**5. The role of adaptive immunity in the development of ANCA autoimmunity and glomerulonephritis**

#### **5.1. The role of humoral immunity in AAV pathogenesis**

crotising glomerulonephritis has been described in patients treated with penicillamine[34] and hydralazine.[35] The rarity of these phenomena has prevented us from learning

Links between infection and ANCA vasculitis have been suggested for some time, with seasonal variation in disease presentation suggesting a correlation with microbial infection. [36] Moreover results from several studies suggested that infection(s) may predate disease initiation and/or relapse in GPA, MPA and pulmonary vasculitis.[37-40] It must be noted that these results are contentious and other studies have not confirmed them.[30] However, nasal colonization with *Staphylococcus Aureus* is significantly increased in patients with GPA and increases the relative risk of relapse over 7 fold.[37] In a key study, published more than 15 years ago, it was shown that prophylactic antibiotic therapy (co-trimoxazole) successfully decreased disease relapses in ANCA vasculitis. This effect was presumed to result from decreased nasal carriage of *Staphylococcus Aureus.*[41] Interestingly, despite this finding longterm maintenance therapy with co-trimoxazole is not the standard of care in many centres, which may reflect concerns about the long-term safety of the drug. Consistent with an infective trigger to the development of AAV; features of vasculitis have been described in patients with bacterial endocarditis.[42-43] Despite the strong evidence linking infection with the develop‐ ment of autoimmunity (MPO/ PR3) and the ensuing organ injury few mechanistic links have

Several mechanisms have been proposed to link infection with the development of AAV, including the use of complementary proteins, molecular mimicry and the ligation of Toll like receptors (TLRs) which heighten innate and adaptive immune responses as well as activating resident kidney cells. A series of clinical and experimental studies have supported each of these concepts, however it is likely that these mechanisms act, at least partially, in combination. Molecular mimicry refers to the development of antibodies to host proteins after (repeat‐ ed) exposure to foreign antigens, this occurs due to structural similarities between host and foreign proteins. Molecular mimicry has been proposed as a reason for the loss of tolerance to self and the subsequent development of autoimmunity.[44] In a series of ele‐ gant experiments it was demonstrated that antibodies to the lysosomal associated mem‐ brane protein-2 (LAMP-2) were highly prevalent in patients with ANCA vasculitis. Furthermore LAMP-2 was pathogenic and administration of polyclonal LAMP-2 to ro‐ dents resulted in a characteristic pattern of AAV, with focal necrotising glomerulonephri‐ tis, similar to that observed in human renal vasculitis. We will discuss LAMP-2 in more detail later in this chapter. There is homology between the immunodominant LAMP-2 epitope and the peptide of FimH, which is a component of the fimbriae of Gram nega‐ tive bacteria. It is hypothesized that certain patients infected with Gram negative bacteria would generate antibodies to LAMP-2 and develop vasculitis, through the process of mo‐ lecular mimicry.[45] This highly plausible theory provides one explanation for the clinical association between infection and the development of ANCAs or LAMP-2 antibodies.

An earlier study reported that a form of molecular mimicry could link *Staphylococcus Aureus* infection with the development of AAV. This process was more complex and involved the use of complementary proteins. The authors observed that patients who were PR3-ANCA positive

more about disease pathogenesis.

38 Updates in the Diagnosis and Treatment of Vasculitis

been provided, until recently.

Since their description in the 1980s antibodies directed against MPO and PR3 have formed the diagnostic hallmark of AAV. While not entirely specific there is a strong association between MPO-ANCA and MPA, while PR3 is commonly associated with GPA. Clinical and experi‐ mental studies have supported the notion that ANCA are pathogenic. Furthermore therapies targeting (humoral immunity and) ANCAs, including plasma exchange[47] and the anti-CD20 monoclonal antibody Rituximab,[48-49] have been successful in clinical practice. Most of the experimental evidence has supported a role for MPO in disease, but more recently an animal model of PR3-associated vasculitis has also been developed. This represents a significant advance and it is anticipated that this model will facilitate an improved understanding of the pathogenesis of PR3-AAV. In this section, we will also discuss other roles for B cells including their function as antigen presenting cells (APCs) and as potential regulators of disease.

