevated concentrations of HMGB1 in the blood have been found in patients with medium vessel vasculitis, KD in particular [155,156], and with small vessel vasculitis, including IgA vasculitis and AAV [157–162]. Plasma HMGB1 concentration increases in the vasculitis active phase [159,162]. Moreover, the concentration of HMGB1 is higher in patients with GPA with a mostly granulomatous disease [160]. In contrast, the accepted disease activity score, Birmingham Vasculitis Activity Score (BVAS) or other inflammatory markers do not discriminate between patients with a predominantly vasculitic or granulomatous involvement [160]. Because HMGB1 is expressed preferentially in the granulomatous tissue [160], systemic levels might reflect local production within granulomas. High HMGB1 levels also identify patients with renal involvement, either active or quiescent. The latter result possibly reflects a low-grade inflammatory response that continues even in the absence of overt clinical manifestations [162].

HMGB1 plays an apparently non-redundant role in controlling the inflammatory response to necrosis, sustaining the repair of injured tissues, on one hand, and controlling the establishment of acquired immune responses on the other hand [149,163–165]. HMGB1 pharmacological blockade in rheumatological diseases is being thoroughly investigated [151], and might prove valuable in the setting of systemic vasculitis.

Inflammasomes and vascular autoinflammation

Inflammasomes are intracellular machineries, assembled on demand after stimulation of innate pattern recognition receptors. Caspases 1 and 5 are ultimately recruited to process and activate cytokines, in particular to convert pro-IL-1β to active IL-1β [166,167] and to regulate cytokine-independent events, such as the acidification of phagosomes that contain bacteria [168]. Several drugs modulate the inflammasome. Colchicine eventually limits the inflammasome-elicited caspase 1 activation [169,170,171]. Anakinra (a recombinant IL-1-receptor antagonist), canakinumab (an anti-IL-1β humanized antibody) and rilonacept (a decoy IL-1-receptor) act downstream of the activation of the inflammasome. IFN-α has a dual inhibitory effect on the inflammasome: direct inhibition occurs through a signal transducer and activator of transcription (STAT)-1-dependent pathway, while the IFN-α-induced IL-10 increase down-regulates pro-IL-1β levels [172].

Inflammasome activation plays a prominent role in autoinflammatory disorders [166]. Low-density lipoprotein receptor-related protein 1 (LRP1) provides a scaffold necessary for the assembly of the inflammasome that activates caspases 1 and 5, required for processing and activation of various inflammatory cytokines: it has been identified as a novel GCA susceptibility gene, information that might hint at a deregulated recruitment of inflammasome in large vessel vasculitis, and suggests that it might represent a therapeutic target [173].

Abnormal expression and circulating levels of IL-1β has also been detected in patients with KD, in particular in those who do not respond to IVIG [174]. A non-redundant role of IL-1β and caspase-1 in coronary arteritis has been established in a mouse model of KD [175]. Genetic and pharmacological evidence supports the issue that IL-1β maturation and secretion are dependent upon the non-obese diabetic (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and are required for the development of coronary lesions. In this model, coronaritis was partially corrected by anakinra [175], providing a rationale for the use of anti-IL-1β treatments to prevent coronary disease in KD patients, even if caution should be used when translating such information in humans.

Elevated levels of IL-1β have been detected in the sera and synovial fluid of patients with Behcet's disease [60] and IL-1β production dominates the response to microbial stimuli of macrophages of patients with active disease [176], possibly providing a clue to the deregulated anti-bacterial response in Behçet's disease. However, caspase-independent pathways might also be involved [177]. Colchicine is recommended for the management of erythema nodosum and arthritis, and IFN-α for a wide range of manifestations of Behcet's disease, including eye, CNS and mucocutaneous involvement. Anakinra and canakinumab have proved to be efficacious in refractory Behcet's disease [178,179] and a pilot study evaluating safety and efficacy of anakinra against placebo is ongoing (NCT01441076).

B cells and vessel inflammation

Immune complex deposition and ANCA generation play a role in the recruitment and activation of innate immune response at sites of vessel inflammation [20,33]. In AAV a positive feedback loop possibly sustains neutrophil and B cell activation [180]. Activated neutrophils are a source of the B cell activating factor BAFF (also known as BLyS, a member of the TNF family) [181,182], whose levels are apparently associated with cryoglobulinaemia in the setting of HCV infection [183].

Marginal zone (MZ) innate-like B lymphocytes potently respond to inflammatory stimuli and their cross-talk with neutrophils results in an enhanced generation of antibodies against conserved microbial antigens, highlighting a B lymphocyte helper activity of neutrophils [184]. Conversely, innate-like CD5⁺ B cells appear to play a homeostatic role in AAV: they are reduced in the blood of patients with active disease, and low numbers after blood repopulation upon depletion with rituximab predict future relapse [185]. The selective expansion of MZ-like B cell clones might play a role in the progression to malignant lymphoproliferation in CV [186]. Factors involved in the regulation of MZ-like B cells such as the Fc receptor-like 5 (FCRL5) pathway [187] are currently under investigation as pathogenic players and potential therapeutic targets in CV [188].

