EPIDEMIOLOGY, GENETIC DIFFERENCES AND CLINICAL OUTCOMES OF AUTOANTIBODY ASSOCIATED SYSTEMIC

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1 EPIDEMIOLOGY, GENETIC DIFFERENCES AND CLINICAL OUTCOMES OF ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODY ASSOCIATED SYSTEMIC VASCULITIS. A thesis submitted to The University of Manchester for the degree of Doctorate in Medicine in the faculty of Medical and Human Sciences 2011 AJAY PRABHAKAR DHAYGUDE 1

2 INDEX TABLE OF CONTENTS 2 LIST OF FIGURES 6 LIST OF TABLES 7 ABSTRACT 9 LIST OF REFERENCES INTRODUCTION: Systemic Vasculitis: Classification:...14 Figure 1: Algorithm of EMEA classification of ANCA associated vasculitis Epidemiology of AASV: Trends: Geographical differences: Aetiology of small vessel vasculitis Anti Neutrophil Cytoplasmic Autoantibodies: Genetic Factors: Environmental factors Pathogenesis of AASV: Activation of neutrophils: Endothelium and neutrophils:...25 Figure 2: Schematic representation of adhesion and transmigration of neutrophils through endothelium Respiratory burst Apoptosis: Complement in AASV: Role of Lymphocytes in AASV: T cells:...29 Figure 3: A) Binding of ANCA with its receptor and release of proinflammatory cytokines B-Cells: The Role of Cytokines in AASV: Polymorphisms of Inflammation related genes in AASV:...33 Table 1: Summary of SNP analysis studies in systemic vasculitis CCR5 and GPA (WG): New insights into pathogenesis of AASV: Neutrophil extracellular traps: Lysosomal Associated Membrane Protein

3 1.7.3 Circulating endothelial cells as potential mediators of disease Endothelial microparticles in ANCA-associated vasculitis Endothelial progenitor cells in ANCA-associated vasculitis Animal models of vasculitis: In vitro models: In vivo models: ANCA negative small vessel vasculitis: Histology: Clinical Features: Wegener s Granulomatosis: Systemic vasculitis (Features common to MPA) Limited GPA (WG): Assessment of GPA (WG): Double positive disease: canca versus panca:...52 Table 2: Differences between canca and panca associated vasculitis Treatment: Induction of remission: Maintenance of remission: Emerging therapies in treatment of AASV:...59 Figure 4: Complex interplay between B cells and T cells and potential targets for therapeutic intervention for AASV HYPOTHESIS, AIMS AND OBJECTIVES Hypothesis: Aims and Objectives: PATIENTS, MATERIALS AND METHODS: Patients: Material And Methods: DNA Extraction, Measurement and Dilution: DNA Measurement and dilution: Taqman PCR technique:...73 Figure 5: Amplification of Target Sequence Figure 6: Allelelic discrimination showing homozygote for alleles X and Y and heterozygote (XY) PCR Methods:...76 Table 3: Details of the genes, SNP positions, RS numbers and ABI kit used for the PCR ELISA Assay for IL10 and IL18: IL 10 Assay: IL 18 Assay: Metabolomics: Sample Preparation: Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) analysis: Data pre-processing: Data analysis: Metabolite Identification: Gene Expression: Collection of whole blood for RNA isolation:

4 3.4.2 RNA concentration and analysis: Gene Expression Chips: EPIDEMIOLOGY OF PAUCIIMMUNE NECROTISING CRESCENTIC GLOMERULONEPHRITIS IN SOUTH-EAST US AND GREATER MANCHESTER UK Introduction: Patients and Methods: Population base (GDCN): Population base (GM): Study Cohort (GM): Statistics: Renal outcomes: Results: Demographics (GM): Ethnicity: (GM):...92 Table 8: Demographics data of Greater Manchester and USA populations...93 Table 9: Incidence of PIGN expressed as cases/million population Annual incidence of PIGN:...94 Figure 7: Changing pattern of incidence in Greater Manchester ANCA serology (GM):...95 Table 10: Details of ANCA serology Renal outcomes (GM):...96 Table 11: Renal Outcomes at the last follow up based on the creatinine at the time of diagnosis for Greater Manchester Study Discussion:...98 Table 12: Reported incidence of PIGN ROLE OF CYTOKINES AND CYTOKINE POLYMORPHISMS AND CYTOKINE PROFILE IN ANCA ASSOCIATED SMALL VESSEL VASCULITIS Introduction: Cytokine: Figure 8: Role of cytokines in the development of autoimmune diseases Single Nucleotide polymorphisms: Cytokine SNPs in AASV: MPO and PR-3 ANCA associated vasculitis: Tumour Necrosis Factor Alpha (TNFα): Actions on Endothelial cells: Actions on neutrophils: Other actions of TNF in the process of inflammation: TNF and AASV: Figure 9: Role of TNF in inflammation Interleukin-8: Figure 10: Biological actions of IL IL-8 and AASV: IL-8 SNPs: Transforming Growth Factor β: CC Chemokine Ligand 5(CCL5):

5 5.5.1 CCL5 and AASV: Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1): Functions of PECAM-1: Interleukin 10 (IL-10): Role of IL-10 in autoimmune disease: IL-10 and AASV: Interaction between PR3 and MPO: IL-10 and SNPs: Table 13: Summary of IL-10 SNPs study in AASV patients Interleukin 18: Figure 11: Different mechanisms for activation of IL-18 with cleavage of pro IL-18 by caspase-1, Chymase, ANCA antigens and Granzyme-B IL-18 Binding protein: Free IL-18: IL-18 and autoimmune diseases: IL-18, IL-17and T H 17 cells; role in AASV: Il-18 and AASV: Il-18 SNP: Table 14: Summary of IL-18 SNPs association with autoimmune diseases Results of clinical characteristics: Patient characteristics: Table 15: ANCA serology by Immunoflouroscence (IIF) Clinical Characteristics: Table 16: Clinical diagnosis of 147 AASV patients Figure 13: Clinical diagnosis of 147 AASV patients Organ involvement: Table 17: Details of organ involvement due to AASV Details of renal function impairment: Table 18: Details of the severity of renal impairment of patients with creatinine greater than 120 µmol/l and not on dialysis Treatment details: Outcome details: Results of Laboratory experiments: Genotype results: TNF α: Table 19: Genotype and allele frequencies of TNFα SNPs IL-8: Table 20: Genotype and allele frequencies of IL-8 SNPs TGFβ: Table 21: Genotype and allele frequencies of TGFβ -800 A/G SNP CCL-5: Table 22: Genotype and allele frequencies for CCL-5-403C/T SNP PECAM C/G SNP: Table 23: Genotyping and allele frequencies of PECAM C/G SNP IL-10: Table 24: Genotype and allele frequencies of IL A/G SNP IL-18:

6 Table 25: Genotype and allele frequencies of IL-18 SNPs Comparison of genotype and allele frequencies between ANCA positive patients and healthy volunteers TNFα -857 C/T SNP: Table 26: Genotype and allele frequencies of TNFα -857 C/T SNP in healthy volunteers and AASV patients PECAM-1 125C/G SNP: Table 27: Genotype and allele frequencies of PECAM C/G SNP in healthy volunteers and AASV patients IL A/G SNP: Table 28: Genotype and allele frequencies of IL A/G SNP in healthy volunteers and AASV patients IL G/T SNP: Table 29: Genotype and allele frequencies of IL G/T SNP in healthy volunteers and AASV patients IL G/T SNP: Table 29: Genotype and allele frequencies of IL G/T SNP in healthy volunteers and AASV patients IL G/A SNP: Table 30: Genotype and allele frequencies of IL G/A SNP in healthy volunteers and AASV patients IL C/G SNP: Table 31: Genotype and allele frequencies of IL C/G SNP in healthy volunteers and AASV patients Summary of results 1: Genotype and allele frequencies and relapses: Results of genotype and allele frequencies of PECAM C/G SNP and association with relapse: Table 32: Genotype and allele frequencies of PECAM C/G SNP and relapses in AASV Table 33: Genotype and allele frequencies of PECAM C/G SNP and relapses in canca positive patients Table 34: Genotype and allele frequencies of PECAM C/G SNP and relapses in panca positive patients Results of genotype and allele frequencies of IL A/G and IL G/T SNPs and association of relapse: Table 35: Genotype and allele frequencies of IL A/G SNP and relapses in AASV patients Table 36: Genotype and allele frequencies of IL A/G SNP and relapses in canca positive patients Table 37: Genotype and allele frequencies of IL A/G SNP and relapses in panca positive patients Table 38: Genotype and allele frequencies of IL G/T (127 C/T) SNP and relapses in AASV patients Table 39: Genotype and allele frequencies of IL G/T (127 C/T) SNP and relapses in canca positive patients Table 40: Genotype and allele frequencies of IL G/T (127 C/T) SNP and relapses in panca positive patients Renal outcomes and Genotype association:

7 Table 41: All AASV patients data regarding IL A/G SNP and development of ESRD Table 42: IL A/G SNP and development of ESRD in canca positive patients Table 43: IL A/G SNP and development of ESRD in panca positive patients Validation of IL-18 SNP in a different population Table 44: Genotype and allele frequencies of IL A/G SNP in combined UK and USA populations Table 45: Genotype and allele frequencies of IL C/T SNP in combined UK and USA populations Circulating cytokine profiles in AASV patients and normal volunteers: Results of circulating IL-10 levels: Table 47: IL-10 levels in patients with active vasculitis, remission state and healthy volunteers Figure 14: Plasma levels of IL-10 in active and remission state of AASV patients and healthy controls IL-10 levels in active versus remission state: IL-10 levels in canca versus panca positive patients: Figure 15: Plasma levels of IL-10 in panca positive, canca positive patients and healthy controls IL-10 levels in paired samples: Table 48: Paired plasma samples during active vasculitis and remission state for circulating IL-10 levels Figure 17: Paired plasma samples during active vasculitis and remission state for circulating IL-10 levels Correlation with IL A/G SNP and IL-10 levels: Table 49: Relation between circulating IL-10 levels and IL A/G SNP Figure 18: Relation between circulating IL-10 levels and IL A/G SNP IL-18 levels in active versus remission state: Table 50: Mean levels of IL-18 in active and remission state of AASV and normal volunteers Figure 19: Plasma levels of IL-18 in active and remission state of AASV patients and healthy controls IL-18 levels in canca versus panca positive patients: Figure 20: Plasma levels of IL-18 in panca positive, canca positive patients and healthy controls IL-18 levels in paired samples: Table 51: IL-18 levels in eleven paired samples during active vasculitis and remission state Figure 21: IL-18 levels during active vasculitis and remission state in paired samples Table 52: Relation between circulating IL-18 levels and IL C/T SNP Figure 23: Relation between circulating IL-18 levels and IL C/T SNP Summary of ELISA results:

