**4. Pathophysiology of ANCA-Associated Systemic Vasculitis (AASV)**

The pathophysiology of AASV remains largely unknown. Clinical and laboratory evidence suggest a multifactorial origin. Although the association between ANCA and pauci-immune small vessel vasculitides has been established, the exact role of ANCA in the pathogenesis of AASV is yet not fully elucidated. It is not known whether ANCA play a direct role in disease manifestations, or whether the antibodies are secondary markers of the disease process. Available data suggest that neutrophils, B- and T- lymphocytes play a key role in the patho‐ physiology of AASV.

#### **4.1. Pathogenic B-cell response and production of ANCA**

**Dominant vessel involved Primary Secondary**

Takayasu's arteritis

Kawasaki disease

Churg-Strauss syndrome\* Microscopic polyangiitis\*

Essential mixed cryoglobulinaemia Cutaneous leukocytoclastic vasculitis

many others. PAN= Polyarteritis Nodosa. RA= Rheumatoid Arthritis. SLE= Systemic Lupus Erythematosus.

(\*) Diseases most commonly associated with ANCA, pausi-immune crescentic glomerulonepghritis and which are most responsive to immunosuppression with cyclophosphamide. (\*\*) e.g. sulphonamides, penicillins, thiazide diuretics, and

ANCA-associated systemic vasculitis (AASV) are a group of diseases classified as small vessel vasculitides that are associated with anti-neutrophil cytoplasmic antibodies. AASV include microscopic polyangiitis, Wegener´s granulomatosis, Churg-Struass syndrome and renal limited vasculitis. Together they are responsible for 5-6% of cases presenting with renal failure. They are characterized histologically by necrotizing vasculitis preferentially affecting small blood vessels and often associated with pauci-immune necrotizing crescentic glomeruloneph‐ ritis. Serologically, these diseases present autoantibodies directed against constituents of

In1990, three independent groups showed that azurophilic granule enzyme proteinase 3 was the target autoantigen recognized by ANCA (PR3-ANCA) [21,22,23]. Together with proteinase 3, another granule protein, myeloperoxidase (MPO) was also identified as a target autoantigen of ANCA (MPO-ANCA) [24]. The discovery of ANCA has been critical to understanding the pathogenesis of the disease, as well as providing a valuable diagnostic tool. The American College of Rheumatology published criteria for classifying vasculitides in 1990, leading to improved categorization of patients for clinical trials [25]. However, these criteria were not adequate for diagnosing patients with ANCA-associated vasculitides. An individual patient could simultaneously meet the criteria for WG, Churg Strauss Syndrome (CSS), Polyarteritis Nodosa (PAN), hypersensitivity vasculitis and Henoch-Schönlein pupura. In 1994 the Chapel Hill Consensus conference (CHCC) adopted standardized names and definitions of vasculi‐

Recently a group of physicians from multiple medical disciplines met at the European Medicines Agency (EMEA) in London in September 2004 and January 2006 and developed a stepwise algorithm for classifying AASV and PAN for epidemiological studies. Their aim was to develop a consensus approach for applying CHCC definitions and ACR criteria to AASV and PAN, in order to facilitate comparison between epidemiological data for different

Aortitis associated with RA Infection (eg. Syphilis)

Infection (eg. Hepatitis B)

Infection (e.g. HIV)

Infection (e.g. Hepatitis B, C)

Drugs

Drugs\*\*

Vasculitis 2 to RA, SLE, Sjögren's syndrome

Large arteries Giant cell arteritis

Small vessels and medium arteries Wegener's granulomatosis\*

Small vessels (leukocytoclastic) Henoch-Schönlein purpura

tides, based on the size of the affected blood vessels [26].

Medium arteries Classical PAN

4 Updates in the Diagnosis and Treatment of Vasculitis

**Table 1.** Classification of systemic vasculitis.

neutrophil granules [20].

vasculitides [27].

