**8. Innate immune responses in ANCA associated vasculitis**

#### **8.1. Neutrophils, key effector cells, in ANCA associated vasculitis**

arthritis,[102] consistent with this finding IL-17A-/- mice are protected from murine experi‐ mental arthritis.[103-104] IL-17A has been implicated in inflammatory bowel disease, both experimental[105] and clinical[106] as well as human inflammatory skin conditions.[107-108]

Early studies performed in gene deficient mice supported a role for Th17 related cytokines in the development of experimental autoimmune glomerulonephritis[109] and sheep anti-mouse glomerular basement membrane disease.[110] A pathogenic role for RORγt, the key IL-17A transcription factor, was also demonstrated in a murine model of crescentic glomeruloneph‐ ritis.[111] A direct role for Th17 cells acting as effectors was subsequently published. The antigen, ovalbumin (OVA), was planted in the kidneys of RAG1-/- mice, after the conjugation of OVA to a non-nephritogenic antibody specific for the glomerular basement membrane. This was followed by the administration of Th-17 polarized ovalbumin specific CD4+ T cells, which resulted in neutrophil mediated proliferative glomerulonephritis.[112] Detailed reviews of the

Th17 cells are a distinct line of CD4+ T helper cells with unique transcription factors and effector cytokines. These cells are active participants in the development of autoimmunity but are also involved as effector cells in autoimmune conditions including rapidly pro‐

In addition to CD4+ effector T cells other T cells are likely to contribute to AAV. Several years ago it was demonstrated that CD4+ effector memory cells (Tem) were increased in the blood of GPA patients in remission, compared to those with active disease.[115] While Tem were decreased in the blood, they were increased in the urine of patients with active disease suggesting that these cells may influence renal injury during active disease.[116] Further *in vitro* studies suggested that in GPA patients these cells could mediate endothelial injury and thus play a role in driving glomerular injury.[117] Fewer studies have assessed potential pathogenic roles of CD8+ T cells in AAV, however it would seem likely that these cells are involved. A study assessing gene expression and outcome in AAV and SLE patients suggested that CD8+ T cell signatures and increased CD8+ T cell memory populations were associated with poorer outcomes.[118] It was hoped that results from these studies would facilitate more individualised treatments. It would seem important that we further explore the role of CD8+

Regulatory T cells (Tregs) represent a subset of CD4+ CD25+ T cells which perform a key role in regulating inflammation and tissue injury. These cells are identified through the expression of FoxP3, which is considered a master regulator of Tregs. In several autoimmune diseases, including Goodpasture's disease, Tregs are required for the maintenance of tolerance and loss of Treg function can result in the development of autoimmunity and organ injury.[119] In GPA clinical studies have shown that although circulating FoxP3-expressing Tregs vary in number their suppressive capacity is reduced.[120] In MPA patients (and experimentally) FoxP3 expressing Tregs display diminished capacity to suppress antigen specific MPO responses an

role of Th17 cells in kidney disease have recently been published.[113-114]

**7. Th17 cells in the kidney**

44 Updates in the Diagnosis and Treatment of Vasculitis

gressive glomerulonephritis.

T cells in AAV.

Neutrophils play a critical role in the pathogenesis of ANCA vasculitis. Not only are neutro‐ phils the primary effector cells in the kidney but neutrophils also contain the target autoantigens, MPO, PR3 (and LAMP-2) and hence are directly involved in the auto-immune process. We will discuss three different aspects of neutrophil involvement in disease, (a) The role of the Neutrophil in the development of Autoimmunity, (b) Neutrophil Activation by ANCAs and (c) Neutrophil Endothelial Interactions, which initiate glomerular injury.

#### *8.1.1. The role of the neutrophil in the development of autoimmunity*

It is well established that ANCAs bind to the autoantigens, MPO or PR3, located on the cell surface of the neutrophil. How and why these autoantigens translocate to the cell surface is poorly understood. We know that neutrophils die through apoptosis or necrosis and data suggests that neutrophil death through apoptosis can promote the loss of tolerance to MPO or PR3. After cell death neutrophils release granule constituents, including MPO and PR3, which translocate to the cell surface[122-123] where they serve as antigenic targets. This phenomenon was thought to occur exclusively after neutrophil death through apoptosis, which is possibly related to a slower mechanism of cell death, although the operational mechanisms of this system require further clarification.

An additional pathway linking neutrophil cell death and autoimmunity has recently been proposed, involving a distinct method of neutrophil death involving neutrophil extracellular traps (NETs). Neutrophils extrude NETs which consist of chromatin structures and include anti-microbial peptides such as; MPO, PR3 elastin, cathepsin, and lactoferrin.[124] Dying neutrophils extrude NETs to kill invading pathogens in a process recently named NETosis. It is understood that neutrophils, through NETosis, contribute to the development of autoim‐ munity, a concept well established in SLE. In SLE, in response to chronic autoantibody stimulation neutrophils and their NETs activate plasmacytoid dendritic cells which secrete IFNα.[125-127] NETosis has been linked with glomerular injury in AAV, through the en‐ hancement of endothelial-leukocyte interaction,[71] however only recently have NETs been implicated in the development of ANCA autoimmunity. NETotic neutrophils interacted with myeloid dendritic cells (mDC). This interaction was not observed when neutrophils died by necrosis or apoptosis. This process was dependent on both TNF and IFNγ and in their absence NETosis did not occur. The interaction between the NETotic neutrophil and the mDC resulted in the transfer of MPO and PR3 to the mDC, which potentially could induce and promote adaptive immune responses. This process was confirmed to be pathogenic *in* vivo. Mice were immunized with mDCs co-cultured with NETotic neutrophils (6 times intraperitoneally) and three months later they developed ANCAs and showed evidence of renal injury. The mice also displayed features consistent with systemic auto-immune disease. A similar process was thought to be present in human AAV. Assessing skin lesions from patients with MPO-ANCA vasculitis revealed an interaction between mDCs and neutrophils, with uploading of the autoantigens.[128] While this process is not yet completely understood, NETosis potentially explains how autoantigens are recognized by antigen presenting cells, activating cellular and humoral autoimmunity in AAV.

injury in rats receiving the treatment.[130] The anti-TNF mAb, Infliximab, has been used with some success in patients with AAV. While Infliximab therapy was useful in treating patients with refractory disease,[131-132] disappointingly the addition of TNF blockade (Inflixi‐ mab[133] or Adalimumab[134]) to standard treatment regimes did not result in an improve‐ ment in clinical outcomes and Etanercept, (a fusion protein TNF inhibitor) could not decrease relapse rates in GPA.[115] Despite the lack of efficacy of TNF blockade in these trials, it would seem that TNF has a central role in the pathogenesis and in selected patients the use of TNF blockade may be associated with clinical improvement. In addition to TNF other cytokines and chemokines are likely to be important in AAV. Interestingly it was first appreciated over 15 years ago that neutrophils stimulated by ANCA produce IL-1β.[135] Few studies have further investigated this observation. Recently the Inflammasome, which is a pattern recog‐ nition receptor which is characterized by the production of IL-1β, has been shown to promote auto-inflammatory and auto-immune disease. It is possible that the Inflammasome is involved in vasculitis. This is clinically relevant because diseases resulting from overstimulation of the Inflammasome have been successfully treated with monoclonal antibody therapy.[136] This