8 5.22 Discussion: TNFα -857C/T: PECAM C/G: IL /AG SNP: IL-18 SNPs: STUDY OF METABOLOMICS AND GENE EXPRESSION IN ANCA ASSOCIATED VASCULITIS Metabolomics: Background: AASV and metabolomics: Material and methods: Results of metabolomics: Figure 24: Separation of canca versus panca using interesting peaks from principal component analysis Figure 25: Separation of PR-3 versus MPO using interesting peaks from principal component analysis Figure 26: Distribution of phenotypic diagnosis of AASV using metabolomics Differences in metabolites between canca and panca positive patients: Figure 27: Different metabolomic profile of active and remission state of vasculitis Figure 28: Different metabolomic profile of active and remission state of vasculitis Discussion: Gene expression: Background: Principles of gene arrays: Gene expression and AASV: Material and Methods: Results: Discussion: DISCUSSION Epidemiology Study Limitations of epidemiology study: Genetics Study: Limitations of genetics study and future direction of research: Metabolomics and gene expression study: Limitations of metabolomics and gene expression study: What is in the future of AASV?: BIBLIOGRAPHY

9 ABSTRACT Introduction: The two subtypes of Antineutrophil Cytoplasmic autoantibody associated systemic vasculitis (AASV) canca and panca associated vasculitis are the commonest causes of rapidly progressive glomerulonephritis. In spite of recent advances in the pathogenesis and development of new therapeutic agents, long term outcomes are still poor with five year mortality of 25%. There are epidemiological, histological, clinical and outcome related differences between these two conditions. This strongly suggests that there must differences between genetic factors and pathogenesis of these two conditions. There was also a perception amongst the clinicians that AASV is more common in Greater Manchester area. Hence in this study I calculated the incidence of pauciimmune glomerulonephritis in Greater Manchester and analysed the genetic differences between canca and panca associated vasculitis. Methods: Five year incidence of pauciimmune glomerulonephritis was calculated in Greater Manchester between 1/1/1999 to 31/12/2003. I recruited 147 patients with ANCA associated vasculitis. Clinical data was collected. I studied single nucleotide polymorphisms (SNPs) of tumour necrosis factor alpha (TNFα), interleukin 8 (IL-8), transforming growth factor beta (TGFβ), platelet endothelial cell adhesion molecule 1 (PECAM-1), Chemokine (CC motif) ligand- 5 C chemokine (CCL-5), interleukin 10 (IL-10) and interleukin 18 (IL-18) genes and compared the frequencies of genotypes and alleles in patients with canca and panca associated small vessel vasculitis and healthy volunteers. I also studied circulating cytokine profiles of IL-10 and IL-18. Results of IL-18 SNPs were validated in AASV cohort from South-East USA. Further I studied the gene expression patterns of active and remission state of AASV and metabolomics profile of canca and panca positive patients during active and remission state of vasculitis. Clinical outcomes (relapses and renal survival) were correlated with the genotypes. Results: I found a significantly higher incidence (9.8/million population) of pauciimmune glomerulonephritis in Greater Manchester compared to the previously published data from UK and USA (2.73 to 4.6/million). Renal function at the time of diagnosis predicted the long term renal survival. I also found a novel genetic association of increased frequency of high producer IL-18 SNPs 113T, 127C and 137G in panca positive patients compared to normal volunteers (p=0.04) and canca positive patients (trend- p=0.08). This was associated with increased levels of circulating IL-18 levels in these patients. This association was further confirmed in an independent cohort of AASV from USA. I also found a lower frequency of low producer GG genotype of IL SNP (p=0.05) and this was associated with lower levels of circulating IL-10 in these patients compared to panca positive patients. I found significant difference in the metabolomics profiles of canca and panca positive patients. In paired plasma samples, levels of some metabolites were high during remission state compared to active vasculitis. Conclusions: These findings strongly support the hypothesis that there is an increased incidence of pauciimmune glomerulonephritis in Greater Manchester. There are genetic differences in canca and panca positive patients which may explain the different observed outcomes. Genomewide association study would strengthen these findings and should guide the vasculitis community to reclassify, assess and perhaps treat these two conditions separately. 9

10 DECLARATION I declare that no portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. COPYRIGHT STATEMENT Copyright in this thesis rests with the author. Copies (by any process)either in full or of extracts may be made only in accordance with the instructions given by the Author and lodged in the John Rylands University Library of Manchester. Details may be obtained from the Librarian. This page must form part of any such copies made. Further copies (by any process) of copies made in accordance with such instructions may not be made without the permission (in writing) of the Author. The ownership of intellectual property rights which may be described in this thesis is vested in the University of Manchester, subject to any prior agreement to the contrary, and may not be made available for use by third parties without the written permission of the University, which will prescribe the terms and conditions of any such agreement. Further information on the conditions under which disclosures and exploitations may take place is available from the head of department of the Manchester Institute of Nephrology and Transplantation at Manchester Royal Infirmary and Head of Medical School. 10

11 To my dear parents and wife for their continued love, support and prayers 11

12 ACKNOWLEDGEMENTS I would like to thank Department of Renal Medicine at Salford Royal Hospital, Renal Unit Endowment fund at South Manchester University Hospitals and Renal Unit Endowment fund at Manchester Royal Infirmary for funding my project. Special thanks are given to my supervisors Professor Brenchley, Dr Venning and Professor Kalra and also my advisor Professor Bruce. I gratefully acknowledge Dr Solomon and Dr Graddon for assistance with recruitment of patients at Royal Preston Hospital and at University Hopsital of Aintree. Special thanks are given to Shelley Harris for assistance with the laboratory methods and laboratory work, statistics and proof reading. I would like to thank Dr Dunn for assitance in the metabolomics project and Dr Hoyle for assistance with the gene expression project. I would like to thank all the Histopathologists in the North West regions for their assistance in providing renal biopsy database for the epidemiology study. I would also like to thank Linda Strickland for helping me with the proof reading. Finally, I would like to thank all the patients who participated in this study. 12

13 1.0 INTRODUCTION 13

14 1.0 INTRODUCTION: 1.1 Systemic Vasculitis: Vasculitis is usually a systemic multi-organ disease and clinical syndromes of vasculitis are heterogeneous. Inflammation of blood vessels both arteries and veins of different calibres and capillaries affecting various organs is common feature. It is associated with inflammatory infiltrate in some or all layers of vessel walls resulting in necrosis and swelling. Healing is associated with fibrosis and compromise of vessel lumen. Vasculitis can be primary or secondary to other pathologic process like connective tissue disorders, infection, malignancies or drugs. It may be associated with deposition of immune complexes in tissues, where immune complexes are absent it is also known is pauciimmune disease. Clinical presentations vary according to size of blood vessels involved and histopathological features. Small to medium size blood vessel systemic involvement without immune complex deposits typically occurs in a triad of Granulomatosis with polyangiitis (GPA) which was previously known as Wegener s Granulomatosis (WG), Microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA) which was previously known as Churg Strauss syndrome (CSS). These potentially life threatening diseases are usually associated with production of anti neutrophil cytoplasmic auto-antibodies (ANCA) and are often described as ANCA associated systemic vasculitis (AASV) Classification: Heterogeneous nature of different vasculitic syndromes and heterogeneous presentations of the same syndrome in different patients has posed a challenge in the classification of vasculitis. The first classification of vasculitis was proposed by The American College of Rheumatology (ACR) in

15 (Hunder et al.). Over a period of 5 years rheumatologists from 48 centres in North America submitted details of 807 patients with vasculitis. Clinical findings which both identified the disease and separated it from others were used to classify diseases. For example to classify GPA (WG), features of 85 patients with GPA (WG) were compared with that of 722 patients with other forms of vasculitis and four criteria were selected; abnormal urinary sediments, abnormalities on chest radiograph, granulomatous inflammation on biopsy and oral ulcers or nasal discharge. This was a clinical features based, research orientated classification. The reliability of these criteria when used in a patient suspected of vasculitis but not yet diagnosed is poor (Rao, Allen, and Pincus). From a nephrologists prospective it did not differentiate between microscopic polyangiitis with crescentic glomerulonephritis and classic polyarteritis nodosa. Subsequently the Chapel Hill Consensus Conference (CHCC), in 1994 proposed a nomenclature method, based on the size of blood vessels involved, clinical and histological parameters (Jennette et al.). This nomenclature provided useful definitions of vasculitic syndromes but was not intended to diagnose or classify patients with vasculitis. It has been widely accepted but it relies heavily on histological criteria which are not always available. Surrogate alternatives for histological evidence have been proposed but are not well defined. More recently in 2006, The European Medical Agency (EMEA) has proposed another classification algorithm which takes in to consideration both clinical and laboratory criteria (Watts et al.) (Fig. 1). This algorithm accepts the Lanham and ACR criteria for diagnosis of CSS. Lanham et al in 1984 propoed that EGPA (CSS) is underdiagnosed as classical histological picture is found in a minority of cases. They proposed clinical criteria comprising of allergic rhinitis, nasal 15

16 polyposis, sinusitis along with essential features of asthma and peripheral eosinophilia (Lanham et al.). They also highlighted the close relation between EGPA (CSS) and other diseases charectarised by eosinophilia and granulomatosis. For the diagnosis of GPA (WG), in absence of ACR and CHCC criteria it proposes surrogate markers such as radiological evidence of granuloma. MPA is diagnosed when clinical parameters and histology are compatible with small vessel vasculitis with the absence of surrogate markers for WG and includes patients with renal limited vasculitis. It remains to be seen if the EMEA algorithm will replace the existing Chapel Hill Consensus Conference nomenclature. Accurate definitions are essential for epidemiological studies and the EMEA algorithm may provide widely acceptable definitions in vasculitis. 16

17 Figure 1: Algorithm of EMEA classification of ANCA associated vasculitis. (Printed with kind permission of BMJ publications, Watts et al, Ann. Rheum Dis. Feb :2; ) 17