Are ANCAs pathogenic? There has been increasing evidence supporting a pathogenic role for ANCAs. Results from *in vitro* studies demonstrate that ANCAs activate primed neutrophils which degranulate and deposit autoantigens in glomeruli. Similarly results from *in vivo* studies, including an expanding number of animal models, have confirmed a pathogenic role for ANCAs. *In vitro* studies have consistently demonstrated that neutrophils from patients with AAV express increased amounts of the target antigens (MPO/PR3) on their cell surface. [50] These auto-antigens are targets for ANCA binding. Furthermore, several cytokines including tumor necrosis factor (TNF), IL-18 and granulocyte macrophage colony stimulating factor can prime neutrophils in AAV, increasing auto-antigen expression which facilitates ANCA binding.[51-53] Binding of ANCA to the neutrophil is associated with increased adherence to the endothelium, superoxide generation and cytokine production.[51, 54] The effect of neutrophils and their interaction with the endothelium will be discussed in greater detail later in this chapter.

traditionally are found to be ANCA negative. Antibodies to LAMP-2 were also pathogenic and administration of human LAMP-2 antibodies to Wistar Kyoto rats resulted in pauci-immune focal necrotizing glomerulonephritis.[45] Subsequently, the authors working with several collaborative groups, have verified the prevalence of antibodies to LAMP-2 in cohorts of ANCA patients from a range of European countries. Three different techniques; enzyme linked immunosorbent assay; western blotting and an indirect immunofluorescence assay were all readily able to detect antibodies. Interestingly antibodies were undetectable shortly after treatment, although they were detectable during clinical relapse, highlighting the potential usefulness of these antibodies in clinical practice.[62] However studies from the United States could not confirm these findings, where the sensitivity of detecting LAMP-2 antibodies was much lower than that seen within the European studies.[63] The divergence of results is interesting and suggests that further work is required to facilitate assays which could result

The Pathogenesis of Antineutrophil Cytoplasmic Antibody Renal Vasculitis

http://dx.doi.org/10.5772/54637

41

Most studies examining the pathogenic role of B cells in AAV have focussed on their role as effector cells, however B cells have a more diverse range of functions than autoantibody production alone. In other scenarios B cells are considered antigen presenting cells, while they

The B cell activating factor (BAFF) has also been shown to be elevated in patients with AAV,[65] which is exciting considering the therapeutic promise shown with BAFF inhibi‐ tors in systemic lupus erythematosus (SLE).[66] B cells may also contribute to disease in other ways and a detailed analysis of renal biopsies from patients with AAV demonstrat‐ ed significant B cell infiltration, including organized B cell clusters.[67] In addition to proinflammatory responses B cell also display regulatory function and produce IL-10, a regulatory cytokine. Interestingly in patients with SLE regulatory B cells (Bregs) are im‐ paired and are unable to suppress effector T cells.[68] While this has not been explored to date in vasculitis, it remains possible that heightened humoral and cellular immunity

In concluding, B cells form the diagnostic hallmarks of ANCA vasculitis and are pathogenic. The success observed in clinical practice with therapies which chiefly target B cells has not been fully elucidated and may extend beyond autoantibody inhibition. Interestingly, Rituxi‐ mab was shown to treat the clinical symptoms of GPA, even when ANCAs were not detectable. [69] An in-depth understanding of the role of humoral immunity is awaited and may help

While ANCAs are diagnostic and pathogenic in AAV, cellular immunity is an essential requirement for the initiation and continued production of auto-reactive B cell responses and for driving effector cell responses in the kidney. Evidence for a key role for cellular autoim‐ munity in AAV comes from several lines of evidence, including observational studies in humans, reports of refractory disease responding to treatments targeting T cells and extensive murine studies showing a pathogenic role for T cells in the development of autoimmunity. Vasculitis involving the glomerular capillary bed has little or no antibody deposition, but

in the development of better diagnostic tools.

possibly influence T cell responses.[64]

occurs as a consequence of impaired Bregs.

**5.2. The role of cellular immunity in AAV pathogenesis**

direct future therapies.

