**5. Are ANCA pathogenic?**

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 antibodies were pathogenic [86]. Neumann et al demonstrated excessive immune deposits in the early stages of life of SCG/Kinjoh mice (that spontaneously develop small vessel vasculitis and p-ANCA), and suggested that immune complex deposition leads to an inflammatory state, which when amplified by ANCA, likely lead to severe vasculitis [87]. In renal biopsies from AASV patients with renal involvement, Bajema et al showed that PR3, MPO, elastase and lactoferrin localized within or around fibrinoid necrotic lesions, and the lesions contained high levels of PR3 and elastase, which were also enriched inside the lesions [88]. Schlieben et al described a case of pulmonary renal syndrome in a newborn who received MPO-ANCA via passive transfer from the mother, supporting the idea that ANCA are pathogenic [89]. Animal models have not been developed to text the pathogenicity of PR3-ANCA, because human and murine PR3 share a low level of homology. However, an animal model of vasculitis and severe segmental and necrotizing glomerulonephritis, similar to WG, was recently developed in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice. In this model, spleno‐ cytes were isolated from NOD mice immunized with recombinant mouse PR3 and transferred into NOD-SCID mice, who developed disease pathology. These findings suggest that PR3- ANCA may play a direct role in PR3-ANCA-associated renal disease; however, in this model, a specific genetic background and autoimmune predisposition for kidney pathology are prerequisites for disease manifestation [90].

production and development of AASV (step two). Finally, environmental and genetic factors

History, Classification and Pathophysiology of Small Vessel Vasculitis

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

11

There are also experimental data that support this developmental model. There is evidence that in an inflammatory environment, autoantigens (nuclear/cytosolic) are presented by the opsonized cells, likely resulting in autoantibody formation. Kettritz et al used high doses of TNF-α to prime neutrophils, and demonstrated that caspase 3 dependent early neutrophil apoptosis was accompanied by increased surface expression of PR3 and MPO. In addition, these early apoptotic neutrophils showed a down-regulation of respiratory burst in response

Interestingly, Patry et al showed that injection of syngenic apoptotic neutrophils, but not freshly isolated neutrophils, into Brown Norway rats resulted in development of P-ANCA, with the majority being specific for elastase, again indicating that apoptotic neutrophils may boost an autoimmune response [102]. In another study, intraperitoneal infusion of live or apoptotic human neutrophils (but not formaline fixed or lysed neutrophils) into C57BL/6J mice resulted in development of ANCA specific for lactoferrin or myeloperoxidase. A second intravenous infusion of apoptotic neutrophils resulted in the development of PR3-specific ANCA. Again no vasculitic lesions were found in those mice developing ANCA [103].

As already known from general molecular biology knowledge, neutrophils migrating to inflamed sites undergo spontaneous apoptosis leading to their clearance without damage to the surrounding tissue. Macrophages in the blood recognize, among other surface membrane signals, the externalized Phosphatidyl Serine (PS) on the apoptotic neutrophils leading to their safe clearance. However, neutrophils that are not cleared in this manner progress to secondary necrosis, a process that triggers the release of pro-inflammatory cytokines. It appears that ANCAs dysregulate the process of neutrophil apoptosis. In an *in vitro* study conducted by Harper et al., ANCAs accelerated apoptosis of TNF--primed neutrophils by a mechanism dependent on NADPH oxidase and the generation of ROS. This was accompanied by uncou‐ pling of the nuclear and cytoplasmic changes from the surface membrane changes. That is, while apoptosis progressed more rapidly, there was no corresponding change in the rate of externalization of PS following activation of neutrophils by ANCAs. This dysregulation created a 'reduced window of opportunity' for phagocyte clearance by macrophages, leading to a more pro-inflammatory environment [104]. It must be noted here that ANCAs were unable to accelerate apoptosis in unprimed neutrophils. Additionally, although there was increased expression of PR3 and MPO as apoptosis progressed, ANCAs were unable to activate these neutrophils. In fact, there was a time-dependent decrease in ROS generation as these neutro‐ phils aged [104]. ANCA accelerates neutrophil apoptosis, in primed neutrophils, via genera‐ tion of ROS that act as amplifying factors for apoptosis. ROS are critical since neutrophils isolated from patients with chronic granulomatous disease (having a defect in ROS production) do not show accelerated apoptosis after ANCA activation [104]. The same authors, in a later study, as well as another independent group showed that ANCA binding to apoptotic neutrophils enhanced phagocytosis by human monocyte-derived macrophages, but at the same time they increased the secretion of pro-inflammatory cytokines like IL-1, IL-8 and TNFα [105,106]. IL-1 and IL-8 are capable of retarding apoptosis and are powerful chemo-attrac‐ tants. The pro-inflammatory neutrophil clearance will result in further cell recruitment and perpetuation of inflammation. The autoimmune response may be promoted by aberrant

can also contribute to disease expression [100].

to ANCA [101].