18 1.2 Epidemiology of AASV: Epidemiological studies of vasculitis are limited and earlier studies especially of MPA and classic polyarteritis nodosa related studies need careful interpretation because of the change in the definitions used to classify vasculitis. In spite of these differences, there are clear geographic differences in the incidence of GPA (WG) and MPA across latitude. The majority of studies are performed in Europe though more data are emerging from China, Japan and North America Trends: There is a broad consensus that the incidence of AASV is increasing. An early study, in 1970 from Bath and Bristol, UK, states the incidence to be 10/million (Scott et al.). Studies from late 80s and early 90s from Norwich have revealed the collective incidence of GPA (WG), MPA and EGPA (CSS) to be 13.3/million (Watts, Carruthers, and Scott). Certainly the introduction of ANCA has increased the awareness of vasculitis in the medical community; increased fitness of the ageing population with changes in clinical practice may explain the apparent rise in the incidence over last three decades. Peak age of onset of AASV is years and it is rare in childhood (Gardner- Medwin et al.) Geographical differences: GPA (WG) is more common in Northern Europe Norway; while MPA is more common in Southern Europe- Spain (Watts et al.). The highest recorded prevalence of AASV is in Southern Sweden (257/million) and the lowest in France (90/million) (Mohammad et al.). A study in a multiethnic, urban population from France revealed a significant difference in prevalence between the white Caucasian (104.7/million) and non Caucasian 18

19 (52.5/million) population. In Japan, although the incidence is similar to that of Europe, ear, nose and throat (ENT) and neurological involvement is less common and majority of the patients are MPO-ANCA positive (Watts et al.). A very high annual incidence of 24/million was reported from Kuwait (El-Reshaid et al.). 1.3 Aetiology of small vessel vasculitis The exact aetiology of AASV is not known. Like many other autoimmune diseases it is likely to be multifactorial and polygenic in nature. A complex interplay between ANCA, predisposing genetic factors and environmental triggers is likely to result in the vasculitic disease process Anti Neutrophil Cytoplasmic Autoantibodies: ANCA were first described by Davies et al in 1982 (Davies et al.). He described a series of eight patients who were generally unwell and presented with focal necrotising glomerulonephritis, weight loss and arthralgia. He describes the presence of a factor, predominantly IgG, in the serum of these patients that stained the cytoplasm of neutrophils. Retrospective serology also confirmed evidence of a past arboviral infection in seven out of eight patients. He further describes the disappearance of this factor a few weeks after starting immunosuppressive treatment in most of the patients. Subsequently in 1985, van der Woude FJ proposed ANCA as a marker of active Wegener s Granulomatosis (van der Woude et al.). Jennette et al demonstrated target antigens as neutrophil components MPO and PR-3 and coined the term panca (MPO ANCA) and canca (PR-3 ANCA) associated vasculitis. (Jennette, Wilkman, and Falk). 19

20 Neutrophils and monocytes when fixed with ethanol, demonstrate either classic diffuse granular cytoplasmic (c-anca) or perinuclear (p-anca) staining pattern in the presence of ANCA. Enzyme linked immunosorbent assay (ELISA) analysis reveals c-anca to be specific for proteinase 3 (PR-3) and p-anca to be specific against myeloperoxidase (MPO), two enzymes which are stored in azurophilic granules of neutrophils and monocytes. p-anca are often detected in non vasculitic conditions such as infections, liver disease, inflammatory bowel disease and are usually against other antigens like the bacterial permeability protein, lactoferrin. Proteinase -3 is a neutral serine proteinase and is stored in α granules, specific granules and secretary granules of neutrophils (Witko-Sarsat et al.). Alpha-1 antitrypsin is physiological inhibitor of PR3. Deficiency of alpha-1 antitrypsin (PiZZ genotype) has been reported to be more frequent in AASV patients compared to normal population (8% versus 1.5%). This low producer genotype was also found to be associated with poor prognosis, disseminated disease and increased mortality with PR-3 positive vasculitis (Callea et al.). Myeloperoxidase in neutrophils facilitates the formation of hypochlorous acid, a strong reducing agent and defence mechanism against micro organisms. c-anca is predominant in GPA (WG) patients (70%) while p-anca is predominant in EGPA (CSS) (60%) with approximately equal distribution in MPA(40% c-anca, 50 % p-anca). Five to 30% of patients with GPA (WG), MPA, and EGPA (CSS) are ANCA negative. A change in ANCA titres over time usually correlates with disease activity but ANCA titres are not infallible and a progressive rising titre requires close monitoring of a patient for possible relapse(boomsma et al.) (Tervaert et al.). Meta-analysis of 15 studies has 20

21 revealed that a positive c-anca by indirect immunofluorescence (IIF) has a sensitivity of 34 to 92% in the diagnosis of WG and a specificity of 88 to 100% (Rao et al.). IIF when combined with ELISA increases the specificity of diagnosis to nearly 100% when compared with healthy controls (Hagen et al.). The role of ANCA in the pathogenesis of systemic vasculitis is debated. Up to 30% of patients with GPA (WG), particularly the limited form, are ANCA negative. ANCA titre doesn t always correlate with disease activity and a persistent positive ANCA titre without clinical evidence of disease activity is sometimes described as titritis. A clinical trial of the prospective treatment with immunosuppression in patients with a four fold raised ANCA titre demonstrated a decrease in relapses with pre emptive immunosuppressant treatment but lead to over treatment of some patients (Tervaert et al.). Animal models using an MPO knock-out mouse, immunised against human MPO, produced an anti MPO antibody. Transfer of these antibodies to another mouse resulted in the development of vasculitis (Xiao et al.). Placental transfer of IgG anti MPO antibody and development of pulmonary renal syndrome in a new born has been described (Schlieben et al.). Direct evidence for the use of anti PR-3 antibody in the treatment of vasculitis is lacking. There is no doubt that introduction of ANCA has highly influenced diagnosis and monitoring of AASV Genetic Factors: There is no evidence that AASV is a single gene disorder. While there have been sporadic case reports of familial clustering of cases of AASV (Hay et al.) a large 21

22 North American study of 701 patients including 12 sets of twins revealed no occurrence of GPA (WG) in the other twin and a low relative risk of developing vasculitis in a first degree relative(abdou et al.). Associations with the HLA system have generally been negative with the exception of a Japanese study of 69 patients with MPO +ve vasculitis which showed a significant association with HLA DRB1*0901 (Tsuchiya et al.) and a German study which showed over expression of HLA-DPB 1*401 (Jagiello et al.). In one study, the short arm of Chromosome 6 was shown to have several haplotypes with a strong association with PR3+ve vasculitis (Gencik et al.). There are several association studies performed looking at single nucleotide polymorphisms (SNPs) and susceptibility or clinical outcomes of AASV. SNPs in the genes encoding IL-10, CTLA4, CD18 and PTPN22 have been confirmed to associate with AASV, in several studies (Bartfai et al.) (Spriewald et al.) (Gencik et al.) (Jagiello et al.) Environmental factors: It has always been thought that an environmental trigger is required on the background of genetic predisposition for developing vasculitis. Data regarding role of environmental factors triggering onset of vasculiti is limited. Few studies have confirmed association with silica exposure and Lane et al showed significant association with occupational exposure to silica and farming to be associated with developing AASV (Lane et al.). Hogan et al alo confirmed the association of silica exposure in the development of AASV (Hogan et al.). Interestingly smoking may reduce the risk of developing AASV (Haubitz et al.). The influence of environmental factors may partly explain clustering of cases in families. 22

23 Drugs like propylthiouracil and minocycline are clearly associated with production of ANCA and the development of vasculitis (Harper et al.). Nasal carriage of Staphylococcus aureus has been associated with relapse in GPA (WG) (Stegeman et al.). There have been case reports of vaccination triggering onset of AASV (Birck et al.) (Konishi et al.). 1.4 Pathogenesis of AASV: The exact pathogenesis of AASV is not known. Most of the understanding is based on in vitro experimental data and it has been difficult to develop animal model of AASV Activation of neutrophils: In vitro data suggests that neutrophils are initially preactivated or primed with low titres of proinflammatory cytokines like TNF-α, IL-1 and IL-8. The source of proinflammatory cytokines in vivo is presumed to be the result of low grade infection or inflammation. Priming of neutrophils results in translocation from the granules to the cell surface of PR3 and MPO, which are target antigens for ANCA (Charles et al.) (Csernok et al.). Expression of PR3 in neutrophils is highly variable within the population and is genetically determined (Schreiber et al.). Increased neutrophil membrane PR-3 expression was found to be significantly more common in AASV patients compared to the healthy population suggesting its role in increasing susceptibility to devlope AASV (Witko-Sarsat et al.). Further more it has also been shown that in AASV patients, those with high membrane P-3 expression are at increased risk of relapse (Rarok et al.). 23

24 The interaction of ANCA with its antigen results in the activation of neutrophils. Activated neutrophils are found in the circulation of patients with active vasculitis and the degree of neutrophil activity correlates with disease activity (Muller Kobold et al.). For activation of neutrophils, not only is binding of Fab-2 fragments of ANCA IgG to MPO or PR-3 essential but also the interaction between Fc fragments with Fc receptors on the neutrophils. It has been reported that binding of FCγ receptors is crucial for activation of neutrophils and blocking of these receptors inhibits ANCA induced superoxide production (Ben-Smith et al.). It has been shown that the IgG3 subclass of ANCA interacts efficiently with Fcγ RIIa receptor and low levels of IgG3 ANCA in ANCA positive patients is associated with clinical remission. SNP studies of Fcγ RIIa have revealed that phenotypes with a high affinity for the ANCA IgG3 subclass are associated with more severe disease and disease relapse (Edberg et al.) (Dijstelbloem et al.) (Tse et al.) although a large prospective study showed that assessment of the IgG3 subclass of ANCA was no better in predicting disease relapse than total IgG PR3 ANCA (Boomsma et al.). The interaction of ANCA with monocytes is less well studied but they are presumed to have an important role in the pathogenesis of AASV. Upregulation of adhesion molecules and enhanced generation of reactive oxygen species from circulating monocytes from patients with AASV has been demonstrated (Bruce, McNally, and Bell). Production of the potent chemoattractant monocyte chemoattractant protein -1 (MCP-1) and interleukin-8 (IL-8) from monocytes has also been proved, suggesting role of monocytes in the pathogenesis of AASV (Casselman et al.) (Ralston et al.). 24