B-cells are the direct precursors of antibody producing plasma cells. B-cells also produce autoantibodies and cytokines (Interleukin IL-6, Tumor Necrosis Factor alpha-TNFα, IL-10), act as antigen presenting cells, and differentiate into long lasting memory B-cells. Csernak et al. have shown that in WG patients, ANCA are produced following B-cell activation [32]. A polyclonal B-cell lymphoid infiltrate in the endonasal granulomatous lesion included PR3-ANCAproducing cells with copy number increase in three VH genes. The granulomatous lesions in WG consist of clusters of PR3 surrounded by an infiltrate consisting of maturing B-cells, antigen-presenting cells (APCs) and Th1-type CD4+CD28− T cells. This suggests that endo‐ nasal B-cell maturation is antigen-driven, and that B-cells generate ANCA via contact with PR3 or an antigenic microbial epitope [33].

B-cells recognize soluble antigens via specific B-cell receptors (BCR) and co-receptor CD19 that augments BCR downstream signaling. CD19 dysregulation has been reported in patients with AASV. Culton et al. showed that CD19 expression is 20% lower in naive B-cells from patients with AASV than from normal controls [34]. In contrast, the memory B-cells from some patients with AASV express more CD19 than normal controls. This subset of B-cells shows evidence of antigenic selection, suggesting that in AASV, mechanisms of self-tolerance may be lost leading to production of auto-reactive B-cells [34]. Experiments in transgenic mice indicate that defective B-cell regulation, specifically in pathways responsible for deletion (central and peripheral) of auto-reactive B-cells, may also play a role in generating autoantibodies in AASV [35]. Interestingly, expression of B-cell activating factor of the TNF family (BAFF) is increased in patients with WG [36]. It is postulated that BAFF may drive B-cell expansion, which then leads to ANCA production. B-cell depletion via rituximab in patients with AASV decreases ANCA levels and induces disease remission [37,38]. Conversely, clinical relapse correlates with increase levels of B cells [39]. These data support the conclusion that B cells play a central role in ANCA production and that ANCA play a significant role in the pathogenesis of AASV.

member of the killer immunoglobulin-like receptor family [51]. A significant increase in the proportion of IL-17 producing CD4+ T cells (Th17 cells) in in vitro stimulated peripheral blood cells from WG patients has also been reported [52]. IL-17 induces secretion of neutro‐ phil-attracting chemokines, and release of pro-inflammatory cytokines (IL-1β, TNF-α) capa‐ ble of increasing expression of PR3 on the surface of neutrophils. Patients with ANCApositive WG are reported to have more PR3-specific Th17 cells than ANCA-negative WG patients and healthy controls [52]. It is, therefore, likely that a Th1 response plays an impor‐

History, Classification and Pathophysiology of Small Vessel Vasculitis

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7

**Figure 1.** Pathophysiology of AASV. The stimulation of neutrophils by TNF-α or IL-1β (priming), e.g. during a preceding infection, leads to the translocation of the ANCA-antigens, PR3 and MPO, from the cytoplasmic granules (specific granules and secretory vesicels) to the cell surface, where they are accessible for ANCA, which leads to a further activa‐ tion of the cell. ANCA-induced neutrophil activation initiates production of ROS, neutrophil degranulation with re‐ lease of inflammatory cytokines and granule contents (e.g., PR3 and HLE) from azurophilic granules, leading to endothelial cell detachment and lysis. Furthermore, neutrophil activation leads to leukocyte adhesion (via ICAM-1, VCAM-1) and transmigration through endothelium (via PECAM-1), and release of ROS and proteases into tissues. Su‐ perantigen (e.g., Staphylococcal exotoxins) or PR3 presented to the T-cells directly or via dendritic cells, are capable of stimulating the proliferation of T-cells, leading to granuloma formation and finally to maturation of PR3-specific au‐ toreactive B-cells, culminating in ANCA production. ROS= Reactive oxygen species. PR3= Proteinase 3, MPO= Myelo‐ peroxidase, HLE= Human Leukocyte elastase, ICAM= Intercellular adhesion molecule-1, VCAM-1=Vascular cell adhesion molecule-1, PECAM-1= Platelet endothelial cell adhesion molecule-1, TCR= T-cell receptor, MHC-II= Major

Histocompatibility complex-II, TNF-α=Tumor necrosis factor-alpha, IL-1β=Interleukin-1 Beta.

tant role in antibody production and granuloma formation in AASV.