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47

The intracellular signalling pathways activated by ANCA neutrophil binding are multiple, although several pathways are shared. Whereas the Fc portion of ANCA IgG activates tyrosine kinase pathways,[137] the F(ab')2 portion activates a G protein pathway.[138] Despite initiating two separate pathways, these pathways converge on the p21ras GTPase, which is essential for many neutrophil functions.[139] The identification of these pathways, combined with the development of antibodies directed against specific components of these pathways, predominantly used in preclinical models, has increased expectations of their potential

The interaction between neutrophils and endothelial cells is important in the initiation of glomerular lesions, including fibrinoid necrosis, which is frequently observed in patients with renal vasculitis. Under normal physiological conditions neutrophils do not interact with the endothelium, however when the endothelium is activated resulting in increased expression of adhesion molecules and chemokines (and neutrophils are activated) neutrophil recruitment, binding and transmigration is increased. Our understanding of this complex dynamic has been improved through the use of in vitro systems, which include flow chambers mimicking blood flow in human capillaries. For these studies neutrophils from healthy controls and patients with AAV have been compared. Further information has been gleaned from experimental

It is likely that TNF production and complement activation in AAV patients results in a persistent low grade activation of neutrophils.[142] Results from in vitro studies have shown that neutrophils exposed to ANCA bind to human umbilical vein endothelial cells (HUVECs), [143-144] with up-regulation of CD11b, an adhesion molecule.[145-146] In a flow system set up to mirror blood flow in human capillaries, ANCA treated neutrophils demonstrated increased adhesion and transmigration which was β2 integrin and CXCR2 (neutrophil cell

therapeutic use in autoimmune diseases including renal vasculitis.[140-141]

models using live imaging of the kidney, including intravital microscopy.

warrants further investigation.

*8.1.3. Neutrophil- endothelial interactions*

While neutrophil apoptosis and NETosis provide some insight into the role of the neutrophil in the development of AAV, there remain several 'gaps' in our knowledge. Why AAV patients develop autoimmunity to MPO/PR3, with an associated clinical syndrome and yet they do not develop autoantibodies to other neutrophil constituents which are released after cell death is unclear. The driving factors behind apoptosis and NETosis have not been well established. Further studies in this area are required before definitive conclusions can be reached. In addition to promoting autoimmunity the ANCA-neutrophil interaction is a key to two other mechanisms of injury, ANCA binding to neutrophils leading to neutrophil activation and an oxidative burst, and the recruitment of ANCA bound neutrophils to the glomerulus where they initiate renal injury.

#### *8.1.2. Neutrophil activation by ANCAs*

The first paper suggesting a role for ANCA in activating neutrophils was published over 20 years ago. In this landmark paper it was shown that both MPO-ANCA and PR3-ANCA could bind to primed neutrophils. After neutrophil priming by tumour necrosis factor (TNF) MPO and PR3 were translocated to the cell surface, providing an autoantibody antigenic target. Neutrophil binding by ANCAs produced an oxidative burst and resulted in degranulation. [115] Other authors have confirmed this process and shown that it is also Fcgamma RIIdependent.[129] Cytokine priming of neutrophils is important for ANCA binding as it increases surface expression of the autoantigens and mobilizes the NADPH oxidase complex, further increasing ANCA binding.[27, 53] In addition to TNF, IL-18 and granulocyte macro‐ phage colony stimulating factor can prime neutrophils and enhance ANCA binding.[52]

The pathogenic role of TNF in AAV has attracted significant interest both experimentally and clinically. In a passive transfer model of MPO-ANCA vasculitis pre-treatment with lipopoly‐ saccharide (LPS) increased systemic inflammation and glomerular injury. The *in vitro* oxidative burst induced by MPO-ANCA required TNF and anti-TNF mAb treatment attenuated renal injury *in vivo.[57]* Similar results were found by other authors who demonstrated that mAb directed against TNF successfully attenuated established glomerulonephritis in a rat model of AAV. Despite intact humoral responses there was a decrease in functional and histological injury in rats receiving the treatment.[130] The anti-TNF mAb, Infliximab, has been used with some success in patients with AAV. While Infliximab therapy was useful in treating patients with refractory disease,[131-132] disappointingly the addition of TNF blockade (Inflixi‐ mab[133] or Adalimumab[134]) to standard treatment regimes did not result in an improve‐ ment in clinical outcomes and Etanercept, (a fusion protein TNF inhibitor) could not decrease relapse rates in GPA.[115] Despite the lack of efficacy of TNF blockade in these trials, it would seem that TNF has a central role in the pathogenesis and in selected patients the use of TNF blockade may be associated with clinical improvement. In addition to TNF other cytokines and chemokines are likely to be important in AAV. Interestingly it was first appreciated over 15 years ago that neutrophils stimulated by ANCA produce IL-1β.[135] Few studies have further investigated this observation. Recently the Inflammasome, which is a pattern recog‐ nition receptor which is characterized by the production of IL-1β, has been shown to promote auto-inflammatory and auto-immune disease. It is possible that the Inflammasome is involved in vasculitis. This is clinically relevant because diseases resulting from overstimulation of the Inflammasome have been successfully treated with monoclonal antibody therapy.[136] This warrants further investigation.

The intracellular signalling pathways activated by ANCA neutrophil binding are multiple, although several pathways are shared. Whereas the Fc portion of ANCA IgG activates tyrosine kinase pathways,[137] the F(ab')2 portion activates a G protein pathway.[138] Despite initiating two separate pathways, these pathways converge on the p21ras GTPase, which is essential for many neutrophil functions.[139] The identification of these pathways, combined with the development of antibodies directed against specific components of these pathways, predominantly used in preclinical models, has increased expectations of their potential therapeutic use in autoimmune diseases including renal vasculitis.[140-141]

#### *8.1.3. Neutrophil- endothelial interactions*

myeloid dendritic cells (mDC). This interaction was not observed when neutrophils died by necrosis or apoptosis. This process was dependent on both TNF and IFNγ and in their absence NETosis did not occur. The interaction between the NETotic neutrophil and the mDC resulted in the transfer of MPO and PR3 to the mDC, which potentially could induce and promote adaptive immune responses. This process was confirmed to be pathogenic *in* vivo. Mice were immunized with mDCs co-cultured with NETotic neutrophils (6 times intraperitoneally) and three months later they developed ANCAs and showed evidence of renal injury. The mice also displayed features consistent with systemic auto-immune disease. A similar process was thought to be present in human AAV. Assessing skin lesions from patients with MPO-ANCA vasculitis revealed an interaction between mDCs and neutrophils, with uploading of the autoantigens.[128] While this process is not yet completely understood, NETosis potentially explains how autoantigens are recognized by antigen presenting cells, activating cellular and