#### **5.1. Role of neutrophil apoptosis in AASV**

Increased neutrophil apoptosis has been observed in AASV. Pathological specimens from patients of WG show clear presence of apoptotic and necrotic neutrophils [91,92]. Leucocytes, with degraded nuclear material, undergoing disintegration and apoptotic cells have been observed in tissue specimens from ANCA-positive renal vasculitis [93]. Histologically, AASV is characterized by leukocytoclasis, with infiltration and accumulation of unscavenged apoptotic and necrotic neutrophils in tissues around blood vessels, and fibrinoid necrosis of the blood vessel walls [94]. E/M studies of the leukocytoclastic lesions, in patients with leukocytoclastic vasculitis, have suggested that there may be a defect in the clearance of apoptotic neutrophils. The minority of neutrophils in this study showed typical apoptotic changes of the condensed and marginated nuclei, while the majority showed intact nuclei with disintegrated cytoplasmic organelles and plasma membranes [95]. Apoptotic neutrophils may, in fact, be a source of immunologically exposed neutrophil antigens that promote the produc‐ tion of ANCAs. It has been speculated that the development of ANCA-positive vasculitis is a three-step pathological process. The first step involves an exogenous stimulus that increases neutrophil and macrophage apoptosis. An example is exposure to an inhaled substance like silica, which is known to induce apoptosis in human peripheral blood lymphocytes and to also induce Fas-ligand expression in lung macrophages (in *vitro* and in *vivo*), promoting Fasdependent macrophage apoptosis in a murine model of silicosis [96,97]. Similarly, other postulated etiological agents for AASV (propylthiouracil, *Streptococcus Pneumoniae*) have also been shown to induce/accelerate apoptosis [98,99]. There is also pathological evidence of leucocytes with degraded nuclear material undergoing disintegration in tissues and apoptotic cells have been observed in AASV. Therefore, it seems logical to suggest that defective clearance/increased exposure to apoptotic neutrophils may be the initiating factor for ANCA

production and development of AASV (step two). Finally, environmental and genetic factors can also contribute to disease expression [100].

antibodies were pathogenic [86]. Neumann et al demonstrated excessive immune deposits in the early stages of life of SCG/Kinjoh mice (that spontaneously develop small vessel vasculitis and p-ANCA), and suggested that immune complex deposition leads to an inflammatory state, which when amplified by ANCA, likely lead to severe vasculitis [87]. In renal biopsies from AASV patients with renal involvement, Bajema et al showed that PR3, MPO, elastase and lactoferrin localized within or around fibrinoid necrotic lesions, and the lesions contained high levels of PR3 and elastase, which were also enriched inside the lesions [88]. Schlieben et al described a case of pulmonary renal syndrome in a newborn who received MPO-ANCA via passive transfer from the mother, supporting the idea that ANCA are pathogenic [89]. Animal models have not been developed to text the pathogenicity of PR3-ANCA, because human and murine PR3 share a low level of homology. However, an animal model of vasculitis and severe segmental and necrotizing glomerulonephritis, similar to WG, was recently developed in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice. In this model, spleno‐ cytes were isolated from NOD mice immunized with recombinant mouse PR3 and transferred into NOD-SCID mice, who developed disease pathology. These findings suggest that PR3- ANCA may play a direct role in PR3-ANCA-associated renal disease; however, in this model, a specific genetic background and autoimmune predisposition for kidney pathology are pre-