#### **4.2. Pathogenic T-response, tissue damage, and granuloma formation**

Under normal conditions, naïve T-cells are activated during an immune response to an antigen stimulus. Antigen-specific T-cells then differentiate into memory T-cells, while effector-T cells undergo apoptosis. Paucity of immunoglobulins in the vasculitic lesions, predominance of IgG1 and IgG4 subclasses of IgG, and the presence of granulomatous lesions indicate that Tcell-mediated immune responses play a role in the pathogenesis of AASV [40]. This is consis‐ tent with the fact that T cell-based treatment strategies produce clinically-relevant remission in AASV patients [41,42].

In patients with active WG, higher proportion of activated T-cells and higher concentration of soluble T cell activation markers (including soluble IL-2 receptor or CD30) are reported to correlate with disease activity [43]. High levels of activation markers also correlate with ANCA-positivity, which suggests persistent T cell activation, likely secondary to a persistent antigenic trigger, as an underlying pathogenic factor. This is consistent with reports of persistent expansion of CD4+ effector memory T-cells (Tem) combined with a decrease in naïve T-cells in patients with AASV [44,45]. A polarization of Th1 and Th2 response has also been reported in AASV. In particular, a Th2-type response is predominant in patients with active generalized WG or CSS, while a Th1 response is dominant in patients with localized WG or MPA, indicating that aberrant T cell response plays a role in the disease process [46,47]. CCR5 is also expressed on T-cells in early, localized WG, which might also favor recruitment of Th1 type cytokine secreting cells into inflammatory lesions in localized WG [48]. Conversion from Th1 to Th2 type response could underlie progression from localized to generalized WG. This shift could reflect B-cell expansion and T-cell-dependent PR3-ANCA production, secondary to interaction between neutrophils and auto-reactive T- and B-cells in inflammatory lesions, Figure 1.

The granulomas in AASV resemble a germinal centre, with a cluster of primed neutrophils surrounded by dendritic cells, T- and B-cells. CD4+ T cells are likely to play an important role in the granulomatous response in AASV. The decrease in CD4+CD28-- Tem subset of Tcells during active disease, in patients with WG, indicates an increased migration of these cells to sites of inflammation [44]. In an experimental model of autoimmune, anti-MPO-asso‐ ciated glomerulonephritis, it was noted that mice depleted of CD4+ T cells, at the time of ad‐ ministration of anti-mouse anti-GBM antibodies, developed significantly less crescent formation and cell response, compared to controls [49]. In patients with ANCA-associated glomerulonephritis, Tem cells are the predominant T-cell subtype in the glomerular infil‐ trate [50]. Together, these observations suggest that a cell mediated immune response con‐ tributes to the pathogenesis of renal lesions. Indeed, CD4+ Tem cells from WG patients lack NKG2A (inhibitory receptor) and demonstrate increased expression of NKG2D, which is a member of the killer immunoglobulin-like receptor family [51]. A significant increase in the proportion of IL-17 producing CD4+ T cells (Th17 cells) in in vitro stimulated peripheral blood cells from WG patients has also been reported [52]. IL-17 induces secretion of neutro‐ phil-attracting chemokines, and release of pro-inflammatory cytokines (IL-1β, TNF-α) capa‐ ble of increasing expression of PR3 on the surface of neutrophils. Patients with ANCApositive WG are reported to have more PR3-specific Th17 cells than ANCA-negative WG patients and healthy controls [52]. It is, therefore, likely that a Th1 response plays an impor‐ tant role in antibody production and granuloma formation in AASV.

in patients with WG [36]. It is postulated that BAFF may drive B-cell expansion, which then leads to ANCA production. B-cell depletion via rituximab in patients with AASV decreases ANCA levels and induces disease remission [37,38]. Conversely, clinical relapse correlates with increase levels of B cells [39]. These data support the conclusion that B cells play a central role in ANCA production and that ANCA play a significant role in the pathogenesis of AASV.