Animal studies have demonstrated a pathogenic role for ANCAs. The model described by Xiao et al was one of the first murine models of AAV, which produced severe renal injury. The observed renal injury bore considerable resemblance to that seen in human rapidly progressive glomerulonephritis. In this model MPO deficient mice were immunized with MPO. Subse‐ quently the spleens of these MPO deficient mice were transferred into recombinant activation gene knockout (RAG2-/-) mice, which lack adaptive immunity. After transfer of splenocytes (from MPO immunized MPO-/- mice) RAG2-/- mice developed humoral autoimmunity with the production of MPO-ANCAs. Kidneys from these mice displayed the hallmarks of severe crescentic glomerulonephritis. The authors also performed a passive transfer experiment, administering MPO-ANCAs to RAG2-/- mice. The passive transfer of MPO-ANCA to RAG2-/ mice resulted in a milder form of glomerular injury compared to that seen after splenocyte transfer.[55] These experiments highlighted the pathogenic role for MPO-ANCAs, however, it should be noted that the severe injury occurring after the transfer of splenocytes could reflect cellular immunity contributing to renal injury. None the less, the passive transfer of ANCAs to mice has consistency resulted in a degree of renal injury, which is neutrophil,[56] lipopo‐ lysaccharide[57], TLR4[58] and complement [59] dependent.

Additional evidence for a pathogenic role for MPO in driving AAV and renal injury was demonstrated in Wistar-Kyoto rats. Rats developed focal necrotizing glomerulonephritis and pulmonary vasculitis after immunization with purified human MPO. Furthermore a patho‐ genic role for the chemokine CXCL1 (the rodent homolog of human IL-8) in neutrophilendothelial interactions was demonstrated, by analysis of neutrophil migration in the capillary beds.[60] Recently Little has described a model of vasculitis, dependent on PR3-ANCA, which develops in mice with a humanised immune system. This model was generated by treating irradiated NOD-scid-IL-2Rγ-/- mice with human haematopoietic cells. In NOD-scid-IL-2Rγ-/ mice there are multiple deficiencies in the function of both innate and adaptive immune cells. These chimeric mice were then treated with human immunoglobulin from patients with PR3- ANCA vasculitis or control serum. In control treated mice no glomerular injury was observed, however mice treated with PR3-ANCA demonstrated (at least mild) glomerulonephritis, while more severe injury was observed in 17% of PR3-ANCA treated mice.[61] While further work is required to confirm that this murine model is robust, it is anticipated that it will provide a good basis to explore the pathogenic nature of PR3-ANCA in clinical practice.

Another potential antigenic target is LAMP-2. Antibodies to LAMP-2 were reliably detected in more than 90% of patients with active ANCA associated necrotising crescentic glomerulo‐ nephritis. LAMP-2 antibodies were detected even when MPO-ANCA and PR3-ANCA could not be detected, suggesting this test may have improved diagnostic sensitivity and could possibly be useful for serological diagnosis in patients with renal limited vasculitis, who traditionally are found to be ANCA negative. Antibodies to LAMP-2 were also pathogenic and administration of human LAMP-2 antibodies to Wistar Kyoto rats resulted in pauci-immune focal necrotizing glomerulonephritis.[45] Subsequently, the authors working with several collaborative groups, have verified the prevalence of antibodies to LAMP-2 in cohorts of ANCA patients from a range of European countries. Three different techniques; enzyme linked immunosorbent assay; western blotting and an indirect immunofluorescence assay were all readily able to detect antibodies. Interestingly antibodies were undetectable shortly after treatment, although they were detectable during clinical relapse, highlighting the potential usefulness of these antibodies in clinical practice.[62] However studies from the United States could not confirm these findings, where the sensitivity of detecting LAMP-2 antibodies was much lower than that seen within the European studies.[63] The divergence of results is interesting and suggests that further work is required to facilitate assays which could result in the development of better diagnostic tools.

including tumor necrosis factor (TNF), IL-18 and granulocyte macrophage colony stimulating factor can prime neutrophils in AAV, increasing auto-antigen expression which facilitates ANCA binding.[51-53] Binding of ANCA to the neutrophil is associated with increased adherence to the endothelium, superoxide generation and cytokine production.[51, 54] The effect of neutrophils and their interaction with the endothelium will be discussed in greater