While neutrophil apoptosis and NETosis provide some insight into the role of the neutrophil in the development of AAV, there remain several 'gaps' in our knowledge. Why AAV patients develop autoimmunity to MPO/PR3, with an associated clinical syndrome and yet they do not develop autoantibodies to other neutrophil constituents which are released after cell death is unclear. The driving factors behind apoptosis and NETosis have not been well established. Further studies in this area are required before definitive conclusions can be reached. In addition to promoting autoimmunity the ANCA-neutrophil interaction is a key to two other mechanisms of injury, ANCA binding to neutrophils leading to neutrophil activation and an oxidative burst, and the recruitment of ANCA bound neutrophils to the glomerulus where

The first paper suggesting a role for ANCA in activating neutrophils was published over 20 years ago. In this landmark paper it was shown that both MPO-ANCA and PR3-ANCA could bind to primed neutrophils. After neutrophil priming by tumour necrosis factor (TNF) MPO and PR3 were translocated to the cell surface, providing an autoantibody antigenic target. Neutrophil binding by ANCAs produced an oxidative burst and resulted in degranulation. [115] Other authors have confirmed this process and shown that it is also Fcgamma RIIdependent.[129] Cytokine priming of neutrophils is important for ANCA binding as it increases surface expression of the autoantigens and mobilizes the NADPH oxidase complex, further increasing ANCA binding.[27, 53] In addition to TNF, IL-18 and granulocyte macro‐ phage colony stimulating factor can prime neutrophils and enhance ANCA binding.[52]

The pathogenic role of TNF in AAV has attracted significant interest both experimentally and clinically. In a passive transfer model of MPO-ANCA vasculitis pre-treatment with lipopoly‐ saccharide (LPS) increased systemic inflammation and glomerular injury. The *in vitro* oxidative burst induced by MPO-ANCA required TNF and anti-TNF mAb treatment attenuated renal injury *in vivo.[57]* Similar results were found by other authors who demonstrated that mAb directed against TNF successfully attenuated established glomerulonephritis in a rat model of AAV. Despite intact humoral responses there was a decrease in functional and histological

humoral autoimmunity in AAV.

46 Updates in the Diagnosis and Treatment of Vasculitis

they initiate renal injury.

*8.1.2. Neutrophil activation by ANCAs*

The interaction between neutrophils and endothelial cells is important in the initiation of glomerular lesions, including fibrinoid necrosis, which is frequently observed in patients with renal vasculitis. Under normal physiological conditions neutrophils do not interact with the endothelium, however when the endothelium is activated resulting in increased expression of adhesion molecules and chemokines (and neutrophils are activated) neutrophil recruitment, binding and transmigration is increased. Our understanding of this complex dynamic has been improved through the use of in vitro systems, which include flow chambers mimicking blood flow in human capillaries. For these studies neutrophils from healthy controls and patients with AAV have been compared. Further information has been gleaned from experimental models using live imaging of the kidney, including intravital microscopy.

It is likely that TNF production and complement activation in AAV patients results in a persistent low grade activation of neutrophils.[142] Results from in vitro studies have shown that neutrophils exposed to ANCA bind to human umbilical vein endothelial cells (HUVECs), [143-144] with up-regulation of CD11b, an adhesion molecule.[145-146] In a flow system set up to mirror blood flow in human capillaries, ANCA treated neutrophils demonstrated increased adhesion and transmigration which was β2 integrin and CXCR2 (neutrophil cell surface receptors) dependent.[146] This is likely to resemble what happens in human AAV, where expression of both β1 and β2 integrins are increased in circulating neutrophils,[147] and the adhesion molecules ICAM and VCAM are expressed on glomerular endothelial cells.[148] Additional results from human studies have implicated IL-8 in leukocyte recruitment which was also shown to correlate with glomerular injury.[51] Neutrophil degranulation with the accompanying release of reactive oxygen species, proteases[149-150] and an oxidative burst[151-152] directly leads to endothelial injury. Evidence for enhanced endothelial injury includes increased levels of endothelial cell microparticles in active disease, which subse‐ quently reduce when the disease remits.[153-154] This is in direct contrast to the restorative endothelial progenitor cells which are decreased when disease is active.[155-156] It has been suggested that the pro-angiogenic protein, angiopoietin-2, may act locally to promote inflam‐ mation and endothelial cell injury.[154] It is likely that several mechanisms combine to result in endothelial injury. We know that neutrophil degranulation also results in deposition of the neutrophil constituents, MPO and PR3 in the glomerular bed,[157] and these deposited autoantigens provide targets for antigen specific T and B cells, which recruit additional effector cells, promoting a vicious cycle of injury.

a unique organ in which neutrophil migration differs from other postcapillary venules. While it is likely that injury in humans with renal vasculitis is a consequence of several mechanisms (discussed above) acting in tandem, direct visualization of the kidney appears to be the best technique to assess glomerulonephritis. In addition to the mechanisms detailed above there are likely to be several other factors which contribute to pathogenic neutrophil-endothelial interaction and the ensuing rapidly progressive glomerulonephritis, several of these are

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49

*8.1.3.1. The role of NETs in neutrophil-endothelial interactions and glomerulonephritis in AAV*

The role of neutrophil extracellular traps (NETs) in the development of autoimmunity to MPO and PR3 has been discussed earlier. A further role for NETs in driving effector responses in renal vasculitis was described in an innovative paper published in 2009. In this manuscript, Kessenbrock et al, found that primed neutrophils cultured with ANCAs resulted in the development of NETs and these chromatin fibres contained the auto-antigens MPO and PR3. When neutrophils were recruited to inflamed glomeruli, degranulation of the neutrophil and NETs resulted in the deposition of these autoantigens in the glomerulus. Furthermore they demonstrated that in human kidney biopsies, from patients with AAV, NET formation was associated with areas of high neutrophil influx and acute injury.[71] It is likely that deposition of MPO and PR3 could directly result in glomerular injury, however these autoantigens could also serve as targets for auto-reactive CD4+ T cells and B cells further increasing the influx of

Microparticles in neutrophils contain an abundance of adhesion molecules and proteases which include the ANCA auto-antigens PR3 and MPO.[162] Recent data has shown ANCAs can induce the release of neutrophil microparticles from primed neutrophils. These micropar‐ ticles bind to endothelial cells through an up-regulation of adhesion molecules and result in increased endothelial reactive oxygen species and released pro-inflammatory cytokines including, IL-6 and IL-8. The clinical relevance of this was supported by data which demon‐ strated that neutrophil microparticles were more readily detected in patients (children) with active AAV, while levels were suppressed in healthy controls and patients with inactive disease.[163] Several other mechanisms of neutrophil microparticle release have been descri‐ bed, including those triggered by the complement system, which is also active in renal vasculitis. These studies further highlight the complex nature of neutrophil induced glomer‐ ular injury in renal vasculitis. It is likely that that the synchrony of many innate immune cells and adaptive immunity result in the severe injury observed in rapidly progressive glomeru‐

**8.2. The role of Toll Like Receptors (TLRs) in ANCA associated vasculitis**

The innate pattern recognition receptors, TLRs, recognise molecular patterns commonly found in bacterial and viral organisms.[164] In response to invading microbes, TLR ligation results in a 'hard-wired' activation of the innate immune system and heightened adaptive immune

discussed later in this chapter.

inflammatory cells and exacerbating glomerular injury.

lonephritis and acute kidney injury.