Under normal conditions, naïve T-cells are activated during an immune response to an antigen stimulus. Antigen-specific T-cells then differentiate into memory T-cells, while effector-T cells undergo apoptosis. Paucity of immunoglobulins in the vasculitic lesions, predominance of IgG1 and IgG4 subclasses of IgG, and the presence of granulomatous lesions indicate that Tcell-mediated immune responses play a role in the pathogenesis of AASV [40]. This is consis‐ tent with the fact that T cell-based treatment strategies produce clinically-relevant remission

In patients with active WG, higher proportion of activated T-cells and higher concentration of soluble T cell activation markers (including soluble IL-2 receptor or CD30) are reported to correlate with disease activity [43]. High levels of activation markers also correlate with ANCA-positivity, which suggests persistent T cell activation, likely secondary to a persistent antigenic trigger, as an underlying pathogenic factor. This is consistent with reports of persistent expansion of CD4+ effector memory T-cells (Tem) combined with a decrease in naïve T-cells in patients with AASV [44,45]. A polarization of Th1 and Th2 response has also been reported in AASV. In particular, a Th2-type response is predominant in patients with active generalized WG or CSS, while a Th1 response is dominant in patients with localized WG or MPA, indicating that aberrant T cell response plays a role in the disease process [46,47]. CCR5 is also expressed on T-cells in early, localized WG, which might also favor recruitment of Th1 type cytokine secreting cells into inflammatory lesions in localized WG [48]. Conversion from Th1 to Th2 type response could underlie progression from localized to generalized WG. This shift could reflect B-cell expansion and T-cell-dependent PR3-ANCA production, secondary to interaction between neutrophils and auto-reactive T- and B-cells in inflammatory lesions,

The granulomas in AASV resemble a germinal centre, with a cluster of primed neutrophils surrounded by dendritic cells, T- and B-cells. CD4+ T cells are likely to play an important role in the granulomatous response in AASV. The decrease in CD4+CD28-- Tem subset of Tcells during active disease, in patients with WG, indicates an increased migration of these cells to sites of inflammation [44]. In an experimental model of autoimmune, anti-MPO-asso‐ ciated glomerulonephritis, it was noted that mice depleted of CD4+ T cells, at the time of ad‐ ministration of anti-mouse anti-GBM antibodies, developed significantly less crescent formation and cell response, compared to controls [49]. In patients with ANCA-associated glomerulonephritis, Tem cells are the predominant T-cell subtype in the glomerular infil‐ trate [50]. Together, these observations suggest that a cell mediated immune response con‐ tributes to the pathogenesis of renal lesions. Indeed, CD4+ Tem cells from WG patients lack NKG2A (inhibitory receptor) and demonstrate increased expression of NKG2D, which is a

**4.2. Pathogenic T-response, tissue damage, and granuloma formation**

in AASV patients [41,42].

6 Updates in the Diagnosis and Treatment of Vasculitis

Figure 1.

**Figure 1.** Pathophysiology of AASV. The stimulation of neutrophils by TNF-α or IL-1β (priming), e.g. during a preceding infection, leads to the translocation of the ANCA-antigens, PR3 and MPO, from the cytoplasmic granules (specific granules and secretory vesicels) to the cell surface, where they are accessible for ANCA, which leads to a further activa‐ tion of the cell. ANCA-induced neutrophil activation initiates production of ROS, neutrophil degranulation with re‐ lease of inflammatory cytokines and granule contents (e.g., PR3 and HLE) from azurophilic granules, leading to endothelial cell detachment and lysis. Furthermore, neutrophil activation leads to leukocyte adhesion (via ICAM-1, VCAM-1) and transmigration through endothelium (via PECAM-1), and release of ROS and proteases into tissues. Su‐ perantigen (e.g., Staphylococcal exotoxins) or PR3 presented to the T-cells directly or via dendritic cells, are capable of stimulating the proliferation of T-cells, leading to granuloma formation and finally to maturation of PR3-specific au‐ toreactive B-cells, culminating in ANCA production. ROS= Reactive oxygen species. PR3= Proteinase 3, MPO= Myelo‐ peroxidase, HLE= Human Leukocyte elastase, ICAM= Intercellular adhesion molecule-1, VCAM-1=Vascular cell adhesion molecule-1, PECAM-1= Platelet endothelial cell adhesion molecule-1, TCR= T-cell receptor, MHC-II= Major Histocompatibility complex-II, TNF-α=Tumor necrosis factor-alpha, IL-1β=Interleukin-1 Beta.