Animal studies have demonstrated a pathogenic role for ANCAs. The model described by Xiao et al was one of the first murine models of AAV, which produced severe renal injury. The observed renal injury bore considerable resemblance to that seen in human rapidly progressive glomerulonephritis. In this model MPO deficient mice were immunized with MPO. Subse‐ quently the spleens of these MPO deficient mice were transferred into recombinant activation gene knockout (RAG2-/-) mice, which lack adaptive immunity. After transfer of splenocytes (from MPO immunized MPO-/- mice) RAG2-/- mice developed humoral autoimmunity with the production of MPO-ANCAs. Kidneys from these mice displayed the hallmarks of severe crescentic glomerulonephritis. The authors also performed a passive transfer experiment, administering MPO-ANCAs to RAG2-/- mice. The passive transfer of MPO-ANCA to RAG2-/ mice resulted in a milder form of glomerular injury compared to that seen after splenocyte transfer.[55] These experiments highlighted the pathogenic role for MPO-ANCAs, however, it should be noted that the severe injury occurring after the transfer of splenocytes could reflect cellular immunity contributing to renal injury. None the less, the passive transfer of ANCAs to mice has consistency resulted in a degree of renal injury, which is neutrophil,[56] lipopo‐

Additional evidence for a pathogenic role for MPO in driving AAV and renal injury was demonstrated in Wistar-Kyoto rats. Rats developed focal necrotizing glomerulonephritis and pulmonary vasculitis after immunization with purified human MPO. Furthermore a patho‐ genic role for the chemokine CXCL1 (the rodent homolog of human IL-8) in neutrophilendothelial interactions was demonstrated, by analysis of neutrophil migration in the capillary beds.[60] Recently Little has described a model of vasculitis, dependent on PR3-ANCA, which develops in mice with a humanised immune system. This model was generated by treating irradiated NOD-scid-IL-2Rγ-/- mice with human haematopoietic cells. In NOD-scid-IL-2Rγ-/ mice there are multiple deficiencies in the function of both innate and adaptive immune cells. These chimeric mice were then treated with human immunoglobulin from patients with PR3- ANCA vasculitis or control serum. In control treated mice no glomerular injury was observed, however mice treated with PR3-ANCA demonstrated (at least mild) glomerulonephritis, while more severe injury was observed in 17% of PR3-ANCA treated mice.[61] While further work is required to confirm that this murine model is robust, it is anticipated that it will provide a

good basis to explore the pathogenic nature of PR3-ANCA in clinical practice.

Another potential antigenic target is LAMP-2. Antibodies to LAMP-2 were reliably detected in more than 90% of patients with active ANCA associated necrotising crescentic glomerulo‐ nephritis. LAMP-2 antibodies were detected even when MPO-ANCA and PR3-ANCA could not be detected, suggesting this test may have improved diagnostic sensitivity and could possibly be useful for serological diagnosis in patients with renal limited vasculitis, who

lysaccharide[57], TLR4[58] and complement [59] dependent.

detail later in this chapter.

40 Updates in the Diagnosis and Treatment of Vasculitis

Most studies examining the pathogenic role of B cells in AAV have focussed on their role as effector cells, however B cells have a more diverse range of functions than autoantibody production alone. In other scenarios B cells are considered antigen presenting cells, while they possibly influence T cell responses.[64]

The B cell activating factor (BAFF) has also been shown to be elevated in patients with AAV,[65] which is exciting considering the therapeutic promise shown with BAFF inhibi‐ tors in systemic lupus erythematosus (SLE).[66] B cells may also contribute to disease in other ways and a detailed analysis of renal biopsies from patients with AAV demonstrat‐ ed significant B cell infiltration, including organized B cell clusters.[67] In addition to proinflammatory responses B cell also display regulatory function and produce IL-10, a regulatory cytokine. Interestingly in patients with SLE regulatory B cells (Bregs) are im‐ paired and are unable to suppress effector T cells.[68] While this has not been explored to date in vasculitis, it remains possible that heightened humoral and cellular immunity occurs as a consequence of impaired Bregs.