B cell-depleting strategies have been tested extensively in clinical settings. A randomized controlled trial and two open-label studies have demonstrated the efficacy of rituximab in CV [28,189,190]. The RAVE and RITUXVAS trials recognized the equivalence of rituximab and cyclophosphamide in inducing remission in AAV [46–48]. According to the RAVE trial, rituximab was significantly more efficient than cyclophosphamide in those patients who relapsed [47,48], and some case reports showed the efficacy of rituximab in four patients with severe and/or refractory IgA vasculitis. Belimumab is an anti-B cell agent with a selective effect on naive B cells and plasma cells, due to blockade of BAFF. Belimumab is currently under investigation in combination with azathioprine for maintenance of remission in GPA and MPA (BREVAS trial: NCT01663623). A randomized placebo-controlled trial is going to begin to evaluate Blisibimod, another BAFF-antagonist, in the setting of AAV (BIANCA-SC trial: NCT01598857). B cell-depleting strategies appear less effective on granulomatous and in general non-vasculitic manifestations of AAV [191,192], pointing to the need for novel therapies targeted at mechanisms responsible for macrophage and eosinophil recruitment and granuloma maintenance, such as T lymphocytes [193].

T cells and vessel inflammation

Enhanced activation of effector T cells and concomitant functional defects in regulatory T cell (T_reg) action characterize AAV [36,194–197], due possibly to genetic predisposition [198] and impairment of peripheral tolerance [193]. A Th1 secretive profile dominates in localized GPA [194], whereas Th2 play a prominent role in EGPA [36] and are involved in generalized GPA [194]. Circulating levels of CD4⁺ effector memory T cells correlate inversely with disease activity in AAV and increase in urine during active renal disease, possibly reflecting their direct involvement in mediating organ inflammation [195] and granuloma formation [199]. Increased circulating levels of Th17 cells as well as IL-23 and IL-17 are detectable in patients with AAV [200–202], possibly as a consequence of PR3 stimulation [200]. The in-vivo role of Th17 in AAV is only partially understood, but possibly relevant mechanisms include:

• activation of macrophages and subsequently

  • ◦ IL-1β/TNF-mediated priming of neutrophils [199],

  • ◦ granuloma formation [36,194];

• activation of the endothelium (and promotion of leucocyte diapedesis) [199,203]; and

• enhancement of B cell function [199,203].

Besides neutrophils, T lymphocytes constitute the predominant leucocyte subgroup in the vascular infiltrate of Behçet's disease [59]. Patients with active Behçet's disease show expansion of the Th1 and Th17 compartment, increased Th17/T_reg ratio in the blood and cerebrospinal fluid [59], lower IL-10 levels and enhanced expression of the receptor for IL-23. The IL-23 pathway stimulates Th17 cells [56,204] which, in turn, promote neutrophil recruitment [205].

GCA is a T cell-dependent disease [206]. Adventitial-recruited Th1 and Th17 cells co-operate to promote [207] macrophage activation and vessel wall remodelling. Corticosteroids are probably only effective on the Th17 branch, whereas IFN-γ-producing Th1 cells are spared [207]. The latter cells could sustain vessel inflammation in refractory disease or induce relapse in apparently remitted patients. A selective cyclo-oxygenase-independent anti-IFN-γ effect has been described for aspirin in a model of GCA [208], possibly providing support for controversial clinical findings [209,210].

Innate-like γδ T cells are highly responsive to TNF [211] and show tropism for the recognition of heat shock proteins (HSP) [212]. Cross-reactivity between human and bacterial HSP could facilitate γδ T cell autoreactivity [213]. Increased expression of HSP occurs in the skin and endothelium of patients with Behçet's disease and TA, respectively [5,214] and is associated with expansion of the γδ T cell subset in the peripheral blood and at sites of inflammatory involvement [5,215–217]. Furthermore, autoreactive γδ T cells exert spontaneous cytotoxicity against aortic endothelial cells in TA [5]. A significant γδ T cell infiltrate has also been reported in GPA with renal involvement and in the skin of patients with cutaneous necrotizing vasculitis [218].

Several studies have tested the role of anti-T drugs in AAV, in particular in the setting of relapsing, refractory or persistent disease, where therapeutic indications are less defined. Anti-thymocyte globulin and 15-deoxypergualin (gusperimus) showed efficacy in open-label trials [219–221]. Alemtuzumab (Campath-1H), an anti-CD52 agent, showed controversial results in an open-label study [222]. A randomized trial (NCT01405807) is ongoing. IFN-α is effective in remission induction in EGPA, possibly by a selective dampening effect on Th2 responses (as well as on eosinophil degranulation) [36]. Productive interactions between adventitial DCs and T lymphocytes are crucial in the pathogenic cascade of large vessel vasculitides, and modulators of lymphocyte activation such as the cytotoxic T lymphocyte antigen (CTLA)-4 analogue abatacept are currently being tested in GCA and TA (NCT00556439). An open-label trial for the efficacy of abatacept in GPA has recently been completed (NCT00468208) and another involving Behçet's disease is expected to end by 2014 (NCT01693640). Blockade of IL-2 signalling (e.g. with the anti-IL-2Rα antibody daclizumab [223]) could perhaps find some application in analogous clinical settings. Anti-Th17-targeted therapies (e.g. the anti-IL-12/IL-23 monoclonal antibody ustekinumab) could prove beneficial in GCA, AAV and Behçet's disease, given the emerging role of Th17 in the pathogenesis of these diseases. In the setting of Behçet's disease, further rationale is provided by the efficacy of anti-Th17 agents in autoimmune diseases with a shared immunogenetic background [224,225].