*8.1.3.2. A pathogenic role for neutrophil microparticles in AAV*

Many of the original studies assessing neutrophil recruitment to the capillaries used intravital imaging of mesenteric and cremasteric vessels. These vessels are more accessible and provide some parallels with leukocyte recruitment seen in renal and lung vasculitis. More recently Michael Hickey's group have pioneered new methods for assessing neutrophil physiology in the inflamed glomerulus, which has considerably improved our understanding of leukocyte behaviour in glomerulonephritis.[158-159].

In vitro studies performed in a flow chamber have shown that human neutrophils treated with ANCA display altered patterns of rolling, adhesion and transmigration.[146, 160] Using intravital microscopy to visualise mesenteric postcapillary venules Little et al found that administration of MPO-ANCA induced neutrophil adhesion and transmigration. Similarly studies using intravital microscopy to visualize murine cremasteric postcapillary venules demonstrated increased neutrophil adhesion and transmigration after the passive transfer of MPO-ANCA. Neutrophil recruitment was both Fcgamma receptor and β2 integrin dependent. [161] While these studies provided valuable insight into neutrophil recruitment and transmi‐ gration in inflamed tissues in AAV, it remained unclear if the observations seen in the postcapillary venules could be replicated in the glomerulus. The use of live imaging of the murine kidney has facilitated the study of leukocyte behaviour in models of glomerular injury. Differences in neutrophil behaviour in the inflamed glomerulus have been noted. In the heterologous phase of renal injury induced after administration of sheep anti-mouse GBM serum, neutrophil recruitment occurred via rapid arrest and occurred in the absence of rolling. [158] Relevant to AAV, in mice treated with LPS and MPO-ANCA glomerular neutrophil recruitment occurred in a lymphocyte function-associated antigen (LFA-1)(a leukocyte integrin) dependent manner. However if an increased dose of MPO-ANCA was used (without LPS priming), neutrophil recruitment was α4-integrin dependent, but β2-integrin independ‐ ent.[18] These studies highlight how MPO-ANCA can induce glomerular neutrophil recruit‐ ment through many different pathways and furthermore demonstrate that the glomerulus is a unique organ in which neutrophil migration differs from other postcapillary venules. While it is likely that injury in humans with renal vasculitis is a consequence of several mechanisms (discussed above) acting in tandem, direct visualization of the kidney appears to be the best technique to assess glomerulonephritis. In addition to the mechanisms detailed above there are likely to be several other factors which contribute to pathogenic neutrophil-endothelial interaction and the ensuing rapidly progressive glomerulonephritis, several of these are discussed later in this chapter.

#### *8.1.3.1. The role of NETs in neutrophil-endothelial interactions and glomerulonephritis in AAV*

The role of neutrophil extracellular traps (NETs) in the development of autoimmunity to MPO and PR3 has been discussed earlier. A further role for NETs in driving effector responses in renal vasculitis was described in an innovative paper published in 2009. In this manuscript, Kessenbrock et al, found that primed neutrophils cultured with ANCAs resulted in the development of NETs and these chromatin fibres contained the auto-antigens MPO and PR3. When neutrophils were recruited to inflamed glomeruli, degranulation of the neutrophil and NETs resulted in the deposition of these autoantigens in the glomerulus. Furthermore they demonstrated that in human kidney biopsies, from patients with AAV, NET formation was associated with areas of high neutrophil influx and acute injury.[71] It is likely that deposition of MPO and PR3 could directly result in glomerular injury, however these autoantigens could also serve as targets for auto-reactive CD4+ T cells and B cells further increasing the influx of inflammatory cells and exacerbating glomerular injury.

#### *8.1.3.2. A pathogenic role for neutrophil microparticles in AAV*

surface receptors) dependent.[146] This is likely to resemble what happens in human AAV, where expression of both β1 and β2 integrins are increased in circulating neutrophils,[147] and the adhesion molecules ICAM and VCAM are expressed on glomerular endothelial cells.[148] Additional results from human studies have implicated IL-8 in leukocyte recruitment which was also shown to correlate with glomerular injury.[51] Neutrophil degranulation with the accompanying release of reactive oxygen species, proteases[149-150] and an oxidative burst[151-152] directly leads to endothelial injury. Evidence for enhanced endothelial injury includes increased levels of endothelial cell microparticles in active disease, which subse‐ quently reduce when the disease remits.[153-154] This is in direct contrast to the restorative endothelial progenitor cells which are decreased when disease is active.[155-156] It has been suggested that the pro-angiogenic protein, angiopoietin-2, may act locally to promote inflam‐ mation and endothelial cell injury.[154] It is likely that several mechanisms combine to result in endothelial injury. We know that neutrophil degranulation also results in deposition of the neutrophil constituents, MPO and PR3 in the glomerular bed,[157] and these deposited autoantigens provide targets for antigen specific T and B cells, which recruit additional effector

Many of the original studies assessing neutrophil recruitment to the capillaries used intravital imaging of mesenteric and cremasteric vessels. These vessels are more accessible and provide some parallels with leukocyte recruitment seen in renal and lung vasculitis. More recently Michael Hickey's group have pioneered new methods for assessing neutrophil physiology in the inflamed glomerulus, which has considerably improved our understanding of leukocyte

In vitro studies performed in a flow chamber have shown that human neutrophils treated with ANCA display altered patterns of rolling, adhesion and transmigration.[146, 160] Using intravital microscopy to visualise mesenteric postcapillary venules Little et al found that administration of MPO-ANCA induced neutrophil adhesion and transmigration. Similarly studies using intravital microscopy to visualize murine cremasteric postcapillary venules demonstrated increased neutrophil adhesion and transmigration after the passive transfer of MPO-ANCA. Neutrophil recruitment was both Fcgamma receptor and β2 integrin dependent. [161] While these studies provided valuable insight into neutrophil recruitment and transmi‐ gration in inflamed tissues in AAV, it remained unclear if the observations seen in the postcapillary venules could be replicated in the glomerulus. The use of live imaging of the murine kidney has facilitated the study of leukocyte behaviour in models of glomerular injury. Differences in neutrophil behaviour in the inflamed glomerulus have been noted. In the heterologous phase of renal injury induced after administration of sheep anti-mouse GBM serum, neutrophil recruitment occurred via rapid arrest and occurred in the absence of rolling. [158] Relevant to AAV, in mice treated with LPS and MPO-ANCA glomerular neutrophil recruitment occurred in a lymphocyte function-associated antigen (LFA-1)(a leukocyte integrin) dependent manner. However if an increased dose of MPO-ANCA was used (without LPS priming), neutrophil recruitment was α4-integrin dependent, but β2-integrin independ‐ ent.[18] These studies highlight how MPO-ANCA can induce glomerular neutrophil recruit‐ ment through many different pathways and furthermore demonstrate that the glomerulus is

cells, promoting a vicious cycle of injury.

48 Updates in the Diagnosis and Treatment of Vasculitis

behaviour in glomerulonephritis.[158-159].