In concluding, B cells form the diagnostic hallmarks of ANCA vasculitis and are pathogenic. The success observed in clinical practice with therapies which chiefly target B cells has not been fully elucidated and may extend beyond autoantibody inhibition. Interestingly, Rituxi‐ mab was shown to treat the clinical symptoms of GPA, even when ANCAs were not detectable. [69] An in-depth understanding of the role of humoral immunity is awaited and may help direct future therapies.

#### **5.2. The role of cellular immunity in AAV pathogenesis**

While ANCAs are diagnostic and pathogenic in AAV, cellular immunity is an essential requirement for the initiation and continued production of auto-reactive B cell responses and for driving effector cell responses in the kidney. Evidence for a key role for cellular autoim‐ munity in AAV comes from several lines of evidence, including observational studies in humans, reports of refractory disease responding to treatments targeting T cells and extensive murine studies showing a pathogenic role for T cells in the development of autoimmunity. Vasculitis involving the glomerular capillary bed has little or no antibody deposition, but rather demonstrates delayed type hypersensitivity responses, including fibrin deposition. This is most likely to be a consequence of auto-reactive CD4+ effector cells recognizing MPO, which is present in glomeruli in both human and experimental ANCA vasculitis [70-72]. In addition to enhancing inflammation, regulatory T cells (Tregs) are likely to have an important role in modulating immune responses and glomerular injury.

progressive glomerulonephritis in MPO-ANCA vasculitis. Subsequent work from this group has supported a role for both Th1 and Th17 cells in disease. Firstly, using IL-17A-/- mice it was shown that the development of cellular autoimmunity and necrotizing glomerulonephritis was IL-17A dependent. Secondly in the absence of IL-17A there was a decrease in glomerular neutrophil and macrophage recruitment and renal injury was attenuated. These results highlight the potential therapeutic benefits of IL-17A blockade in AAV.[24] This group has also elucidated that both IL-17A and IFNγ can drive nephritogenic autoimmunity and renal injury in AAV. Interestingly ligation of different TLRs dictated the pattern of cytokine production, TLR2 ligation promoted the development of Th17 autoimmunity, while TLR9 ligation drove Th1 autoimmunity. Mice which developed Th17 induced renal injury were successfully treated with anti-IL-17A monoclonal antibody (mAb). Conversely in mice that developed predominant Th1 driven injury, administration of anti-IFNγ mAb attenuated renal injury.[82] Work from Richard Kitching's group has further refined our understanding of the role of CD4+ T cells in the pathogenesis of AAV. Using 20 amino acid sequence peptides they identified the immunodominant MPO CD4+ T cell epitope. Subsequently they produced T cell clones which were specific for this immunodominant MPO epitope, which were then injected into mice. Using three different techniques it was demonstrated that when the MPO peptide (or whole MPO) was deposited in glomeruli focal necrotising glomerulonephritis was driven by antigen specific CD4+ T cells.[83] These key studies have helped define how effector T cells

The Pathogenesis of Antineutrophil Cytoplasmic Antibody Renal Vasculitis

http://dx.doi.org/10.5772/54637

43

**6. The role of Th17 cells in autoimmunity and glomerulonephritis**

The original description of Th1, IFNγ producing and Th2, IL-4 producing, T helper cells by Mosmann and Coffman [84] has been expanded to include a new subset of Th cells, the IL-17A producing Th17 cells.[79, 85-86] While the prototypic cytokine produced by Th17 cells is IL-17A, these cells produce numerous other cytokines, including the ubiquitous IL-6, TNF and IL-1β.[85] Two transcription factors are critical for the development of Th17 cells; STAT3 and Rorγt.[87-88] For the induction and maintenance Th17 cells, several cytokines are required, these include; IL-23,[89] IL-6, TGF-β,[90-93] while IL-21 is required for amplification of Th17

Prior to the discovery of Th17 cells, autoimmunity was believed to be predominantly a Th1 mediated phenomenon. There were inconsistencies, however, in this paradigm, for example IFNγ-/- mice developed exaggerated organ inflammation and injury in experimental autoim‐ mune models.[97-98] Subsequently it was demonstrated that organ injury (in the most common autoimmune model, experimental autoimmune encephalomyelitis [EAE]) was unchanged in IL-12p35-/- mice (functionally Th1 deficient), while injury was significantly attenuated in IL-12p40-/- (functionally Th1 and Th17 deficient) and IL-12p19-/- (functionally Th17 deficient) mice.[99] Similarly IL-17A-/- mice were protected from EAE, [100] while increased IL-17 expression was seen in patients with multiple sclerosis, [101] a common autoimmune disease seen in clinical practice, which is the human equivalent of EAE. Further studies have implicated Th17 cells in several autoimmune diseases including rheumatoid

drive glomerular injury.