25 1.4.2 Endothelium and neutrophils: The binding of FCγ receptors and MPO/PR3 with ANCA result in the activation of neutrophils and release of reactive oxygen products. Activation of neutrophils is more efficient when they are attached to the endothelium. Adherence of neutrophils to endothelial surface is facilitated by the expression and activation of adhesion molecules on neutrophils and endothelial cells. Increased expression of both β1 (CD29) and β2 integrins (CD18) has been described on both neutrophils and monocytes isolated from patients with GPA (WG) compared with normal controls (Haller et al.). Increased levels of circulating adhesion molecules (intracellular adhesion molecules ICAM and vascular cell adhesion molecules VCAM) have been described in the sera of patients with GPA (WG) (Mrowka and Sieberth) (Mrowka and Sieberth). PR-3 is the major autoantigen in GPA (WG) and has been shown to bind to endothelial cells (Taekema-Roelvink et al.) and incubation of human umbilical vein endothelial cells (HUVEC) with PR3 results in detachment and lysis of endothelial cells. PR3 is also known to induce the production of IL-8, a strong chemotactic and activating factor for neutrophils (Berger et al.). Further studies have confirmed that PR3 to induce the expression of MCP-1 and ICAM-1 both at the protein and m-rna level (Taekema-Roelvink et al.). Incubation of HUVEC with PR3 for 24 hours significantly increases adhesion of neutrophils and this can be blocked by monoclonal antibodies to ICAM-1 or CD18 (Taekema-Roelvink et al.). Adhesion of neutrophils to the endothelium is assumed to occur in the small vessels and biopsies from patients with crescentic GN have revealed activated neutrophils adherent to endothelium (Brouwer et al.). In vitro flow models using intra-vital microscopy show clear interaction between neutrophils, endothelial cells and ANCA (Fig-2). After 25

26 treatment with TNF α endothelial cells capture neutrophils from flow and support rolling adhesion via selectin molecules (P, E and L selectins). Perfusion of ANCA over rolling neutrophils induces stationary adhesion of neutrophils to the endothelium and ten fold increased transmigration. Both transmigration and adhesion are β2 integrin dependent but transmigration alone can be reduced by blocking of chemokines receptor CXCR2 (Radford et al.) (Smith et al.) (Calderwood et al.). Figure 2: Schematic representation of adhesion and transmigration of neutrophils through endothelium. (Printed with kind permission of Elsevier Limited T. Pankhurst et al. Current Opinion in Pharmacology : ) It has been demonstrated that PR3 and human neutrophil elastase (HNE) can directly enter endothelial cells and stimulate the apoptotic pathway through an extracellular signal regulated kinase like Jun N- terminal kinase and p38 MAP 26

27 protein kinase. Entry of these proteases in the endothelial cells results in the cleavage of p65 nuclear factor κ B and diminishes its transcription. Furthermore these processes of cleavage of p65 nuclear factor κ B are not inhibited by caspases. This shows that proteases like HNE and PR3 play a specific role in the cross talk between endothelial cells and neutrophils and PR-3 can control the fate of endothelial cells (Preston et al.). As in vitro studies suggest detachment of endothelial cells when incubated with PR3, attempts have been made to isolate circulating endothelial cells as a possible marker of active vasculitis. In one study, the number of circulating endothelial cells was significantly higher in the active vasculitic state, compared with healthy controls, other glomerulonephritis and infection (Woywodt et al.). In this study with a small number of patients the number of endothelial cells had a higher sensitivity and specificity for the diagnosis of AASV, compared with ANCA. Furthermore a declining number of circulating endothelial cells was seen during clinical remission and may therefore, provide a useful marker of vasculitic activity in planning treatment. Autoantibodies to circulating endothelial cells (AECA) have been found in patients with AASV and other vasculitic conditions and may offer evidence of further mechanisms of tissue injury in vasculitis. They are a heterogeneous group of antibodies and are not specific to endothelial cells but also react against fibroblasts. They have been isolated from various vasculitic syndromes and are not specific to AASV. The titre of AECA does reflect disease activity but are a common feature to vasculitic syndromes which suggests pathogenic role. The antibodies react against constitutively expressed antigens on endothelial cells. HUVEC when incubated with AECA, show increased expression of adhesion molecules, chemokines and cytokines and may promote 27

28 leukocyte recruitment across endothelium (Carvalho et al.). In vitro, AECA have also been shown to mediate complement and cellular dependent cytotoxicity against a cultured monolayer of endothelial cells (Del et al.). Further studies suggest that AECA promote thrombogenic events with increased production of tissue factor and von Willebrand factor (Frampton et al.). A definitive animal model supporting the pathophysiological role of AECA in the development of vasculitis has not yet been discovered but injecting autoantibodies to known endothelial antigens like angiotensin converting enzyme and factor VIII-von Willebrand complex, results in pulmonary and renal vasculitis in rats and rabbits (Belizna and Tervaert). These findings are suggestive of a role in the pathogenesis of vasculitis Respiratory burst: Activation of neutrophils by ANCA is not fully understood but it is likely to involve two separate pathways of signal transduction leading to oxidative burst. The F(ab2) portion of ANCA binds with MPO or PR3 and activates a G protein coupled receptor pathway resulting in the binding of Fcγ receptor with Fc portion of ANCA which leads to the activation of Syk kinase, protein kinase C and calcium release pathway Fig.4 (Williams et al.;hewins et al.). However, Lu et al proposed that EC release adenosine, which inhibits the respiratory burst of ANCA activated neutrophils and protects the endothelial surface from the potentially deleterious effects of superoxide (Lu et al.). This observation lead the authors to conclude that ANCA induced superoxide release was an unlikely mechanism for the EC injury in vivo. 28

29 1.4.5 Apoptosis: The rate of apoptosis is found to be higher in neutrophils from patients with AASV compared to healthy controls and apoptotic neutrophils highly express MPO and PR3. This in turn binds with ANCA and may promote their uptake by macrophages (Harper et al.). In in vitro studies where neutrophils are primed with ANCA and mixed with macrophages they show increased apoptosis and secondary necrosis however, after 12 hours the phagocytic activity appears to be diminished. This lack of safe clearance of necrosed neutrophils may contribute to local tissue damage (Harper et al.) Complement in AASV: Direct evidence for the involvement of the complement system in the pathogenesis of AASV is lacking (Mathieson and Peters). Complement factor C3dg has been shown to interact with MPO and may be important for the clearance and localisation of MPO at the inflammatory locus (Segelmark et al.). An increased gene frequency of C3F has been reported in the PR3+ ANCA subgroup (Persson et al.). 1.5 Role of Lymphocytes in AASV: The majority of research into AASV has been focused on neutrophils and ANCA, however, the role lymphocytes is emerging T cells: Immunohistological studies as early as 1983 showed involvement of T cells in perivascular lymphoid infiltrate in patients with GPA (WG) (Gephardt, Ahmad, and Tubbs). Isotype switching between the two predominant forms of ANCA in AASV, IgG1 and IgG4 is dependent on IL-4, a T cell cytokine which suggests that the antibody production is T cell dependent. 29

30 Furthermore, disease activity in GPA (WG) correlates well with concentration of IL-2, which is a marker of T cell activation (Schmitt et al.). T cells from patients with AASV, whether active or in remission, when cultured with ANCA antigens (PR-3 and MPO) show significant proliferation (King et al.). The cytokine secretion profile of T cells from granulomatous inflammatory sites in GPA (WG), when compared with that of peripheral T cells, shows that a T helper-1 (T H 1 ) pattern predominates (Csernok et al.). Other studies have also confirmed increased production of IFNγ by T cells in patients with GPA (WG), suggesting T H 1 pattern (Ludviksson et al.). T cell activation requires the help of a costimulatory pathway. In GPA (WG) patients, it has been shown that the expression of CD28 is significantly decreased and the expression of B7 is raised compared to normal (Moosig et al.). CD28 costimulation is associated with Th2 response but its absence T cells default to a Th1 response (Webb and Feldmann). These studies strongly suggest a role for T cells in the pathogenesis of AASV but the exact mechanism is unknown. Understanding the mechanisms resulting in the loss of tolerance in AASV may be of importance in prognosis and the development of new immunotherapies. 30

31 Figure 3: A) Binding of ANCA with its receptor and release of proinflammatory cytokines. B) Initiation of two separate pathways of signal cascade by ANCA IgG F(ab2) and Fc portion. C) Transmigration of neutrophil and release of proteolytic granules and superoxide. (With kind permission of The Biochemical Society, J. Williams et al. Clinical science 2005 I ) 31

32 1.5.2 B-Cells: The role of ANCA in pathogenesis AASV has been reviewed above (Section 1.3.1). B cells are direct precursors of antibody producing plasma cells. Mathieson et al showed that B cells from vasculitis patients produced auto antibodies against MPO and monitoring of sequential samples showed reduction in antibody production with immunosuppressive therapy (Mathieson, Lockwood, and Oliveira). In addition, B cells are likely to contribute to the pathogenesis of AASV by other mechanisms such as co-stimulation and antigen presentation within granuloma (Voswinkel et al.). B cells are highly effective in antigen presentation and cytokine production (IL6, IL10 and TNFα). It has been shown that there is an increased level of B cell activating factor (BAFF) in patients with GPA (WG) and it inversely correlates with ANCA titre (Krumbholz et al.) (Bader, Koldingsnes, and Nossent). 1.6 The Role of Cytokines in AASV: Cytokines and chemokines play a crucial role in any inflammatory and immune mediated processes. Infiltration of neutrophils and monocytes in renal tissue, oxidative burst and release of reactive oxygen species (ROS) start a cascade of pro-inflammatory cytokines. TNFα has been shown in several in vitro studies to prime neutrophils, a crucial step in the pathogenesis of AASV (Falk et al.) (Rarok, Limburg, and Kallenberg). However, in the presence of TNFα blocking antibodies neutrophils can be primed with other cytokines such as IL-18 (Hewins et al.). Plasma or serum levels of IL-18 are also increased in patients with AASV, irrespective of disease activity (Hultgren et al.). Urinary excretion of several cytokines such as TNFα, IL-6, IL-8 is increased in active vasculitis compared to the remission state (Tesar et al.) (Tesar et al.). Urinary levels of MCP-1 have also been shown to correspond with 32