Microparticles in neutrophils contain an abundance of adhesion molecules and proteases which include the ANCA auto-antigens PR3 and MPO.[162] Recent data has shown ANCAs can induce the release of neutrophil microparticles from primed neutrophils. These micropar‐ ticles bind to endothelial cells through an up-regulation of adhesion molecules and result in increased endothelial reactive oxygen species and released pro-inflammatory cytokines including, IL-6 and IL-8. The clinical relevance of this was supported by data which demon‐ strated that neutrophil microparticles were more readily detected in patients (children) with active AAV, while levels were suppressed in healthy controls and patients with inactive disease.[163] Several other mechanisms of neutrophil microparticle release have been descri‐ bed, including those triggered by the complement system, which is also active in renal vasculitis. These studies further highlight the complex nature of neutrophil induced glomer‐ ular injury in renal vasculitis. It is likely that that the synchrony of many innate immune cells and adaptive immunity result in the severe injury observed in rapidly progressive glomeru‐ lonephritis and acute kidney injury.

#### **8.2. The role of Toll Like Receptors (TLRs) in ANCA associated vasculitis**

The innate pattern recognition receptors, TLRs, recognise molecular patterns commonly found in bacterial and viral organisms.[164] In response to invading microbes, TLR ligation results in a 'hard-wired' activation of the innate immune system and heightened adaptive immune responses. While TLRs are required for protection from invading microbes, inappropriate stimulation can result in the development of autoimmunity and organ injury,[165] including renal disease.[166] In several experimental models of kidney disease, including acute kidney injury,[167] lupus nephritis[168] and crescentic glomerulonephritis,[169] we and others have demonstrated pathogenic roles for TLRs. However their role in AAV is likely to be dual. Firstly ligation of TLRs heightens innate and adaptive immune response, which in turn leads to the loss of tolerance and the development of autoimmunity. Secondly, TLRs activate both effector cells and resident kidney cells, increasing glomerular inflammation and renal injury. This is important as infections are known to promote injury in AAV, with TLRs providing a link between infection and the development of AAV and disease relapses. While many TLRs have been implicated in autoimmunity studies in AAV have largely concentrated on the surface receptors, TLR2 and TLR4, as well as the intracellular TLR9.

We used bone marrow chimeras to define the relative contributions of bone marrow cell TLR4 and intrinsic renal cell TLR4 to the disease process. We found that both bone marrow and resident kidney cell TLR4 were required for maximal neutrophil recruitment and renal injury. [58] These studies highlighted the importance of TLR4 (and potentially other TLRs) in driving

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**Figure 1.** TLR9 staining in a kidney biopsy from a patient with PR3-ANCA vasculitis. While TLR9 staining was not de‐ tectable in normal kidney samples stained with a mAb directed against TLR9 and visualized under immunofluorescent light, TLR9 staining was readily detectable in patients with crescentic glomerulonephritis and PR3-ANCA vasculitis.

The complement system is recognized as one of the phylogenetically oldest components of human immune defence. This highly regulated system of proteins (together with their regulatory inhibitors) compromise an important part of host defence. In response to either innate or adaptive stimuli activation of the complement system results in a cascade of ampli‐ fication and cleavage steps with the generation of anaphylatoxins (C5a and C3a) and a terminal attack complex capable of lying cells.[177] Three complement pathways are well described, namely, the classical pathway, the alternate pathway which is initiated by recognition of foreign surfaces and the mannose binding lectin pathway.[178] More recently a pathway which is initiated by coagulation and fibrinolytic proteins has been described.[179] In addition to its role in host defence, activation of the complement cascade can result in tissue injury and has been implicated in many forms of glomerulonephritis and kidney injury. Traditionally complement was not considered critical to the pathogenesis of AAV as renal injury was considered 'pauci immune' in nature and hence free from complement (and immune complex) deposition. Interestingly complement is frequently observed in renal and skin biopsies from patients with AAV,[180-182] while in vitro studies have demonstrated a role for complement

From historical data we know that when neutrophils are activated by ANCA, the complement cascade is triggered and C3a is produced.[183] We also know that priming neutrophils with C5a enhances ANCA-neutrophil interactions,[184] an effect mediated by p38 mitogen-

**8.3. The role of complement in ANCA associated vasculitis**

in ANCA-neutrophil interactions.

effector responses in ANCA vasculitis.

There is clinical evidence implicating TLRs in the loss of tolerance in AAV. Stimulation of peripheral blood mononuclear cells (PBMCs) from GPA patients with a TLR9 ligand resulted in increased ANCA production.[170] Moreover in patients with AAV in remis‐ sion TLR9 expression is increased on B lymphocytes and when these B lymphocytes were cultured with a TLR9 ligand they produced ANCA.[171] These studies support a role for infection (through ligation of TLR9) promoting humoral autoimmunity. Expression of TLR2, TLR4 and TLR9 on B lymphocytes, T lymphocytes, natural killer (NK) cells, mono‐ cytes and granulocytes from AAV patients (and controls) was assessed. Amongst AAV patient's monocytes and NK cells had increased TLR expression.[172] We have provided supporting evidence for a pathogenic role for TLRs using experimental models of AAV. Immunization of WT mice with a TLR ligand and MPO resulted in the loss of tolerance with the development of cellular and humoral autoimmune responses and later necrotis‐ ing glomerulonephritis. Interestingly immunization with a TLR9 ligand and MPO result‐ ed in T-bet dependent IFNγ production and macrophage mediated renal injury. Conversely autoimmunity induced by a TLR2 ligand and MPO resulted in ROR-γ de‐ pendent Th17 autoimmunity and neutrophil mediated renal injury.[82]

However, TLRs are also likely to be involved in effector responses. While TLRs are expressed at low and often undetectable levels in normal kidney biopsies, increased expression has been seen in glomerulonephritis. In lupus nephritis glomerular and tubular TLR9 expression was shown to be increased in both children and adults.[173-174] In studies assessing patterns of TLR2, TLR4 and TLR9 in glomerulonephritis, strong TLR2 and TLR4 staining was seen in the inflammatory infiltrates of patients with AAV.[175] We have stained renal biopsies from patients with AAV and found that TLR9 staining is positive in the glomeruli (unpublished data), as illustrated.

Further studies have supported an interaction between AAV and TLRs, when epithelial cells, from kidney and lung, primed with PR3-ANCA serum produced exaggerated cytokine levels after TLR stimulation.[176] While early studies suggested that lipopolysaccharide (LPS) enhanced effector responses in AAV,[57] our understanding of this process has increased. We demonstrated that highly purified LPS, a pure TLR4 ligand, increased neutrophil recruitment and glomerular injury after the passive transfer of MPO-ANCA, in a TLR4 dependent manner. We used bone marrow chimeras to define the relative contributions of bone marrow cell TLR4 and intrinsic renal cell TLR4 to the disease process. We found that both bone marrow and resident kidney cell TLR4 were required for maximal neutrophil recruitment and renal injury. [58] These studies highlighted the importance of TLR4 (and potentially other TLRs) in driving effector responses in ANCA vasculitis.