#### **4.3. Monocyte activation and production of pro-inflammatory cytokines**

Wickman et al compared monocytes and cytokine profiles in patients with acute anti-PR3 vasculitis and normal controls; monocytes from patients were reported to have a reduced capacity to produce oxygen radicals [53]. Ohlsson et al., from our group, reported a positive correlation between circulating levels of IL-8 and monocyte IL-8 mRNA in patients with AASV, suggesting prolonged immune activation [54]. Pathological analysis of renal tissue from patients with AASV revealed the presence of monocytes in the glomerular crescents and granulomas [55]. In-vitro studies demonstrated that ANCA are capable of stimulating monocytes, leading to release of cytokines including IL-8, MCP-1, TNF-, IL-1, IL-6 and thromboxane A2 [56,57]. On the other hand, membrane PR3 expression on monocytes does not correlate with disease activity. There are many possible explanations for the presence of activated monocytes in glomerular crescents. For example, it is possible that monocytes are activated by direct physical interaction with components of glomerular lesions once they reach site of lesion; alternatively, dysfunctional apoptosis may stimulate monocyte activation [58].

Beaudreuil et al showed that exposure to silica is associated with a nearly seven-fold increased risk of being ANCA-positive [69]. ANCA, both PR3 and MPO, are detected in sera of patients with protracted infections; however, in most infections, ANCA are directed against a wide repertoire of antigens and tend to be dual [70]. Stegeman et al. described an association between nasal S. aureus and relapses of PR3-AAV [71]. Chronic infections may prime neutrophils, which can be further activated by PR3-ANCA, leading to vasculitis. It is also possible that some exogenous non-self proteins (i.e., bacterial, viral, fungal) mimic auto-antigens, which generates ANCA and an ANCA response. For example, PR3-ANCA has been detected in sera of patients with bacterial endocarditis [72]. Long standing exposure of the immune system to specific antigens, may set the stage for development of ANCA and subsequent AASV. Many theories have been made in line of this thought, including anti-complementary PR3 antibody theory [73] and Anti-LAMP (Lysosomal associated membrane protein) anatibody theory [74], which

History, Classification and Pathophysiology of Small Vessel Vasculitis

http://dx.doi.org/10.5772/55238

9

In general, autoimmune diseases display familial inheritance, suggesting that affected individuals carry genetic variation that contributes to disease susceptibility. Case reports show clusters of WG in siblings and close relatives, and specific HLA associations (DR1-DQw1) in AASV patients also suggest the existence of genetic susceptibility loci [75,76,77]. In patients

than in healthy controls, leading to a skewed bimodal distribution of mPR3 towards a high

periods of time, it also runs in families and is similar between twins [79]. Furtherore, patients with WG carry a polymorphism that disrupts a putative transcription factor binding site in the PR3 promoter region [80]. This polymorphism may lead to increased expression of PR3

(affecting T cell activation), alpha-1 antitrypsin level (protease inhibitor of PR3), and other

The subject of pathogenicity of ANCA is controversial. ANCA are absent in some patients with small vessel vasculitis, while MPO-ANCA are detected in patients with rheumatoid arthritis and other disorders [85]. Also, a paucity of immune complexes at sites of pathological lesions argues against a direct role for ANCA. However, animal models of small vessel vasculitis provide convincing evidence that ANCA are pathogenic in AASV. Xiao et al demonstrated that Rag2-/- mice, which are completely deficient in T- and B-lymphocytes with antigen receptors, developed a severe necrotizing glomerulonephritis and small vessel vasculitis when they were injected with anti-MPO splenocytes, while mice that received anti-BSA or normal splenocytes remained disease-free. Similarly, Rag2-/- and WT B6-mice injected with anti-MPO IgG developed focal glomerular necrosis and crescent formation, clearly indicating that the

phenotype in WG [78]. This phenomenon may be genetically determined, because the

neutrophils is a stable phenotype in the same individual over prolonged

phenotype. Additional polymorphisms involving CTLA-4

) are more abundant

with WG, neutrophils with positive expression of membrane-PR3 (mPR3+

genes/proteins have been reported in AASV patients [81,82,83,84].

are out of the scope of this study.