Selected immune targets in vascular inflammation

TNF has a central role in a wide range of inflammatory conditions, including fever, neutrophil priming, endothelial activation and granuloma formation, which are all key events in the pathogenesis of vasculitis. TNF blockade has been tested extensively. An action on granulomatous lesions could be involved in the efficacy of anti-TNF agents in TA [226,227] and in naive or refractory AAV [51]. In the latter conditions, inhibition of neutrophil and endothelial activation could also occur. A similar mechanism could also be active in severe Behçet's disease, PAN and in the acute phase of KD. A study of the safety and effectiveness of infliximab in GCA (NCT00076726) was terminated, due to an interim analysis showing that infliximab did not reduce the number of first relapses in GCA or cumulative glucocorticosteroid dosage.

IL-5 and IL-25 are key players in EGPA [36]. IL-5, a well-characterized eosinophilic stimulating factor, is part of the Th2-associated cytokine array that characterizes EGPA. Conversely, eosinophil-derived IL-25, which is increased in the blood of patients with EGPA and expressed selectively in the vasculitic lesions, sustains Th2 responses [228]. Mepolizumab, an anti-IL-5 monoclonal antibody appears promising in EGPA [42,229], and anti-IL-25 agents are under development.

IL-6 is a pleiotropic cytokine secreted by monocytes and T cells and acts as a major inducer of systemic inflammation and local neoangiogenesis. Increased levels of IL-6 are detectable in the serum and cerebrospinal fluid of patients with neuro-Behçet and correlate with disease activity [60]. Serum concentrations of IL-6 are raised in GCA and correlate with disease activity. Moreover, IL-6 is expressed by infiltrating macrophages and promotes Th17 polarization [207], suggesting a direct involvement of the cytokine in the pathogenesis of the disease even if a protective effect of IL-6 in GCA could not be excluded [230]. Some studies have reported the efficacy of IL-6 blockade in Behçet's disease [231–233], in GCA [234,235] and in TA [13,236–238]. Randomized placebo-controlled trials are now ongoing (NCT01791153, NCT01450137, NCT01693653).

IL-21 is produced by activated central memory CD4⁺ T cells and promotes Th17 and Th1 in spite of T_reg differentiation [59]. High levels of IL-21 are present in circulating blood of patients with Behçet's disease and active GCA [239,240] and IL-21 blockade influences the Th17/T_reg balance in vitro [239,240], suggesting that anti-IL-21 agents (currently under development) might be effective [59].

Summary of potential therapeutic targets in vasculitides

A novel generation of agents has enhanced our ability to regulate the humoral and cellular immune response. As a consequence, better clinical outcomes and reduced drug-related toxicities have been obtained. However, we are just beginning to decipher the events associated with the persistent, self-maintaining vessel inflammation that is the hallmark of most systemic vasculitis. Specifically, we are just beginning to be aware of the protective role of vessel remodelling, necrosis and thrombosis in immune physiology that are the key features of most systemic vasculitis.

Extinguishing the priming events in vessel inflammation could be achieved by targeting the mechanisms involved in intravascular immunity, including inflammasome activation, leucocyte recruitment, cytokine- or thrombin-mediated endothelial and platelet activation. DAMPs, PAMPs and the innate receptor machinery involved in their recognition represent a particularly attractive area of work in which strategies aimed at eliciting even relatively subtle changes in the microenvironmental conditions might impact substantially on the outcome of the immune response. NETs are attracting growing interest, both as structures endowed with direct biological actions involved in vasculitis and as relevant sources of autoantigens, which could be involved in maintaining the vicious circle leading to unrelenting vascular inflammation.

The identification of the immunoregulatory properties of various subpopulations of innate leucocytes, and specifically innate B lymphoctes, and pericytes could further extend our ability to reset vascular inflammatory processes from their very beginning. Multiple pathways are also potentially involved in the remodelling of the vessel wall, and evidence derived from vasculitic and non-vasculitic settings suggests the potential efficacy of agents either with a direct neutralizing or agonist effect towards cytokines/growth factors that are implicated at a pre- and post-receptor level. Finally, identification of the protective action of molecules involved in the humoral innate immune response on vascular integrity and in guiding effective vascular repair suggests that we could rely upon the signals involved in the physiological homeostatic response of the vessels to injury as a novel frontier in drug development.

Disclosure

The authors declare that there is no conflict of interest.