**Figure 1.** TLR9 staining in a kidney biopsy from a patient with PR3-ANCA vasculitis. While TLR9 staining was not de‐ tectable in normal kidney samples stained with a mAb directed against TLR9 and visualized under immunofluorescent light, TLR9 staining was readily detectable in patients with crescentic glomerulonephritis and PR3-ANCA vasculitis.

#### **8.3. The role of complement in ANCA associated vasculitis**

responses. While TLRs are required for protection from invading microbes, inappropriate stimulation can result in the development of autoimmunity and organ injury,[165] including renal disease.[166] In several experimental models of kidney disease, including acute kidney injury,[167] lupus nephritis[168] and crescentic glomerulonephritis,[169] we and others have demonstrated pathogenic roles for TLRs. However their role in AAV is likely to be dual. Firstly ligation of TLRs heightens innate and adaptive immune response, which in turn leads to the loss of tolerance and the development of autoimmunity. Secondly, TLRs activate both effector cells and resident kidney cells, increasing glomerular inflammation and renal injury. This is important as infections are known to promote injury in AAV, with TLRs providing a link between infection and the development of AAV and disease relapses. While many TLRs have been implicated in autoimmunity studies in AAV have largely concentrated on the surface

There is clinical evidence implicating TLRs in the loss of tolerance in AAV. Stimulation of peripheral blood mononuclear cells (PBMCs) from GPA patients with a TLR9 ligand resulted in increased ANCA production.[170] Moreover in patients with AAV in remis‐ sion TLR9 expression is increased on B lymphocytes and when these B lymphocytes were cultured with a TLR9 ligand they produced ANCA.[171] These studies support a role for infection (through ligation of TLR9) promoting humoral autoimmunity. Expression of TLR2, TLR4 and TLR9 on B lymphocytes, T lymphocytes, natural killer (NK) cells, mono‐ cytes and granulocytes from AAV patients (and controls) was assessed. Amongst AAV patient's monocytes and NK cells had increased TLR expression.[172] We have provided supporting evidence for a pathogenic role for TLRs using experimental models of AAV. Immunization of WT mice with a TLR ligand and MPO resulted in the loss of tolerance with the development of cellular and humoral autoimmune responses and later necrotis‐ ing glomerulonephritis. Interestingly immunization with a TLR9 ligand and MPO result‐ ed in T-bet dependent IFNγ production and macrophage mediated renal injury. Conversely autoimmunity induced by a TLR2 ligand and MPO resulted in ROR-γ de‐

However, TLRs are also likely to be involved in effector responses. While TLRs are expressed at low and often undetectable levels in normal kidney biopsies, increased expression has been seen in glomerulonephritis. In lupus nephritis glomerular and tubular TLR9 expression was shown to be increased in both children and adults.[173-174] In studies assessing patterns of TLR2, TLR4 and TLR9 in glomerulonephritis, strong TLR2 and TLR4 staining was seen in the inflammatory infiltrates of patients with AAV.[175] We have stained renal biopsies from patients with AAV and found that TLR9 staining is positive in the glomeruli (unpublished

Further studies have supported an interaction between AAV and TLRs, when epithelial cells, from kidney and lung, primed with PR3-ANCA serum produced exaggerated cytokine levels after TLR stimulation.[176] While early studies suggested that lipopolysaccharide (LPS) enhanced effector responses in AAV,[57] our understanding of this process has increased. We demonstrated that highly purified LPS, a pure TLR4 ligand, increased neutrophil recruitment and glomerular injury after the passive transfer of MPO-ANCA, in a TLR4 dependent manner.

receptors, TLR2 and TLR4, as well as the intracellular TLR9.

50 Updates in the Diagnosis and Treatment of Vasculitis

pendent Th17 autoimmunity and neutrophil mediated renal injury.[82]

data), as illustrated.

The complement system is recognized as one of the phylogenetically oldest components of human immune defence. This highly regulated system of proteins (together with their regulatory inhibitors) compromise an important part of host defence. In response to either innate or adaptive stimuli activation of the complement system results in a cascade of ampli‐ fication and cleavage steps with the generation of anaphylatoxins (C5a and C3a) and a terminal attack complex capable of lying cells.[177] Three complement pathways are well described, namely, the classical pathway, the alternate pathway which is initiated by recognition of foreign surfaces and the mannose binding lectin pathway.[178] More recently a pathway which is initiated by coagulation and fibrinolytic proteins has been described.[179] In addition to its role in host defence, activation of the complement cascade can result in tissue injury and has been implicated in many forms of glomerulonephritis and kidney injury. Traditionally complement was not considered critical to the pathogenesis of AAV as renal injury was considered 'pauci immune' in nature and hence free from complement (and immune complex) deposition. Interestingly complement is frequently observed in renal and skin biopsies from patients with AAV,[180-182] while in vitro studies have demonstrated a role for complement in ANCA-neutrophil interactions.

From historical data we know that when neutrophils are activated by ANCA, the complement cascade is triggered and C3a is produced.[183] We also know that priming neutrophils with C5a enhances ANCA-neutrophil interactions,[184] an effect mediated by p38 mitogenactivated protein kinase, extracellular signal-regulated kinase and phosphoinositol 3-kinase. [185] Results from clinical studies have shown that serum and urine levels of C5a are elevated in patients with active disease strongly supporting the notion that the complement cascade is activated in active AAV.[186] Strong support for a pathogenic role for complement has also been provided from experimental studies. In an extensive set of experiments, the North Carolina group robustly demonstrated that complement depletion (achieved through the use of cobra venom serum) abrogated disease, an effect mediated through C5 and Factor B.[183] Factor B is critical for alternative pathway activation. Similarly inhibition of C5 using a mAb successfully attenuated experimental anti-MPO induced glomerulonephritis.[59] These studies detailing a pathogenic role for the alternative pathway in ANCA induced glomerulo‐ nephritis have helped improve our understanding of the disease. More recently a mAb directed against C5, Eculuzimab (also known as Soliris and manufactured by Alexion Pharmaceuticals) has been licensed for the treatment of several complement mediated diseases, including paroxysmal nocturnal hemoglobinuria. There is growing interest that C5 inhibition could be used for the treatment of glomerulonephritis and organ injury induced by AAV and this has formed the basis of a clinical trial currently underway in the United States.

#### **8.4. Dendritic cells as antigen presenting cells and effector cells in AAV**

Evidence from experimental models has supported a role for dendritic cells (DCs) in initiating and promoting immune responses in autoimmune diseases.[187] These specialised antigen presenting cells (APCs) recognise antigens through pattern-recognition receptors and coordinate the initiation and maintenance of the immune response.[188] Little is currently known about antigen presentation and the subsequent development of autoimmunity in AAV. It is likely that DCs are involved in two processes, firstly in the development of autoimmunity through interaction with dying neutrophils and also, acting locally, promoting kidney injury where their presence in renal biopsy samples positively correlates with injury.