cells.[94-96]

T cells are active participants in the loss of tolerance and the development of autoimmunity in AAV. Firstly we know that ANCAs are class switched high affinity antibodies which are (therefore) dependent on T cells for their generation.[73] Secondly, in proliferation studies, it has been demonstrated that auto-reactive T cells from patents with AAV respond to MPO and PR3,[74] while markers of T cell activity are increased in parallel with disease activity.[75-76] Furthermore, in renal biopsy samples from patients with AAV, the number of infiltrating T cells correlates with the severity of injury. Additional evidence supporting a pathogenic role for T cells was provided when 15 patients with refractory vasculitis, resistant to other therapies, were successfully treated with anti-thymocyte globulin, which targets T cells.[77]

Early studies supported a role for T helper (Th) 1 (and possibly Th2) cells in the pathogenesis of AAV. Peripheral blood lymphocytes from patients with MPO-ANCA were shown to produce IFNγ when stimulated.[78] The more recently defined Th17 cells represent a distinct lineage of CD4+ T cells, which are characterized by the production of IL-17A.[79] Two key human studies supported a role for Th17 cells in ANCA vasculitis. Firstly it was demonstrated that when peripheral blood from GPA patients was stimulated with PR3, there was an increased percentage of IL-17A producing CD4+ T cells (Th17). After stimulation no difference in IFNγ production was seen, suggesting that Th1 cells were not involved. The authors proposed that this skewed Th17 response supported a role for Th17 cells in disease.[80] A subsequent study demonstrated that sera from patients with active AAV consistently dis‐ played a Th17 phenotype. Cytokines associated with Th17 cells, including IL-17A and IL-23, were increased in patients with acute AAV, while levels of IFNγ were unchanged. Interestingly immunosuppressive therapy did not consistently decrease IL-23 or IL-17 production.[23] In a study of human ANCA biopsies it has been shown that IL-17A producing CD4+ T cells constitute part of the inflammatory infiltrate and correspond with disease severity.[81] In addition, murine models have provided strong evidence for a pathogenic role for CD4+ T cells in glomerulonephritis.

An MPO-dependent murine model which demonstrates considerable homology to human ANCA vasculitis, where mice develop autoimmunity to MPO and focal necrotising glomeru‐ lonephritis was described. Immunization of C57BL/6 wild type mice with MPO results in cellular and humoral autoimmunity to MPO. A small dose of sheep anti-mouse glomerular basement membrane serum is subsequently administered. Treatment of chicken ovalbumin (OVA) immunized mice with this dose of sheep anti-mouse glomerular basement membrane serum does not result in significant renal injury. However in mice immunized with MPO and then sheep anti-mouse glomerular basement membrane serum significant renal injury is seen. Depletion of CD4+ effector cells significantly attenuated glomerular injury in this model, while experiments performed in B cell-deficient mice did not show renal protection.[72] These results provide strong evidence for a pathogenic role for CD4+ effector cells contributing to rapidly progressive glomerulonephritis in MPO-ANCA vasculitis. Subsequent work from this group has supported a role for both Th1 and Th17 cells in disease. Firstly, using IL-17A-/- mice it was shown that the development of cellular autoimmunity and necrotizing glomerulonephritis was IL-17A dependent. Secondly in the absence of IL-17A there was a decrease in glomerular neutrophil and macrophage recruitment and renal injury was attenuated. These results highlight the potential therapeutic benefits of IL-17A blockade in AAV.[24] This group has also elucidated that both IL-17A and IFNγ can drive nephritogenic autoimmunity and renal injury in AAV. Interestingly ligation of different TLRs dictated the pattern of cytokine production, TLR2 ligation promoted the development of Th17 autoimmunity, while TLR9 ligation drove Th1 autoimmunity. Mice which developed Th17 induced renal injury were successfully treated with anti-IL-17A monoclonal antibody (mAb). Conversely in mice that developed predominant Th1 driven injury, administration of anti-IFNγ mAb attenuated renal injury.[82] Work from Richard Kitching's group has further refined our understanding of the role of CD4+ T cells in the pathogenesis of AAV. Using 20 amino acid sequence peptides they identified the immunodominant MPO CD4+ T cell epitope. Subsequently they produced T cell clones which were specific for this immunodominant MPO epitope, which were then injected into mice. Using three different techniques it was demonstrated that when the MPO peptide (or whole MPO) was deposited in glomeruli focal necrotising glomerulonephritis was driven by antigen specific CD4+ T cells.[83] These key studies have helped define how effector T cells drive glomerular injury.