33 disease activity in renal vasculitis and can serve as a non-invasive biomarker (Tam et al.) Polymorphisms of Inflammation related genes in AASV: Single nucleotide polymorphisms (SNPs) are the most common genetic abnormality, occurring genomewide with a frequency of about 1 per kb. Replacement of one nucleotide with other may have a functional significance in that it may alter the rate of gene transcription and therefore alter protein synthesis or if it falls within the coding sequence it may cause an amino acid change which may alter the function of the mature peptide. Several association studies have been published in human diseases including AASV. In AASV focus has been mainly been on inflammation related genes with respect to the pathogenesis of the disease. A summary of these SNPs study is presented in Table: 1 33

34 Table 1: Summary of SNP analysis studies in systemic vasculitis Gene/SNP Ref Disease Description IL A/G (Bartfai et al.) WG and MPA 161 patients, increased frequency AA homozygote in both-wg (p<0.005) and MPA (p< ) patients. AA genotype more common in female patients with MPA (P<.05) IL-10, A/G (Murakozy et WG 39 WG patients, significant shift to al.) AA genotype. IL-10/ -592,-819,- (Spriewald et WG WG patients without ESRD had high 1082 haplotype al.) frequency of GCC/ACC promoter haplotype; (OR 0.13) IL10 CA repeat (Zhou et al.) WG 36 Swedish patients. CA repeat microsatellite in the polymorphism in IL10 gene promoter region associated with disease. TNF α Ncol (Huang et al.) WG 32 Swedish patients. No significant association. TNFα -308 and 1 st (Mascher et WG 35 patients. Both SNPs: no intron of TNFβ al.) significant association. TNF α promoter, (Tsuchiya et AASV 69 Japanese patients, HLA- TNF RII and HLA II al.) DRB1*0901 significant association. (MPO) TNF α -238 G/A (Spriewald et WG 32 patients. Increased frequency of al.) G/A genotype (OR 5.01) CTLA 4 coding (Spriewald et WG 32 patients. Increased frequency of sequence +49 A/G al.) heterozygote C/T genotype at and promoter region promoter site in patients ( P<.05) 318 C/T CTLA4 microsatellite (AT)n (Huang et al.) WG 32 Swedish patients. Strong association of decreased frequency of shortest CTLA4 allele. ( P<0.0001) 34

35 CTLA4 (AT]n (Zhou et al.) WG 117 American patients. Lower microsatellite frequency of homozygote for shortest allele of CTLA4 microsatellite, p< CTLA4 promoter - (Spriewald et WG 32 patients. CTLA4-318 C/C 318 C/C al.) genotype more common in WG patients with ESRD (OR 2.25) codon 25 of TGF-β 1 (Bartfai et al.) AASV 161 patients. No association found. codon 25 of TGF-β 1 (Murakozy et WG 39 patients, trend to genotype CG al.) PTPN R620W (Jagiello et al.) WG 199 German, patients. Increased frequency of 620 W allele ( p<0.001) CD18 gene entire (Meller et al.) AASV 76 PR-3 and 31 MPO associated coding and vasculitis patients. 10 SNPs regulatory region identified, 4 with significant association with MPO + ve disease. 1 SNP at alternate transcription initiation site (T65C) IFN γ +874 T/A (Spriewald et WG 32 WG Patients. Increased freq. of al.) T/T genotype (OR 3.4); Increased frequency of A/A genotype in WG with ESRD CD18- exon 11 (Gencik et al.) MPO RFLP in exon 11 of CD18 associated with MPO associated vasculitis. IL , -1082, -592 TAC haplotype (Wieczorek et al.) AASV 403 WG and 107 CSS German patients. Significant association with ANCA negative CSS but not with WG. (CSS p>0.0003) IL-1 β and IL-1 RA (Borgmann et al.) AASV 109 patients. Increased secretion of IL1 β and decreased secretion of its antagonist IL1-RA associated with ESRD in PR-3 +ve patients. 35

36 Fcγ R 2a/3a (Dijstelbloem et al.) WG 91 patients. Homozygote for R131 form of Fcγ RIIa and F158 form of Fcγ IIIa is associated with increased rate of relapse. Chromosome 6p (Gencik et al.) AASV 102 patients. Several haplotypes on chromosome 6p strongly associated with PR3+ve patients IL-2, IL-5RA (Gencik et al.) AASV 102 patients. No association PR3 Entire coding and promoter region BPI- ANCA A645G SNP MBL gene 6 SNPs in promoter and coding region Phospholipase C γ 2 gene. 3 exons in coding region. ACE insertion/deletion polymorphism intron 16 NOD2/CARD 15 (Gencik et al.) WG 79 patients. 7 SNPs including 1 non synonymous SNP, one 84 bp insertion/deletion and 1 microsatellite identified. Functional SNP (A-564G)- no significant association. (Jagiello et al.) WG 201 patients. No significant association. (Kamesh et al.) AASV 170 patients. No significant association. (Jagiello et al.) WG No significant association (Murakozy et WG 39 patients. No association al.) (Newman, WG No significant association. Rubin, and Siminovitch) 36

37 serine protease gene cluster alpha 1 antichymotrypsisn gene (Borgmann et al.) (Borgmann, Haubitz, and Schwab) WG 79 Patients. Increased PI*Z in WG is known- Protease Inhibitor (PI) is a part of cluster of serine protease inhibitor gene. Centromeric and telomeric simple tandem repeat polymorphism is in linkage disequilibrium with defective PI*Z allele in all patients. AASV 109 Patients. No significant association. 202 microsatellites on apoptosis related genes CD226 Gly307Ser 22 SNPs previously studied in rheumatoid arthritis, systemic lupus erythematosus and Sjogren s syndrome (Jagiello et al.) WG 150 German patients. 202 microsatellites screened - 6 significant associations, multivariate - 1 strong association retinoid x receptor beta gene RXRB, on MHC, close to HLA-DPB1. Further analysis showed overrepresentation of HLA DPB 1*401 and extended haplotype HLA DPB1*401/RXRB03 showed even stronger association. (OR 6.41) (Wieczorek et WG 640 German patients. 307 Ser allele al.) associated with increased risk of WG (Wieczorek et WG >600 German patients. Nominally al.) significant differences in allele distribution of TNFAIP3 and CDK6 genes but strong association for functionally relevant 4 SNPs haplotype of IRF5 (p=0.0012) 37

38 1.6.2 CCR5 and GPA (WG): CCR5 is a receptor for a β chemokine previously known as RANTES (regulated upon activation normal T cell expressed and secreted). It is an 8-10 kda protein and is a cell type selective chemoattractant, expressed on macrophages, T cells and dendritic cells. Increased concentrations of CCR5 ligands and cells enriched with CCR5 in affected organs have been reported in several autoimmune diseases (Cameron et al.) (Balashov et al.) (Sorensen et al.). These findings suggest a role for a CCR5 signalling pathway in the recruitment of T cells and macrophages. The presence of macrophages and T cells in the granuloma of GPA (WG) and the T H 1 type of response in GPA (WG) point to the importance of CCR5 in the pathogenesis of GPA (WG) (Loetscher et al.) (Qin et al.). Zhou et al showed in an immunohistological study of lung biopsies from patients with GPA (WG) that they have increased expression of RANTES and an increased number of CCR5 + ve cells in the inflamed lung tissue compared to adjacent normal lung tissue (Zhou et al.). Interestingly numerous CCR5+ve cells were found to be bound to the pulmonary vascular endothelium, compared with predominantly CCR5-ve cells free in the lumen, suggesting that CCR5 is involved in the transmigration of cells across the endothelium. A 32 base-pair deletion in CCR5 gene (CCR5 32) has been described and has been shown to give rise to a truncated, non-functional CCR5 protein. The CCR5 32 has been demonstrated by several groups confer resistance to human immunodeficiency virus (HIV) as CCR5 is essential for membrane binding and cell penetration by the HIV virus (Alkhatib et al.). In vasculitis this deletion is significantly under represented in ANCA negative patients, conversely, CCR5 32 is associated with persistent ANCA positivity. Lack of this deletion in ANCA negative patients suggests the importance of 38

39 CCR5 in the pathogenesis of GPA (WG) and it can be postulated that absence of a functional CCR5 pathway and a negative ANCA status will dramatically reduce development of GPA (WG) (Zhou et al.). 1.7 New insights into pathogenesis of AASV: Neutrophil extracellular traps: Recently Kessenbrock et al provided an exciting new insight into a hitherto unknown mechanism of endothelial damage in ANCA-associated vasculitis (Kessenbrock et al.). It has been known for quite some time that neutrophils are capable of releasing gummous DNA upon cell death, a mechanism stimulated by Toll-like receptor 4 (TLR4) (Clark et al.). This DNA in turn can trap bacteria (Brinkmann et al.) hence the term neutrophil extracellular traps (NET) has been coined. Eventually the gummous DNA can stick to the endothelium and causes tissue damage during sepsis. The release of NET thus characterizes a unique type of neutrophil-related cell death that is linked to innate immunity. Kessenbrock et al. were able to demonstrate that NETs can trigger vasculitis and showed that ANCA stimulated neutrophils release NETs, which contain MPO and PR3 (Kessenbrock et al.). NETs have also been shown to activate plasmacytoid dendritic cells and B cells. Interestingly, they also showed that the neutrophils ability to form NETs was enhanced by bacterial infection with S. Aureus, which has long been linked to vasculitis (Brinkmann et al.) and that NETs can in turn bind Staphylococcus. Their results also suggest that PR-3/MPO co-localize with NETs, which may in turn present PR-3/MPO as antigens to the remainder of the immune system, thus fuelling a vicious circle (Bosch). The importance of these new results is twofold: 39

40 First, they provide a whole new pathogenetic mechanism for vasculitis. Moreover, they provide a new link between infection and vasculitis Lysosomal Associated Membrane Protein-2 It is not known what provokes autoantibody formation in pauciimmune glomerulonephritis. Three possible explanations for this autoantibody formation are: infection induced stimulation of neutrophils, bacterial superantigens provoking autoantibody production and finally a molecular mimicry between microbial antigens and host proteins. Recently Kain et al has proposed molecular mimicry between lysosomal membrane protein 2 (LAMP2) and bacterial adhesin FimH (Kain et al.). In neutrophils LAMP2 is integrated in to membranes of MPO and PR3 containing vescicles, hence autoantibodies to LAMP2 are detected as positive in ANCA assays. In their experiments, exposure to FimH induced autoantibodies to human LAMP2, and these autoantibodies could initiate pauciimmune glomerulonephritis in rats immunised with FimH. They also showed that prevalence of these autoantibodies to human LAMP2 was more than 90% in patients with active pauciimmune glomerulonephritis. As onset of vasculitis is often preceded by infections with FimH expressing bacteria, they conclude that FimH triggers the autoimmunity to human LAMP2, which is responsible for the tissue injury in vasculitis syndrome Circulating endothelial cells as potential mediators of disease Several lines of evidence have long suggested that circulating endothelial cells (CEC) themselves could be proinflammatory (Woywodt et al.). Damaged eukaryotic cells have been shown to release a variety of proinflammatory factors which initiate a Toll-like-receptor-2/NFPB-dependent reaction in monocytes and 40