A pathogenic role for DCs in human AAV was recognised several years ago. When immature DCs were isolated from GPA patients and cultured with PR3, markers of DC activation, CD80 and CD86 were increased. These antigen primed DCs were able to produce IFNγ, consistent with a Th1 phenotype.[189] In an experimental model of MPO induced ANCA vasculitis, we have shown that pulsing DCs with MPO is an effective means of inducing cellular and humoral autoimmunity directed against MPO. Furthermore, using our murine model of focal necrot‐ ising glomerulonephritis, these mice developed severe functional and histological renal injury (unpublished data). It is likely that up-regulation of DCs is (at least partially) TLR mediated and in AAV this could result from infection. After immunizing WT mice with a TLR2 or TLR9 ligand and MPO we found an increase in DC maturation (assessed as an increase in CD86 expression), compared to mice immunized with MPO alone.[82] In additional unpublished work, pilot studies have shown that stimulation of DCs with a TLR9 ligand and MPO results in increased CD40, CD80 and CD86 expression, compared to DCs stimulated with MPO alone, this is demonstrated below. These clinical and experimental studies implicate DCs in the loss of tolerance to MPO.

**Figure 2.** Dendritic cell (DC) activation after culturing DCs with MPO and a TLR9 ligand. After culturing DCs with MPO and control or a TLR 9 ligand, there was an increase on CD40+ DCs shown schematically in the top figure. In the mid‐ dle figure we see representative CD40 expression from DCs treated with control and MPO, while the bottom figure

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A limitation of both the clinical and experimental studies is that these studies have largely focussed on myeloid DCs and not plasmacytoid DCs. In other diseases, including systemic lupus erythematosus, plasmacytoid DCs have been shown to be potent inducers of Type 1 IFNs and drive the development of autoimmunity.[190] In addition to their role in the

shows the increase in CD40 expression after treatment with MPO and a TLR9 ligand.

activated protein kinase, extracellular signal-regulated kinase and phosphoinositol 3-kinase. [185] Results from clinical studies have shown that serum and urine levels of C5a are elevated in patients with active disease strongly supporting the notion that the complement cascade is activated in active AAV.[186] Strong support for a pathogenic role for complement has also been provided from experimental studies. In an extensive set of experiments, the North Carolina group robustly demonstrated that complement depletion (achieved through the use of cobra venom serum) abrogated disease, an effect mediated through C5 and Factor B.[183] Factor B is critical for alternative pathway activation. Similarly inhibition of C5 using a mAb successfully attenuated experimental anti-MPO induced glomerulonephritis.[59] These studies detailing a pathogenic role for the alternative pathway in ANCA induced glomerulo‐ nephritis have helped improve our understanding of the disease. More recently a mAb directed against C5, Eculuzimab (also known as Soliris and manufactured by Alexion Pharmaceuticals) has been licensed for the treatment of several complement mediated diseases, including paroxysmal nocturnal hemoglobinuria. There is growing interest that C5 inhibition could be used for the treatment of glomerulonephritis and organ injury induced by AAV and this has

52 Updates in the Diagnosis and Treatment of Vasculitis

formed the basis of a clinical trial currently underway in the United States.

**8.4. Dendritic cells as antigen presenting cells and effector cells in AAV**

where their presence in renal biopsy samples positively correlates with injury.

of tolerance to MPO.

Evidence from experimental models has supported a role for dendritic cells (DCs) in initiating and promoting immune responses in autoimmune diseases.[187] These specialised antigen presenting cells (APCs) recognise antigens through pattern-recognition receptors and coordinate the initiation and maintenance of the immune response.[188] Little is currently known about antigen presentation and the subsequent development of autoimmunity in AAV. It is likely that DCs are involved in two processes, firstly in the development of autoimmunity through interaction with dying neutrophils and also, acting locally, promoting kidney injury

A pathogenic role for DCs in human AAV was recognised several years ago. When immature DCs were isolated from GPA patients and cultured with PR3, markers of DC activation, CD80 and CD86 were increased. These antigen primed DCs were able to produce IFNγ, consistent with a Th1 phenotype.[189] In an experimental model of MPO induced ANCA vasculitis, we have shown that pulsing DCs with MPO is an effective means of inducing cellular and humoral autoimmunity directed against MPO. Furthermore, using our murine model of focal necrot‐ ising glomerulonephritis, these mice developed severe functional and histological renal injury (unpublished data). It is likely that up-regulation of DCs is (at least partially) TLR mediated and in AAV this could result from infection. After immunizing WT mice with a TLR2 or TLR9 ligand and MPO we found an increase in DC maturation (assessed as an increase in CD86 expression), compared to mice immunized with MPO alone.[82] In additional unpublished work, pilot studies have shown that stimulation of DCs with a TLR9 ligand and MPO results in increased CD40, CD80 and CD86 expression, compared to DCs stimulated with MPO alone, this is demonstrated below. These clinical and experimental studies implicate DCs in the loss

**Figure 2.** Dendritic cell (DC) activation after culturing DCs with MPO and a TLR9 ligand. After culturing DCs with MPO and control or a TLR 9 ligand, there was an increase on CD40+ DCs shown schematically in the top figure. In the mid‐ dle figure we see representative CD40 expression from DCs treated with control and MPO, while the bottom figure shows the increase in CD40 expression after treatment with MPO and a TLR9 ligand.

A limitation of both the clinical and experimental studies is that these studies have largely focussed on myeloid DCs and not plasmacytoid DCs. In other diseases, including systemic lupus erythematosus, plasmacytoid DCs have been shown to be potent inducers of Type 1 IFNs and drive the development of autoimmunity.[190] In addition to their role in the development of autoimmune responses DCs represent a component of the characteristic inflammatory infiltrate see on renal biopsy samples from patients with AAV. Increased numbers of immature (CD209+) and mature (CD208+) DCs were found in renal biopsies from patients with AAV.[191] While these studies do not prove that DCs are pathogenic in renal vasculitis, their association with the inflammatory infiltrate suggests that they may be involved in the promotion of renal inflammation and injury.

Our studies strongly supported a role for glomerular endothelial cells in this disease process and the role of the endothelium in promoting inflammation and injury is well known. It is likely that other glomerular cell types contribute to injury and immunofluorescent staining of kidney biopsies from patients with AAV has demonstrated that podocytes and tubulointerstitial cells are major producers of IL-18, which is involved in neutrophil recruitment.[52] Similarly after staining human biopsies from AAV patients with crescentic glomerulonephritis the pathogenic isoform of the stress response protein kinase p38MAPK was detected in the podocyte, further implicating the role of this specialised cell in driving glomerular injury.[192] In addition to the glomerular injury observed in ANCA associated renal vasculitis tubular lesions, most notably peritubular inflammatory capillaritis, are common and are associated with a poor prognosis.[193] The interstitium is a prime target for inflammatory cells as many of the tubular epithelial cells express MHCII, TLRs and complement receptors, with which they can interact.[194] Furthermore peritubular capillaries display physiological characteris‐ tics similar to postcapillary venules which further increases the recruitment of inflammatory cells commonly observed in crescentic glomerulonephritis.[195-196] In conclusion it is apparent that the kidney harbours a particular environment which facilitates the recruitment of inflammatory cells and subsequent renal injury making it the key target for injury in AAV.