**4.6. Genetic predisposition**

mPR3+

proportion of mPR3+

and explain the high mPR3+

**5. Are ANCA pathogenic?**

#### **4.4. Endothelial cell activation and enhanced expression of adhesion molecules**

Endothelial damage, neutrophil invasion and necrosis are histopathological features of AASV [59]. Activated endothelial cells express high levels of adhesion molecules. Increased circulat‐ ing levels of endothelial proteins (thrombomodulin, vWF), and adhesion molecules (soluble intercellular adhesion molecule (sICAM)-1 and the soluble endothelial cell-leukocyte adhesion molecule (sELAM)-1) have been reported in vasculitis [60]. Woywodt et al. reported the presence of significant number of circulating endothelial cells and necrotic endothelial cell fragments in patients of active AASV [61]. A significant proportion of the circulating endo‐ thelial cells (EC) stain positive for tissue factor (TF), which links proinflammatory mechanisms with thrombosis [61]. Interestingly, TF expression can be induced in ECs by the release of PR3 and elastase from neutrophils; this may be mediated via PR3 receptors on the endothelial cell surface [62]. Endothelial cell necrosis, and release of TF, may play a role in development of vasculitic lesions. The mechanism of endothelial cell necrosis is not yet fully elucidated. Although anti-endothelial cell antibodies have been detected in AASV, their significance in this regard is not clear [63]. ANCA antigens, PR3 and MPO, can bind to endothelial cells via endothelial cell receptors [64,65]. ANCA can bind to endothelial cell bound antigens, leading to EC activation. It is possible that ANCA-induced neutrophil activation induces release of cytotoxic enzymes that damage endothelial cells. In AASV patients with renal involvement, the levels of circulating angiopoietin-2 (Ang-2) correlate with the increased number of circulating ECs. In-vitro studies suggeset that the endothelial-specific angiopoietin (Ang)-Tie ligand-receptor system regulates endothelial cell detachment. By analogy, Ang-2 might regulate endothelial cell detachment in AASV [66].

#### **4.5. Environmental factors**

Clinical and epidemiological evidence demonstrate that environmental factors, including silica, asbestos, drugs (anti-thyroid medications), and various infections (bacterial endocardi‐ tis, hepatitis C visrus), correlate with circulating ANCA and development of AASV [67,68]. Beaudreuil et al showed that exposure to silica is associated with a nearly seven-fold increased risk of being ANCA-positive [69]. ANCA, both PR3 and MPO, are detected in sera of patients with protracted infections; however, in most infections, ANCA are directed against a wide repertoire of antigens and tend to be dual [70]. Stegeman et al. described an association between nasal S. aureus and relapses of PR3-AAV [71]. Chronic infections may prime neutrophils, which can be further activated by PR3-ANCA, leading to vasculitis. It is also possible that some exogenous non-self proteins (i.e., bacterial, viral, fungal) mimic auto-antigens, which generates ANCA and an ANCA response. For example, PR3-ANCA has been detected in sera of patients with bacterial endocarditis [72]. Long standing exposure of the immune system to specific antigens, may set the stage for development of ANCA and subsequent AASV. Many theories have been made in line of this thought, including anti-complementary PR3 antibody theory [73] and Anti-LAMP (Lysosomal associated membrane protein) anatibody theory [74], which are out of the scope of this study.

#### **4.6. Genetic predisposition**

**4.3. Monocyte activation and production of pro-inflammatory cytokines**

8 Updates in the Diagnosis and Treatment of Vasculitis

**4.4. Endothelial cell activation and enhanced expression of adhesion molecules**

regulate endothelial cell detachment in AASV [66].