41 fibroblasts (Li et al.). Evidence has also emerged that ANCA accelerate apoptosis in neutrophils and impaired clearance of apoptotic neutrophils (van Rossum, Limburg, and Kallenberg). Kirsch et al. showed in a recent study that apoptotic and necrotic endothelial cells and their fragments are rapidly internalized by healthy endothelium (Kirsch et al.). Support for these findings came from other studies demonstrating the phagocytic capability of endothelial cells (Chen et al.). Kirsch et al. also demonstrated that endothelial cells exposed to apoptotic and necrotic cells exhibit enhanced adhesion properties for leukocytes and that isolated CECs from patients with vasculitis even aggravated these effects. These effects on binding properties could be explained, at least in part, by the release of the pro-inflammatory chemo-attractants IL-8 and MCP1, enhanced levels of which have been detected in the serum of patients with active vasculitis (Yang et al.). Interestingly, apoptotic and necrotic cells induced different patterns of effects in healthy endothelium. Endothelial synthesis of these mediators triggered by ANCA and circulating endothelial cells may contribute to the pro-inflammatory state associated with vasculitis (Mayet et al.). Kirsch et al. have recently investigated this topic further and became interested in the role of thrombospondin (TSP-1) (Kirsch et al.). This multi-domain, multifunctional glycoprotein modulates cell adhesion and proliferation (Bonnefoy, Moura, and Hoylaerts). Kirsch and co-workers were able to show that apoptotic cells induce enhanced expression of TSP-1 in human endothelial cells and demonstrated that TSP-1 facilitates engulfment of apoptotic cells by phagocytes. It is tempting to speculate that endothelial-derived elevated TSP-1 may serve as a signal for the promotion of enhanced phagocytic clearance of apoptotic cells. 41

42 1.7.4 Endothelial microparticles in ANCA-associated vasculitis In general, microparticles (MP) are sub-micrometric fragments derived from plasma membranes. Increased numbers occur in response to a variety of events, such as activation, injury or apoptosis. Loss of phospholipid asymmetry is a crucial event during this process (Brogan et al.). On their surface microparticles express antigens that reflect their cellular origin. These surface markers permit their enumeration and characterisation by flow cytometry. Microparticles have attracted considerable interest in vascular disease although a consensus definition of these particles and a uniformly accepted protocol for their enumeration is still lacking (Burnier et al.). In this regard the situation resembles that of CEC in that several techniques compete and progress is hampered by lack of standardised criteria for definition and enumeration. Furthermore, endothelial microparticles only represent a small subgroup of all MP found in human plasma (Jy et al.). Specific endothelial microparticles were first described in 1990 by Hamilton and colleagues (Hamilton et al.). Erdbruegger et al studied endothelial microparticles (EMP) by FACS analysis and found elevated EMP in active vasculitis (Erdbruegger et al.). Particle counts also correlated with disease activity (Erdbruegger et al.). It is probably safe to assume that CECs and microparticles do not reflect the same disease process. In other words, CEC, EMP and also soluble markers, such as thrombomodulin, each reflect different facets of vascular activation and damage although some degree of overlap may exist. EMP may also have pathogenetic importance in vasculitis. They are now regarded as crucial players at the interface of atherosclerosis and inflammation (Ardoin, Shanahan, and Pisetsky) (Mesri and Altieri). Leukocyte MP induce endothelial 42

43 IL-6 and MCP-1 production (Mesri and Altieri). It has been demonstrated that EMP are tissue-factor positive (Sabatier et al.) and very recent evidence suggests that they can also convert plasminogen into plasmin (Lacroix et al.). Finally, elegant studies in flow chambers have demonstrated that MP enhance leukocyte rolling (Forlow, McEver, and Nollert). Taken together, current data suggest that EMP may not only be a surrogate marker of vasculitis but that they may contribute to the pro-inflammatory and pro-coagulant status of the endothelium Endothelial progenitor cells in ANCA-associated vasculitis The role of endothelial progenitor cells (EPC) in vascular disease and their potential role for therapy have been reviewed recently (Mobius-Winkler et al.). Of note, the field of EPC is particularly hampered by lack of standardisation (Fadini et al.). Our knowledge about the kinetics of CEC detachment and EPC mobilisation as well as their interaction is equally limited. Very recently, the margins between endothelial progenitor cells and haematopoietic stem cells have become somewhat blurred after proof that endothelial cells can be haematopoietic in mice (Eilken, Nishikawa, and Schroeder). de Groot et al studied numbers of circulating CD34+ progenitor cells and EPCs in vasculitis and demonstrated that these cells increased significantly after the institution of immunosuppressive therapy and with disease remission (de et al.). In contrast to de Groot and co-workers, other studies postulate an imbalance between CECs and EPCs in patients with vasculitis (Holmen et al.) (Zavada et al.). What make these studies so difficult to compare is, again, the lack of standardisation and the use of different assays and surface markers. Therefore, these studies provide interesting food for thought but require independent confirmation. Certain drugs like erythropoietin and statins are known to influence number of EPCs 43

44 (Bahlmann et al.) (Matsumura et al.). EPCs are capable of homing in to sites of vascular damage. It is probably fair to say that EPCs will receive further scientific attention in vasculitis while a standard as to their definition and enumeration is eagerly awaited. 1.8 Animal models and further evidence for role ANCA in pathogenesis of vasculitis: More definitive evidence of the pathogenesis of AASV and the pathogenic role of ANCA in development of AASV may come from the establishment of an animal model of the disease. However, because of the lack of a robust animal model for AASV this has not yet been achieved In vitro models: The glomerulus is a complex three dimensional structure consisting of various cell types, and in vitro models of glomerulonephritis do not exist. Some experiments have been performed using cell lines to study the interaction between ANCA and neutrophils. Falk et al in 1990 first demonstrated ANCA causes human neutrophils to undergo an oxidative burst and degranulate in presence of TNFα (Falk et al.). Subsequently Lockwood s group showed that incubation of ANCA or F(AB)2 with neutrophils interferes with fmet-leu-phe (fmlp] induced inositol phosphate metabolism and protein C kinase translocation, both important steps in signal transduction pathways leading to neutrophil activation (Lai and Lockwood). Furthermore, techniques using intravital microscopy have demonstrated neutrophil adhesion to endothelium and transmigration in response to ANCA (Radford et al.) (Calderwood et al.) (Smith et al.). 44

45 1.8.2 In vivo models: Animal models of glomerulonephritis have been classified in to four categories based on the nature of the initiating autoantigen. Oliveira et al in early nineties showed that auto immunity could be induced in to Brown- Norway rats using mercury chloride (Mathieson, Thiru, and Oliveira). Their mercury chloride model of Brown-Norway mouse model was associated with glomerulonephritis, antibodies against MPO (Mathieson et al.). They further demonstrated expression of IL-4 mrna in mast cells. (Oliveira et al.). Subsequently in 1997 Kiely et al produced an animal model of vasculitis was based on exposure to mercury chloride in Brown Norway rats (Kiely, Pecht, and Oliveira). This model demonstrated arthritis, caecal vasculitis, raised IgE and production of multiple autoantibodies including anti MPO antibodies. It did not reflect disease pattern found in human disease. Subsequent attempts to induce vasculitis in rodents with anti-mpo antibodies were unsuccessful. Neumann et al developed SCG/Kinjoh, a new strain of mouse which developed spontaneous vasculitis and crescentic glomerulonephritis with ANCA but it was associated with significant immune deposits in the kidney (Neumann et al.). Xiao et al subsequently came out with novel MPO knock-out mice and immunised it against murine MPO. Transfer of splenocytes from these immunised mice in to RAG2 -/- mice induced vasculitis and pauciimmune crescentic glomerulonephritis.(xiao et al.). This has been the most robust model of AASV to date although the antigen in this study is not an autoantigen but a neoantigen and the immune response may not reflect that found in an autoimmune disease. Wistar Kyoto rats when immunised with purified human MPO develop pauciimmune crescentic GN and pulmonary haemorrhage (Little et al.). Using a similar method to develop animal model of PR3 associated vasculitis has not 45

46 resulted in disease, demonstrating that different mechanisms are responsible for loss of tolerance to these autoantigens. The ANCA epitope on human PR3 is not shared by the murine homolog. ANCA generated against recombinant murine PR3 in PR3 deficient mice recognises murine antigen on neutrophil surface. Local injection of TNF, after passive systematic transfer of PR3 ANCA, results in severe panniculitis, compared with passive transfer of mock immune serum. Although there is no evidence of kidney or lung injury in this animal model it may suggest a pathogenic role for PR3 (Pfister et al.). 1.9 ANCA negative small vessel vasculitis: Up to 20% patients with pauciimmune RPGN never have circulating ANCA and 30 % patients with pauciimmune RPGN are persistently ANCA positive even when in clinical remission (Hedger et al.). Mei Ding et al proposed hypothesis offering alternative mechanism responsible for vasculitis in this patients. In their experiments deletion of Von Hippel-Lindau gene (VHL) from intrinsic glomerular cells was sufficient to initiate crescentic pauciimmune RPGN in mice (Ding et al.). VHL is a negative regulator of Hypoxia inducible factor (HIF) which is a master regulator of oxygen homeostasis, angiogenesis and vascular remodelling (Hirota and Semenza). Loss of VHL leads to stabilisation of 1 α subunit of HIF and upregulation of hypoxia response downstream genes. TNFα and VEGFA, molecular targets of HIF are increased in RPGN (Galban et al.). Furthermore, Ding et al also found strikingly increased expression of chemokine receptor CXCR4 in podocytes. CXCR4 binds to stromal derived factor 1(SDF1), a growth factor secreted by mesangial cells and increased expression has also been noted in glomeruli (Ding et al.). Increased SDF1 production may recruit 46