Increased neutrophil apoptosis has been observed in AASV. Pathological specimens from patients of WG show clear presence of apoptotic and necrotic neutrophils [91,92]. Leucocytes, with degraded nuclear material, undergoing disintegration and apoptotic cells have been observed in tissue specimens from ANCA-positive renal vasculitis [93]. Histologically, AASV is characterized by leukocytoclasis, with infiltration and accumulation of unscavenged apoptotic and necrotic neutrophils in tissues around blood vessels, and fibrinoid necrosis of the blood vessel walls [94]. E/M studies of the leukocytoclastic lesions, in patients with leukocytoclastic vasculitis, have suggested that there may be a defect in the clearance of apoptotic neutrophils. The minority of neutrophils in this study showed typical apoptotic changes of the condensed and marginated nuclei, while the majority showed intact nuclei with disintegrated cytoplasmic organelles and plasma membranes [95]. Apoptotic neutrophils may, in fact, be a source of immunologically exposed neutrophil antigens that promote the produc‐ tion of ANCAs. It has been speculated that the development of ANCA-positive vasculitis is a three-step pathological process. The first step involves an exogenous stimulus that increases neutrophil and macrophage apoptosis. An example is exposure to an inhaled substance like silica, which is known to induce apoptosis in human peripheral blood lymphocytes and to also induce Fas-ligand expression in lung macrophages (in *vitro* and in *vivo*), promoting Fasdependent macrophage apoptosis in a murine model of silicosis [96,97]. Similarly, other postulated etiological agents for AASV (propylthiouracil, *Streptococcus Pneumoniae*) have also been shown to induce/accelerate apoptosis [98,99]. There is also pathological evidence of leucocytes with degraded nuclear material undergoing disintegration in tissues and apoptotic cells have been observed in AASV. Therefore, it seems logical to suggest that defective clearance/increased exposure to apoptotic neutrophils may be the initiating factor for ANCA

requisites for disease manifestation [90].

10 Updates in the Diagnosis and Treatment of Vasculitis

**5.1. Role of neutrophil apoptosis in AASV**

There are also experimental data that support this developmental model. There is evidence that in an inflammatory environment, autoantigens (nuclear/cytosolic) are presented by the opsonized cells, likely resulting in autoantibody formation. Kettritz et al used high doses of TNF-α to prime neutrophils, and demonstrated that caspase 3 dependent early neutrophil apoptosis was accompanied by increased surface expression of PR3 and MPO. In addition, these early apoptotic neutrophils showed a down-regulation of respiratory burst in response to ANCA [101].

Interestingly, Patry et al showed that injection of syngenic apoptotic neutrophils, but not freshly isolated neutrophils, into Brown Norway rats resulted in development of P-ANCA, with the majority being specific for elastase, again indicating that apoptotic neutrophils may boost an autoimmune response [102]. In another study, intraperitoneal infusion of live or apoptotic human neutrophils (but not formaline fixed or lysed neutrophils) into C57BL/6J mice resulted in development of ANCA specific for lactoferrin or myeloperoxidase. A second intravenous infusion of apoptotic neutrophils resulted in the development of PR3-specific ANCA. Again no vasculitic lesions were found in those mice developing ANCA [103].

As already known from general molecular biology knowledge, neutrophils migrating to inflamed sites undergo spontaneous apoptosis leading to their clearance without damage to the surrounding tissue. Macrophages in the blood recognize, among other surface membrane signals, the externalized Phosphatidyl Serine (PS) on the apoptotic neutrophils leading to their safe clearance. However, neutrophils that are not cleared in this manner progress to secondary necrosis, a process that triggers the release of pro-inflammatory cytokines. It appears that ANCAs dysregulate the process of neutrophil apoptosis. In an *in vitro* study conducted by Harper et al., ANCAs accelerated apoptosis of TNF--primed neutrophils by a mechanism dependent on NADPH oxidase and the generation of ROS. This was accompanied by uncou‐ pling of the nuclear and cytoplasmic changes from the surface membrane changes. That is, while apoptosis progressed more rapidly, there was no corresponding change in the rate of externalization of PS following activation of neutrophils by ANCAs. This dysregulation created a 'reduced window of opportunity' for phagocyte clearance by macrophages, leading to a more pro-inflammatory environment [104]. It must be noted here that ANCAs were unable to accelerate apoptosis in unprimed neutrophils. Additionally, although there was increased expression of PR3 and MPO as apoptosis progressed, ANCAs were unable to activate these neutrophils. In fact, there was a time-dependent decrease in ROS generation as these neutro‐ phils aged [104]. ANCA accelerates neutrophil apoptosis, in primed neutrophils, via genera‐ tion of ROS that act as amplifying factors for apoptosis. ROS are critical since neutrophils isolated from patients with chronic granulomatous disease (having a defect in ROS production) do not show accelerated apoptosis after ANCA activation [104]. The same authors, in a later study, as well as another independent group showed that ANCA binding to apoptotic neutrophils enhanced phagocytosis by human monocyte-derived macrophages, but at the same time they increased the secretion of pro-inflammatory cytokines like IL-1, IL-8 and TNFα [105,106]. IL-1 and IL-8 are capable of retarding apoptosis and are powerful chemo-attrac‐ tants. The pro-inflammatory neutrophil clearance will result in further cell recruitment and perpetuation of inflammation. The autoimmune response may be promoted by aberrant phagocytosis of apoptotic neutrophils by dendritic cells. In a recent study it has been shown that anti-PR3 antibody can also penetrate into human neutrophils (*in vitro*) and lead to enhancement of the apoptotic process [107].