rather demonstrates delayed type hypersensitivity responses, including fibrin deposition. This is most likely to be a consequence of auto-reactive CD4+ effector cells recognizing MPO, which is present in glomeruli in both human and experimental ANCA vasculitis [70-72]. In addition to enhancing inflammation, regulatory T cells (Tregs) are likely to have an important role in

T cells are active participants in the loss of tolerance and the development of autoimmunity in AAV. Firstly we know that ANCAs are class switched high affinity antibodies which are (therefore) dependent on T cells for their generation.[73] Secondly, in proliferation studies, it has been demonstrated that auto-reactive T cells from patents with AAV respond to MPO and PR3,[74] while markers of T cell activity are increased in parallel with disease activity.[75-76] Furthermore, in renal biopsy samples from patients with AAV, the number of infiltrating T cells correlates with the severity of injury. Additional evidence supporting a pathogenic role for T cells was provided when 15 patients with refractory vasculitis, resistant to other therapies,

Early studies supported a role for T helper (Th) 1 (and possibly Th2) cells in the pathogenesis of AAV. Peripheral blood lymphocytes from patients with MPO-ANCA were shown to produce IFNγ when stimulated.[78] The more recently defined Th17 cells represent a distinct lineage of CD4+ T cells, which are characterized by the production of IL-17A.[79] Two key human studies supported a role for Th17 cells in ANCA vasculitis. Firstly it was demonstrated that when peripheral blood from GPA patients was stimulated with PR3, there was an increased percentage of IL-17A producing CD4+ T cells (Th17). After stimulation no difference in IFNγ production was seen, suggesting that Th1 cells were not involved. The authors proposed that this skewed Th17 response supported a role for Th17 cells in disease.[80] A subsequent study demonstrated that sera from patients with active AAV consistently dis‐ played a Th17 phenotype. Cytokines associated with Th17 cells, including IL-17A and IL-23, were increased in patients with acute AAV, while levels of IFNγ were unchanged. Interestingly immunosuppressive therapy did not consistently decrease IL-23 or IL-17 production.[23] In a study of human ANCA biopsies it has been shown that IL-17A producing CD4+ T cells constitute part of the inflammatory infiltrate and correspond with disease severity.[81] In addition, murine models have provided strong evidence for a pathogenic role for CD4+ T cells

An MPO-dependent murine model which demonstrates considerable homology to human ANCA vasculitis, where mice develop autoimmunity to MPO and focal necrotising glomeru‐ lonephritis was described. Immunization of C57BL/6 wild type mice with MPO results in cellular and humoral autoimmunity to MPO. A small dose of sheep anti-mouse glomerular basement membrane serum is subsequently administered. Treatment of chicken ovalbumin (OVA) immunized mice with this dose of sheep anti-mouse glomerular basement membrane serum does not result in significant renal injury. However in mice immunized with MPO and then sheep anti-mouse glomerular basement membrane serum significant renal injury is seen. Depletion of CD4+ effector cells significantly attenuated glomerular injury in this model, while experiments performed in B cell-deficient mice did not show renal protection.[72] These results provide strong evidence for a pathogenic role for CD4+ effector cells contributing to rapidly

were successfully treated with anti-thymocyte globulin, which targets T cells.[77]

modulating immune responses and glomerular injury.

42 Updates in the Diagnosis and Treatment of Vasculitis

in glomerulonephritis.