47 inflammatory cells and contribute to the formation of glomerular crescents (Burger, Burger, and Kipps). Interestingly treatment of mice with neutralising antibodies to CXCR4 results in the delayed onset and reduced severity of RPGN in mice highlighting its importance in the pathological mechanisms underlying the disease. Ding et al further strengthens these findings by demonstrating increased expression of HIF1α target genes and CXCR4 protein in glomeruli from humans with pauciimmune RPGN. This study provides an important road map for further evaluation to determine if increased HIF1α expression and its targets are critical in the pathogenesis of pauciimmune RPGN and blocking antibodies to CXCR4 or HIF1α may prove to have a therapeutic role Histology: Kidney biopsy typically shows focal segmental necrotising glomerulonephritis. Extracapillary proliferation with crescent formation is seen in patients with rapidly progressive glomerulonephritis. The lesions are of varying ages compared to that of anti-gbm disease where all the lesions are of same age. Chronicity is more common with MPO ANCA associated disease, suggesting a less aggressive course and which may cause a delay in diagnosis. Vasculitis is characterised by infiltration of inflammatory cells in the vessel wall and granuloma with monocytes and macrophages are not always present. Interstitium shows inflammatory infiltrate consisting of neutrophils, monocytes, lymphocytes and macrophages. A large study of 134 kidney biopsies from patients of GPA (WG) revealed extracapillary proliferation to be the commonest lesion occurring in 70% biopsies followed by fibrinoid necrosis of the glomerular tuft in 54% samples. Granuloma was found in only 7 of 134 samples (Bajema et al.). 47

48 ANCA-associated vasculitides may not be as pauci-immune as previously believed, (Jennette and Falk). Traditionally immuno-fluorescence indicated the presence of few, if any, immune complexes within vasculitic lesions. More recent research, however, demonstrated that the alternative complement pathway is indeed crucial to the pathogenesis in animal models (Xiao et al.) (Schreiber et al.) and that some immune complexes are present on careful examination (Brons et al.) Clinical Features: Kussmaul and Meyer in 1866 first described a syndrome of fever, muscle disease, gastrointestinal and renal disease, associated with nodular swellings of arteries. They described it as a periarteritis nodosa, subsequently it was confirmed that the swelling process started from within the vessel wall and in twentieth century polyarteritis nodosa replaced periarteritis nodosa. GPA (WG), MPA and EGPA (CSS) are three major ANCA associated conditions. Though there are overlapping features, it is often possible to differentiate between the three, using Chapel Hill nomenclature method, ACR criteria and EMEA algorithm Wegener s Granulomatosis: Friedrich Wegener, a German pathologist described clinical and pathological findings in three cases with similar pattern in As more cases were described in the 1940s it was referred as Wegener s Granulomatosis. The first description of nephritis, uraemia, and necrotising sinusitis with systemic vasculitis was first reported by Klinger in 1931 and in 1954, pathological findings were reviewed by Godman and Churg. They 48

49 described the presence of necrotising granuloma in respiratory tract, systemic vasculitis affecting arteries and veins, focal glomerulonephritis with granulomatous inflammation in patients with GPA (WG). Though such a widespread organ involvement is not always present and limited form of GPA (WG), mainly involving upper airways and /or Ear/Nose/Throat (ENT) is common (Carrington and Liebow). Upper airways: These are involved in upto 90% patients of GPA (WG) (Fauci et al.). Epistaxis, rhinorrhoea, nasal crusting, obstruction of paranasal sinuses, sinusitis, otitis media and conductive deafness are common symptoms. Destruction of nasal septal cartilage leads to characteristic deformity of nose. Inflammation can involve larynx and trachea, sometimes healing with subglottic stenosis. Hoarseness of voice and haemoptysis are other features. Pulmonary disease: Upto 90% patients of GPA (WG) have involvement of lung parenchyma. Granuloma formation is a relatively benign disease presenting as dyspnoea, cough, haemoptysis and abnormal chest radiograph. A more malignant presentation is that of an alveolar haemorrhage as a result of pulmonary capillaritis (Bosch et al.). It often presents as a Goodpasture Syndrome with pulmonary haemorrhage and rapidly progressive glomerulonephritis. Clinically, cough, dyspnoea, haemoptysis and anaemia are associated with alveolar shadow on chest radiograph; increased transfer factor of carbon monoxide is diagnostic. Intercurrent infections and fluid overload can precipitate pulmonary haemorrhage. Other organ involvement: Palpable purpuric rash, nail bed infarcts and splinter haemorrhages are typical of skin involvement but non healing ulcers with 49

50 granulomatous inflammation are a rare presentation of GPA (WG). Necrotising leuckocytoclastic vasculitis is a histological feature. Eye involvement occurs in 50% patients and has a wide spectrum of presentation. It can cause conjunctivitis, episcleritis or more severe forms include retinal vasculitis, optic neuropathy and destructive granuloma affecting orbit leading to enucleation. (Bullen et al.). Arthralgia and myalgia are common but arthritis with erosive changes is less common. Gastrointestinal (GI) tract involvement presents as abdominal pain and GI bleeding. Endoscopy reveals purpuric lesions of the GI tract. Cardiac involvement in GPA (WG) is rare and takes form of arrhythmia, cardiomyopathy, pericarditis and valvulitis mimicking infective endocarditis (Gerbracht, Savage, and Scharff) Nervous system: This takes forms of cerebral vasculitis characterised by confusion, seizure and neurological deficit, peripheral neuropathy usually mononeuritis multiplex and isolated cranial nerve involvement may occur. Radiology and CSF analysis can be helpful but not pathognomonic. ANCA negative primary CNS vasculitis is a well recognised entity. Other symptoms: Constitutional symptoms consisting of weight loss, fever and night sweats are common in AASV Systemic vasculitis (Features common to MPA) Renal disease: It is common and often asymptomatic. Active urinary sediments (haemato-proteinuria) raise the possibility of renal vasculitis even in absence of 50

51 impaired glomerular filtration rate (GFR). Hypertension is absent in more than half of patients (Coward et al.). Asthma, peripheral neuropathy and medium size vessel involvement is more common in CSS Limited GPA (WG): Not all patients have disseminated disease and Carrington Liebow in 1966 described series of patients with involvement of respiratory tract, and constitutional symptoms but no evidence of extrapulmonary disease (Carrington and Liebow). They did have severe disease in that they all died directly due to pulmonary GPA (WG), but many patients with limited GPA (WG) do not develop renal disease even without immunosuppressive therapy (Luqmani et al.). De Remee proposed ELK classification to indicate involvement of organs ENT/Lungs/Kidney. (e.g. LK disease means involving lungs and kidney) (DeRemee et al.) Assessment of GPA (WG): Serial uniform assessment of GPA (WG) is possible with Birmingham Vasculitis scoring system (B-VAS) (Luqmani et al.). This scoring system was intended to be used in any vasculitic disease and has some limitations. BVAS was modified specifically to assess GPA (WG) (BVAS/WG) by international network for the study of systemic vasculitis in 2001 (Stone et al.) Double positive disease: In patients with anti glomerular basement antibodies (GBM) both circulating and linear deposition in glomeruli, circulating ANCA have been reported. In fact 10-20% patients with anti-gbm disease have 51

52 circulating ANCA and anti GBM antibodies specific to the Goodpasture s antigen (α 3 chain of type 4 collagen) may be found in 2-3% of patients with AASV (Jayne et al.). These patients usually have anti MPO antibodies, extra renal disease and an outcome of AASV with a significant risk of relapse (Bosch et al.) canca versus panca: Franssen et al elegantly summarised the differences between canca and panca associated vasculitis (Franssen et al.). These are summarised in Table-2. Table 2: Differences between canca and panca associated vasculitis canca associated vasculitis panca associated vasculitis Male preponderance (66%) Female preponderance (62%) More extrarenal manifestations (3.9 organs) Granulomas common Renal lesions more acute Rapid worsening of renal function Upper airways commonly affected CXR: granuloma- nodules and cavities Extrarenal involvement less (2.2 organs) Granulomas are rare Renal lesions more chronic Slow deterioration Lung involvement similar CXR: haemorrhagic capillaritis patchy shadows 1.12 Treatment: General Principals: A common feature of the ANCA-associated vasculitides is marked heterogeneity regarding disease manifestation, severity and prognosis, 52

53 which affects treatment strategies. Briefly, non-organ or non-life-threatening disease, which can be localized or early systemic, is distinguished from organ threatening disease, which can be generalized, severe or refractory. Treatment strategies have evolved since the introduction of glucocorticoids in Methotrexate, azathioprine and cyclophosphamide were introduced in the 1960 s, and plasma exchange in It is worthwhile to remember that long-term survival and remission only became feasible with the introduction of Cyclophosphamide by Fauci (Fauci, Haynes, and Katz). Before the introduction of cyclophosphamide-based therapeutic regimens, mortality of (GPA) WG at 1 year was 80% (Walton). Until the landmark NIH trial of cyclophosphamide, treatment of GPA (WG) was limited to corticosteroids (Fauci et al.). However corticosteroids increased median survival of GPA (WG) patients only to 7.5 months compared to median survival of 5 months of untreated GPA (WG) (Hollander and Manning). Since then much has been learned through numerous studies including multi-centre, international and prospective trials over the last 20 years. It is through this body of evidence that ANCA-associated vasculitis has now become a chronic and treatable disease. 5-year survival rates now approach 80% (Mukhtyar et al.). The cytotoxic drug regimens currently used have a significant toxicity, which can contribute to morbidity and mortality. A considerable share of the current mortality is indeed due to infections. Malignancy is of similar concern and cyclophosphamide is usually implicated whereby the cumulative dose seems to be of greatest importance. A large study from Scandinavia reported a twofold increased risk of cancer when compared to the general population (Knight, Askling, and Ekbom). Cancer of the urothelium and skin as well as lymphoma and leukemia are most common. Life-long 53