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As new concepts of autoimmunity and cellular functions are elucidated in both innate and humoral immunity our scope of understanding of this complex disease entity continues to expand. Whilst an appreciation of the involvement of the adaptive immune dysfunction that contributes to AAV is well established new and varied innate immune system mechanisms of pathogenesis are emerging. Recent work investigating neutrophil functions and life cycle including the newly identified and described NETosis, along with imaging modalities allowing accurate characterisation of neutrophil trafficking and interactions with endothelial cells of the vessel wall provide us with a better understanding of the important role these cells have to play in this multifactorial disease process. The huge range of new biologic agents and advancing therapeutic technologies bring with them the possibilities of more effective,

Sharon Lee Ford1,2, Stephen Roger Holdsworth1,2 and Shaun Andrew Summers1,2

2 Department of Nephrology, Monash Medical Centre, Australia

1 Centre for Inflammatory Diseases, Department of Medicine, Monash University, Australia

**10. Conclusions**

**Author details**

targeted, less toxic therapies for our patients.

**Figure 3.** Summary of the Pathogenesis of MPO-ANCA Vasculitis. A summary of the events which contribute to the loss of tolerance to MPO, with the development of autoimmunity resulting in rapidly progressive crescentic glomeru‐ lonephritis.

#### **9. The role of resident kidney cells in ANCA associated renal vasculitis**

Results from many of the studies discussed above have demonstrated that he kidney is not an 'innocent bystander' in the disease process. Rather the kidney provides an anatomic and physiological milieu which is well suited to recruit inflammatory cells. In our studies we have found that TLR ligation increased ANCA induced glomerular neutrophil recruitment and injury, which required contributions from both bone marrow and resident kidney cells.[58] Our studies strongly supported a role for glomerular endothelial cells in this disease process and the role of the endothelium in promoting inflammation and injury is well known. It is likely that other glomerular cell types contribute to injury and immunofluorescent staining of kidney biopsies from patients with AAV has demonstrated that podocytes and tubulointerstitial cells are major producers of IL-18, which is involved in neutrophil recruitment.[52] Similarly after staining human biopsies from AAV patients with crescentic glomerulonephritis the pathogenic isoform of the stress response protein kinase p38MAPK was detected in the podocyte, further implicating the role of this specialised cell in driving glomerular injury.[192] In addition to the glomerular injury observed in ANCA associated renal vasculitis tubular lesions, most notably peritubular inflammatory capillaritis, are common and are associated with a poor prognosis.[193] The interstitium is a prime target for inflammatory cells as many of the tubular epithelial cells express MHCII, TLRs and complement receptors, with which they can interact.[194] Furthermore peritubular capillaries display physiological characteris‐ tics similar to postcapillary venules which further increases the recruitment of inflammatory cells commonly observed in crescentic glomerulonephritis.[195-196] In conclusion it is apparent that the kidney harbours a particular environment which facilitates the recruitment of inflammatory cells and subsequent renal injury making it the key target for injury in AAV.

### **10. Conclusions**

development of autoimmune responses DCs represent a component of the characteristic inflammatory infiltrate see on renal biopsy samples from patients with AAV. Increased numbers of immature (CD209+) and mature (CD208+) DCs were found in renal biopsies from patients with AAV.[191] While these studies do not prove that DCs are pathogenic in renal vasculitis, their association with the inflammatory infiltrate suggests that they may be involved

**Figure 3.** Summary of the Pathogenesis of MPO-ANCA Vasculitis. A summary of the events which contribute to the loss of tolerance to MPO, with the development of autoimmunity resulting in rapidly progressive crescentic glomeru‐

**9. The role of resident kidney cells in ANCA associated renal vasculitis**

Results from many of the studies discussed above have demonstrated that he kidney is not an 'innocent bystander' in the disease process. Rather the kidney provides an anatomic and physiological milieu which is well suited to recruit inflammatory cells. In our studies we have found that TLR ligation increased ANCA induced glomerular neutrophil recruitment and injury, which required contributions from both bone marrow and resident kidney cells.[58]

in the promotion of renal inflammation and injury.

54 Updates in the Diagnosis and Treatment of Vasculitis

lonephritis.

As new concepts of autoimmunity and cellular functions are elucidated in both innate and humoral immunity our scope of understanding of this complex disease entity continues to expand. Whilst an appreciation of the involvement of the adaptive immune dysfunction that contributes to AAV is well established new and varied innate immune system mechanisms of pathogenesis are emerging. Recent work investigating neutrophil functions and life cycle including the newly identified and described NETosis, along with imaging modalities allowing accurate characterisation of neutrophil trafficking and interactions with endothelial cells of the vessel wall provide us with a better understanding of the important role these cells have to play in this multifactorial disease process. The huge range of new biologic agents and advancing therapeutic technologies bring with them the possibilities of more effective, targeted, less toxic therapies for our patients.

### **Author details**

Sharon Lee Ford1,2, Stephen Roger Holdsworth1,2 and Shaun Andrew Summers1,2

1 Centre for Inflammatory Diseases, Department of Medicine, Monash University, Australia

2 Department of Nephrology, Monash Medical Centre, Australia

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57

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**Chapter 3**

**Immunological Mechanisms and Clinical Aspects in**

McGraw-Hill Concise Dictionary of Modern Medicine [1] defines pulmonary-renal syndrome (PRS) as an idiopathic condition characterized by pulmonary hemorrhage, rapid progressive glomerulonephritis, and positive autoantibodies. Pulmonary-renal syndrome may be also defined as a heterogeneous group of multisystem diseases – e.g. Goodpasture syndrome, Wegener's granulomatosis, collagen vascular disease – in particular systemic lupus erythematosus, polyarteritis nodosa, Henoch-Schönlein purpura, and various other conditions, which all have prominent pulmonary and renal components. According to Sanders [2] the strict definition of pulmonary-renal syndrome is the combined clinical picture of rapid progressive glomerulonephritis and pulmonary capillaritis requiring histological

If we translate the definition into pathological nomenclature, pulmonary-renal syndrome is defined as combination of diffuse alveolar hemorrhage (DAH) and immune crescent glomerulonephritis. The essential substrate of all these changes is vasculitis, which is according to contemporary nomenclature based mostly on morphological and histopathological criteria. These criteria for the most common forms of vasculitides were introduced in 1994 by Jennette et al. at the Chapel Hill Consensus Conference organized by the American College of Rheumatology [3]. The inflammation of small vessels (microangiopathic vasculitis) restricts blood flow to various organs and damages them. If correct diagnosis and appropriate treatment are delayed the condition can be fatal. Prognosis is good when treatment begins before onset of respiratory and renal failure. Because of the similarity in clinical picture,

The pathophysiology of the vasculitides is based on immunologic mechanisms. These appear to play an active role in mediating the inflammatory response, but their exact mechanisms still

and reproduction in any medium, provided the original work is properly cited.

© 2013 Lukán; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

differential diagnosis of these diseases at the bedside can be challenging.

**Pulmonary-Renal Syndrome: A Review**

Additional information is available at the end of the chapter

N. Lukán

http://dx.doi.org/10.5772/55181

**1. Introduction**

confirmation.