Understanding the pathogenesis of neutrophil apoptosis and clearance in AASV can help to rationalize existing therapies and indicate new approaches to therapy [108].

#### **5.2. The role of netting Neutrophils (NETs)**

A novel form of PMN death named "NETosis", characterized by the active release of chro‐ matin, has been described recently [109]. Neutrophil extracellular traps (NETs) are extrusions of plasma membrane and nuclear material, containing granule components and histones. These structures bind gram-positive and negative bacteria, as well as fungi. In vitro, NETs have been shown to bind and kill extracellular microorganisms; *in vivo*, they have been documented in conditions, including appendicitis, sepsis, pre-eclampsia and experimental models of shigellosis [110]. The changes leading to NET formation follow a specific pattern, which is initiated by the loss of nuclear segregation into eu- and heterochromatin. Once the chromatin and granular components are mixed, NETs are released from the cell after cyto‐ plasmic membrane rupture by a process distinct from necrosis or apoptosis, termed NETosis. NADPH oxidase plays a role in this process, via generation of ROS, which act as signaling molecules. Fuchs et al demonstrated that NET formation is a part of active cell death, and that NETs are released when the activated neutrophils dies [111].

Kessenbrock et al. demonstrated that ANCA-stimulated neutrophils release NETs, which contain PR3 and MPO in addition to chromatin and LL37 (an antimicrobial peptide with capabilities of activating dendritic cells) [112]. In-vivo presence of NETs was shown in tissues (kidney biopsies from patients with small vessel vasculitis), with maximal concentration in areas showing neutrophilic infiltration, which suggests that NET formation occurs predomi‐ nantly during active disease [112]. In patients of AASV, increased levels of circulating nucleosomes has been reported [113]. It is likely that these may, in fact, be derived from and reflect NET formation in AASV. In short, NETs may incite production of ANCA, via presen‐ tation of antigen-chromatin complexes to the immune system, or ANCA may incite production of NETs, which then could aggravate the immune response, leading to perpetuation of the auto-immune response, Figure 2.

A significant recent finding is that mPR3 and CD177 are co-expressed on the same subset of circulating neutrophils in healthy subjects as well as in AASV patients [118,119]. Our group

**Figure 2.** Pathophysiological model of neutrophil extracellular traps (NETs) in ANCAassociated vasculitis. ANCA can induce TNF-α-primed neutrophils to produce NETs. The deposition of NETs may activate plasmacytoid dendritic cells that produce large amounts of interferon-α driving the autoimmune response. In this context, NETs may activate au‐ toreactive B cells to the production of ANCA, which results in a vicious circle of NET production that maintains the delivery of antigen–chromatin complexes to the immune system. Moreover, NETs may also stick to the endothelium

patients as compared to healthy controls, which suggests a distinct pathophysiological neutrophil phenotype in AASV [116]. Interestingly, higher CD177–mRNA, but not PR3– mRNA was found to correlate with a higher proportion of mPR3+/CD177+cells, suggesting that overproduction of CD177 could lead to an increase in the proportion of mPR3+/

It is likely that these two subpopulations have distinct functions, which may have a direct bearing on pathophysiological processes. Membrane CD177 helps neutrophils adhere to the endothelium, while m-PR3 helps this positive subpopulation to migrate through the endothe‐

neutrophil subpopulation was larger in AASV

History, Classification and Pathophysiology of Small Vessel Vasculitis

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

13

/CD177+

has demonstrated that the mPR3+

CD177+neutrophils [116].

and cause endothelial damage.