**4.5. Environmental factors**

Endothelial damage, neutrophil invasion and necrosis are histopathological features of AASV [59]. Activated endothelial cells express high levels of adhesion molecules. Increased circulat‐ ing levels of endothelial proteins (thrombomodulin, vWF), and adhesion molecules (soluble intercellular adhesion molecule (sICAM)-1 and the soluble endothelial cell-leukocyte adhesion molecule (sELAM)-1) have been reported in vasculitis [60]. Woywodt et al. reported the presence of significant number of circulating endothelial cells and necrotic endothelial cell fragments in patients of active AASV [61]. A significant proportion of the circulating endo‐ thelial cells (EC) stain positive for tissue factor (TF), which links proinflammatory mechanisms with thrombosis [61]. Interestingly, TF expression can be induced in ECs by the release of PR3 and elastase from neutrophils; this may be mediated via PR3 receptors on the endothelial cell surface [62]. Endothelial cell necrosis, and release of TF, may play a role in development of vasculitic lesions. The mechanism of endothelial cell necrosis is not yet fully elucidated. Although anti-endothelial cell antibodies have been detected in AASV, their significance in this regard is not clear [63]. ANCA antigens, PR3 and MPO, can bind to endothelial cells via endothelial cell receptors [64,65]. ANCA can bind to endothelial cell bound antigens, leading to EC activation. It is possible that ANCA-induced neutrophil activation induces release of cytotoxic enzymes that damage endothelial cells. In AASV patients with renal involvement, the levels of circulating angiopoietin-2 (Ang-2) correlate with the increased number of circulating ECs. In-vitro studies suggeset that the endothelial-specific angiopoietin (Ang)-Tie ligand-receptor system regulates endothelial cell detachment. By analogy, Ang-2 might

Clinical and epidemiological evidence demonstrate that environmental factors, including silica, asbestos, drugs (anti-thyroid medications), and various infections (bacterial endocardi‐ tis, hepatitis C visrus), correlate with circulating ANCA and development of AASV [67,68].

Wickman et al compared monocytes and cytokine profiles in patients with acute anti-PR3 vasculitis and normal controls; monocytes from patients were reported to have a reduced capacity to produce oxygen radicals [53]. Ohlsson et al., from our group, reported a positive correlation between circulating levels of IL-8 and monocyte IL-8 mRNA in patients with AASV, suggesting prolonged immune activation [54]. Pathological analysis of renal tissue from patients with AASV revealed the presence of monocytes in the glomerular crescents and granulomas [55]. In-vitro studies demonstrated that ANCA are capable of stimulating monocytes, leading to release of cytokines including IL-8, MCP-1, TNF-, IL-1, IL-6 and thromboxane A2 [56,57]. On the other hand, membrane PR3 expression on monocytes does not correlate with disease activity. There are many possible explanations for the presence of activated monocytes in glomerular crescents. For example, it is possible that monocytes are activated by direct physical interaction with components of glomerular lesions once they reach site of lesion; alternatively, dysfunctional apoptosis may stimulate monocyte activation [58].

> In general, autoimmune diseases display familial inheritance, suggesting that affected individuals carry genetic variation that contributes to disease susceptibility. Case reports show clusters of WG in siblings and close relatives, and specific HLA associations (DR1-DQw1) in AASV patients also suggest the existence of genetic susceptibility loci [75,76,77]. In patients with WG, neutrophils with positive expression of membrane-PR3 (mPR3+ ) are more abundant than in healthy controls, leading to a skewed bimodal distribution of mPR3 towards a high mPR3+ phenotype in WG [78]. This phenomenon may be genetically determined, because the proportion of mPR3+ neutrophils is a stable phenotype in the same individual over prolonged periods of time, it also runs in families and is similar between twins [79]. Furtherore, patients with WG carry a polymorphism that disrupts a putative transcription factor binding site in the PR3 promoter region [80]. This polymorphism may lead to increased expression of PR3 and explain the high mPR3+ phenotype. Additional polymorphisms involving CTLA-4 (affecting T cell activation), alpha-1 antitrypsin level (protease inhibitor of PR3), and other genes/proteins have been reported in AASV patients [81,82,83,84].