#### **5.3. Recent updates**

Experiments performed by our group, have shown that the plasma levels of mature PR3 as well as pro-PR3 are elevated in AASV [114,115,116]. It was also observed that mPR3+ neutro‐ phils are more abundant in AASV compared to healthy donors, which agrees with previous studies suggesting that a high percentage of mPR3+ cells may be a risk factor for vasculitis [78,115]. Circulating neutrophils and monocytes from patients with AASV display upregulat‐ ed transcription of the PR3 gene [117]. It is likely that aberrant PR3/mPR3 expression may reflect, or be a marker of a specific functional defect in neutrophils. A possible origin of high plasma levels of PR3 is shedding of membrane PR3.

History, Classification and Pathophysiology of Small Vessel Vasculitis http://dx.doi.org/10.5772/55238 13

phagocytosis of apoptotic neutrophils by dendritic cells. In a recent study it has been shown that anti-PR3 antibody can also penetrate into human neutrophils (*in vitro*) and lead to

Understanding the pathogenesis of neutrophil apoptosis and clearance in AASV can help to

A novel form of PMN death named "NETosis", characterized by the active release of chro‐ matin, has been described recently [109]. Neutrophil extracellular traps (NETs) are extrusions of plasma membrane and nuclear material, containing granule components and histones. These structures bind gram-positive and negative bacteria, as well as fungi. In vitro, NETs have been shown to bind and kill extracellular microorganisms; *in vivo*, they have been documented in conditions, including appendicitis, sepsis, pre-eclampsia and experimental models of shigellosis [110]. The changes leading to NET formation follow a specific pattern, which is initiated by the loss of nuclear segregation into eu- and heterochromatin. Once the chromatin and granular components are mixed, NETs are released from the cell after cyto‐ plasmic membrane rupture by a process distinct from necrosis or apoptosis, termed NETosis. NADPH oxidase plays a role in this process, via generation of ROS, which act as signaling molecules. Fuchs et al demonstrated that NET formation is a part of active cell death, and that

Kessenbrock et al. demonstrated that ANCA-stimulated neutrophils release NETs, which contain PR3 and MPO in addition to chromatin and LL37 (an antimicrobial peptide with capabilities of activating dendritic cells) [112]. In-vivo presence of NETs was shown in tissues (kidney biopsies from patients with small vessel vasculitis), with maximal concentration in areas showing neutrophilic infiltration, which suggests that NET formation occurs predomi‐ nantly during active disease [112]. In patients of AASV, increased levels of circulating nucleosomes has been reported [113]. It is likely that these may, in fact, be derived from and reflect NET formation in AASV. In short, NETs may incite production of ANCA, via presen‐ tation of antigen-chromatin complexes to the immune system, or ANCA may incite production of NETs, which then could aggravate the immune response, leading to perpetuation of the

Experiments performed by our group, have shown that the plasma levels of mature PR3 as

phils are more abundant in AASV compared to healthy donors, which agrees with previous

[78,115]. Circulating neutrophils and monocytes from patients with AASV display upregulat‐ ed transcription of the PR3 gene [117]. It is likely that aberrant PR3/mPR3 expression may reflect, or be a marker of a specific functional defect in neutrophils. A possible origin of high

neutro‐

cells may be a risk factor for vasculitis

well as pro-PR3 are elevated in AASV [114,115,116]. It was also observed that mPR3+

rationalize existing therapies and indicate new approaches to therapy [108].

enhancement of the apoptotic process [107].

12 Updates in the Diagnosis and Treatment of Vasculitis

**5.2. The role of netting Neutrophils (NETs)**

NETs are released when the activated neutrophils dies [111].

auto-immune response, Figure 2.

studies suggesting that a high percentage of mPR3+

plasma levels of PR3 is shedding of membrane PR3.

**5.3. Recent updates**

**Figure 2.** Pathophysiological model of neutrophil extracellular traps (NETs) in ANCAassociated vasculitis. ANCA can induce TNF-α-primed neutrophils to produce NETs. The deposition of NETs may activate plasmacytoid dendritic cells that produce large amounts of interferon-α driving the autoimmune response. In this context, NETs may activate au‐ toreactive B cells to the production of ANCA, which results in a vicious circle of NET production that maintains the delivery of antigen–chromatin complexes to the immune system. Moreover, NETs may also stick to the endothelium and cause endothelial damage.

A significant recent finding is that mPR3 and CD177 are co-expressed on the same subset of circulating neutrophils in healthy subjects as well as in AASV patients [118,119]. Our group has demonstrated that the mPR3+ /CD177+ neutrophil subpopulation was larger in AASV patients as compared to healthy controls, which suggests a distinct pathophysiological neutrophil phenotype in AASV [116]. Interestingly, higher CD177–mRNA, but not PR3– mRNA was found to correlate with a higher proportion of mPR3+/CD177+cells, suggesting that overproduction of CD177 could lead to an increase in the proportion of mPR3+/ CD177+neutrophils [116].

It is likely that these two subpopulations have distinct functions, which may have a direct bearing on pathophysiological processes. Membrane CD177 helps neutrophils adhere to the endothelium, while m-PR3 helps this positive subpopulation to migrate through the endothe‐ lium and interstitial tissues. It may be inferred that the mPR3+/CD177+ cells possess greater killing capabilities, including higher NET and ROS production, than the mPR3– /CD177– subpopulation. In simplistic terms, the mPR3+ /CD177+ neutrophils may be the designated "fighting" neutrophils, designed to migrate from blood into tissues and promote pro-inflam‐ matory, microbicidal functions, while mPR3-negative neutrophils are destined to stay in the intra-vascular compartment, and function as anti-inflammatory cells, until they are needed for resolution of inflammation to produce anti-inflammatory mediators or to phagocytose tissue debris and other dead neutrophils at the site of inflammation.

Investigations addressing polymorphisms in genes encoding for proinflammatory cytokines (TNF-α, IL-1b, IL-8, TGF-β and VEGF) have so far not revealed any predisposing factors for

History, Classification and Pathophysiology of Small Vessel Vasculitis

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

15

Familial mediterranean fever (FMF) is an autoinflammatory disease caused by a mutation in the MEFV gene, which in 7 % of cases is associated with HSP [137]. There is a high prevalence of children with MEFV mutations among HSP patients in countries with relative abundance of FMF [138,139]. The implication this association has on the general pathogenesis of HSP is,

HSP is usually preceded by infections, in up to 95 % of cases localized in the upper respiratory tract, and appears in clusters in families [140,141,142]. The incidence of HSP is highest during early childhood and shows distinct seasonal variations with a peak during autumn and winter [6]. Both early childhood and the autumn-winter season are periods with frequent infections. Thus, clinical observations suggest an important role of infections in the etiology and patho‐

Several studies have shown a circumstantial relation of infections with group A streptococci and the development of HSP [143,144,145]. Others found serological evidence for an associa‐ tion with infections with other bacteria such as Bartonella henselae or viruses such as parvo‐

Non-infectious agents have been found to be associated with the development of HSP especially in adults. These include certain drugs such as angiotensin-converting enzyme inhibitors, angiotensin II-receptor antagonists, antibiotics, and non-steroidal anti-inflamma‐

IgA deposits in HSP are composed of immune-complexes mainly consisting of IgA1 [150].

Serum samples from HSN patients were found to have elevated levels of underglycosylated polymeric IgA1 compared to controls [151]. However, in children with HSP without renal involvement the levels were not higher than those of controls [152]. Underglycosylated polymeric IgA1 has been found to exhibit an inflammatory and proliferative effect on mesan‐ gial cells (see IgA1 in IgAN). Taken together, underglycosylated polymeric IgA1 seems to be involved in the development of HSN, but its role in the pathogenesis of HSP per se remains

The acute phase of systemic vasculitis is generally characterized by vascular leukocytic infiltration and activation of innate immunity. Elevated levels of inflammatory cytokines are

tory drugs as well as insect bites, vaccinations or food allergies [149].

usually detectable in the serum and affected tissues in these diseases.

HSP [135,136].

if at all, unclear.

genesis of HSP.

**6.3. IgA1 in HSP**

unclear.

**6.4. Mediators of inflammation**

**6.2. Infectious and non-infectious agents**

virus B19 and hepatitis C virus [146,147,148].

Our group is the first to demonstrate a lower rate of spontaneous apoptosis and *longer in vitro* survival in neutrophils from AASV patients in remission as compared to neutrophils from healthy blood donors [120].

Contrary to our results, Harper et al. showed that neutrophils from AASV patients, especially those with active disease, have an accelerated rate of apoptosis [106].