54 vigilance regarding malignancy, and urothelial neoplasms in particular, is advised in all patients who have been treated with cyclophosphamide. However prospective studies on screening strategies in these high risk patients are lacking. Immunosuppression also increases the risk of infertility, whereby the risk is particularly high with cyclophosphamide. In the NIH study, cyclophosphamide therapy in women resulted in a 57 percent incidence of amenorrhea lasting more than one year or inability to become pregnant (Hoffman et al.). Data in men treated with cyclophosphamide are not nearly as good in vasculitis but studies using cyclophosphamide to treat tumours show there is no reason to believe their fertility should be spared by alkylating agents such as cyclophosphamide in vasculitis. Cryopreservation of sperm should therefore be considered if appropriate. Inflammatory vascular disease also confers an increased risk of cardiovascular disease although the evidence is stronger for lupus than for ANCA-associated vasculitis (Kaplan) (Morgan et al.). Given this considerable toxicity of treatment, more recent studies have attempted to establish treatment strategies with reduced toxicity that are more tailored to the individual patient s characteristics. Therapeutic concepts are also changing from permanent cytotoxic treatment to sequential treatment strategies using less toxic drugs after remission has been achieved. This approach has surely reduced mortality and morbidity and must be used as a reference to test the efficacy and safety of newer drugs. A recent paper from the European Vasculitis Study Group has summarised current recommendations for the management of ANCAassociated small and medium vessel vasculitis (Mukhtyar et al.). Nevertheless it remains sobering that some forty years after the introduction of 54

55 cyclophosphamide this toxic drugs still remains the mainstay of treatment in severe disease. Treatment of AASV is divided in to two phases, initial aggressive phase of induction of remission and subsequent phase of maintenance of remission with weaker immunosuppresants Induction of remission: This is the initial intense phase of therapy to induce remission and usually lasts for at least three months. Typically it consists of high dose corticosteroids (CS) and cytotoxic drugs like cyclophosphamide or methotrexate in less severe non renal disease (NORAM) (de Groot et al.). Corticosteroids used are high dose methyl prednisolone (250 to 1000 mg/ day for three days) and replaced with oral prednisolone 1-2 mg/kg which is gradually tapered over 3-6 months to maintenance dose of 5-10mg/day. Methyl prednisolone may not be used at all and high dose oral prednisolone is given instead. Plasma exchange is traditionally used for induction of remission in patients with severe disease associated with pulmonary haemorrhage. A recent prospective randomised controlled trial comparing methyl prednisolone with plasma exchange in patients with severe renal vasculitis (serum creatinine >500 µ mol/l) showed plasma exchange to be superior to methyl prednisolone in preserving GFR (24% reduction in the risk of development of ESRD) and dialysis independence (Jayne et al.). However unpublished data suggests that this difference is maintained only for 12 months and five years follow up shows no difference between the two groups for renal survival. Plasma exchange remains the fastest way of removing circulating ANCA. Typically 1.5x plasma volume exchange removes 75% macromolecules though only 45% of total IgG is 55

56 intravascular and level returns to 40% by 48 hours post plasma exchange. One to 1.5 plasma volume exchanged every 48 hours for 5-7 treatments helps to remove significant autoantibodies and offers disease control until immunosuppressive action of cyclophosphamide starts. Cyclophosphamide is an alkylating agent that prevents cell division by cross linking of the DNA thereby decreasing DNA synthesis. Cyclophosphamide can be used as a daily oral regime or intravenous pulsed regime. Both the regimes have been proven highly successful in achieving the induction of remission. Mesna is usually administered with intra venous pulse cyclophosphamide to reduce bladder toxicity. It provides free thiol which binds to and inactivates acrolein, a toxic metabolite of cyclophosphamide. Methotrexate interferes with folate metabolism and, therefore interferes with synthesis of purine, required for DNA synthesis and cell division. Use of methotrexate for induction of remission as an alternative to cyclophosphamide in milder presentations of AASV (serum creatinine <150 µ mol/l) is useful in achieving remission but is associated with a high rate of relapse after withdrawal suggesting need of prolonged period of immunosuppression (de Groot et al.) Maintenance of remission: Maintenance of remission is a less aggressive but a prolonged phase of immunosuppressive therapy, which is continued for at least two years and corticosteroids remain key drugs in the maintenance of remission phase. Use of an appropriate bone protection strategy is used in early phase to prevent steroid induced osteoporosis. Other drugs used in maintenance of remission are further cyclophosphamide, methotrexate and azathioprine. The original NIH trial used daily oral cyclophosphamide 2mg/kg for upto two years. This regime was excellent with remission rate of 79 % and with an 56

57 average follow up of eight years was associated with a relapse rate of 44% (Hoffman et al.). Though cyclophosphamide transformed the long-term outcomes of AASV, treatment associated morbi-mortality remains major concern. Major side effects of cyclophosphamide include bone marrow suppression, infections, infertility, and haemorrhagic cystitis and nine to 45 fold increase risk of bladder cancer. Cyclophosphamide has been used as intravenous pulses with success in treatment of proliferative lupus nephritis. These regimes reduced the cumulative cyclophosphamide dose significantly. A meta-analysis of 19 prospectively conducted clinical trials comparing various immunosuppressive regimes, has revealed intravenous pulse cyclophosphamide (ivcyp) to be as effective as daily oral cyclophosphamide (ccyp) (Bansal and Beto). Positive data from lupus nephritis encouraged use of ivcyp in patients with AASV. De Groot et al performed a meta-analysis of three randomised controlled trials (RCT) comparing ccyp with ivcyp in AASV (de Groot, Adu, and Savage). She confirmed that ivcyp was at least as effective as ccyp in achieving remission in AASV and it was associated with less infective complications. The rate of relapse was slightly higher in ivcyp group compared with ccyp group though it did not reach statistical significance. Two of the three RCTs actually had less relapses with ivcyp regime compared with ccyp but a third RCT had less relapses with ccyp. The regimes used, duration of treatment and adjunctive treatment was different in all three RCTs making it difficult to draw direct comparisons. Our own retrospective study of prolonged low dose ivcyp showed an excellent rate of remission, low rate of infections and mortality, and a decreased rate of relapse (Dhaygude et al.). The European Vasculitis Study 57

58 Group (EUVAS) is a European network of renal physicians, rheumatologists and other medical practitioners involved in the management of AASV. EUVAS has organised several clinical trials addressing key issues in treatment of AASV. One of the key trial to compare daily oral versus intermittent pulse cyclophosphamide regimes; CYCLOPS trial confirmed that intermittent pulsed Cyp was associated with fewer complications like leucopenia and infections. This trial did not show any difference between rates of remissions and relapses in the two groups (de Groot et al.). Unpublished data for long term follow up, however does show increased relapses in intermittent pulse group. CYCAZAREM was the first randomised trial conducted by EUVAS, comparing cyclophosphamide against azathioprine for maintenance of remission. In this study cyclophosphamide was used until remission was achieved (at least three months) and subsequently patients were randomised to either receive further cyclophosphamide or azathioprine for the maintenance of remission. At 18 months follow up, there was no difference in disease free survival in either group (Jayne et al.). A major criticism of this study was the short follow up duration. Unpublished data from a retrospective Dutch study suggests that use of cyclophosphamide for maintenance of remission prevents late relapses in PR3 associated vasculitis. This difference is significant even at ten years follow up. Long-term follow up outcomes of CYCAZAREM are expected in near future and will help to resolve this issue. Azathioprine: Azathioprine is a member of the mercaptourine group of drugs and inhibits purine synthesis, essential for synthesis of DNA and RNA. It has been established in the management of many autoimmune diseases and in solid organ transplantation. CYCAZAREM has proven the potency of azathioprine in the 58

59 management of the maintenance phase of vasculitis. More recently high dose intravenous pulse (1200 mg/24 hrs) along with daily 100mg azathioprine has been used successfully in treatment of retro orbital GPA (WG), resistant to cyclophosphamide and rituximab (Aries et al.) Emerging therapies in treatment of AASV: Empiric cytotoxic treatment has improved the survival of patients with AAV significantly and newer regimens have reduced treatment-related toxicity and morbidity. However by today s standards many of these drugs, such as cyclophosphamide, seem crude and not targeted on the pathogenetic mechanisms of vasculitis. In addition, relapsing and refractory disease remain challenging to treat. It must be assumed that preventing the initiation of the inflammatory cascade is the most effective treatment in vasculitis. If this is impossible, the goal is to attenuate subsequent immune responses by targeting crucial pathogenetic mechanisms or inflammatory mediators. Directing therapies at these sequential targets will go beyond non specific immunosuppression and hopefully expose patients to less toxicity. Most of the newer drugs are used either as additional or adjunctive treatment, only some of them are investigated as an alternative to the standard of care. Mycophenolate mofetil (MMF): Mycophenolic acid is an active metabolite of MMF which inhibits inosine monophosphate dehydrogenase (IMPDH). T and B lymphocytes are dependant on IMPDH pathway for guanosine nucleotide synthesis. MMF has well-established role in the management of solid organ transplantation. Data regarding use of MMF in AASV is limited to a few small 59

60 open label clinical trials. Results are contradicting in terms of rate of relapse and EUVAS is conducting a multicentre study to compare MMF with cyclophosphamide for the induction of remission in AASV (MYCYC). They recently published a large RCT to study efficacy of this drug in maintenance of AASV, however it failed to show that MMF was superior to azathioprine. The major side effects of MMF are GI intolerance, leucopoenia and opportunistic infections. Use of monoclonal antibodies in systemic vasculitis was pioneered in 1990 by Mathieson et al. They used anti CD52 monoclonal antibody Campath-1H and CD-4 monoclonal antibody of rat origin for successful treatment of refractory vasculitis (Mathieson et al.). Anti-TNF antibodies: As TNF plays major role in priming of neutrophils, blocking TNF was thought to be able to prevent subsequent interaction with ANCA. Etanercept, a soluble TNFα inhibitor was used in addition to standard therapy for maintenance of remission in a large randomised controlled trial of 180 patients with GPA (WG). After the initial period of standard therapy (cyclophosphamide or methotrexate) only etenarcept was continued. This study revealed that etenarcept was not associated with improved, disease free survival and six patients developed solid tumours in etenarcept arm (Etanercept plus standard therapy for Wegener's granulomatosis 3). Rituximab: Rituximab is a chimeric monoclonal antibody against CD20. CD20 is expressed on B lymphocytes which are precursors of plasma cells, antibody producing cells. The exact mechanism of action of rituximab in ANCA vasculitis is incompletely understood, since CD-20 is not expressed on plasma cells 60

61 themselves, but ANCA titres decrease following rituximab treatment. An open label study of eleven patients (ten with GPA (WG)) published in 2005, revealed 100% remission rate with rituximab. All these patients were either resistant to cyclophosphamide or use of cyclophosphamide was contraindicated. Patients also received plasma exchange and/ or high dose corticosteroids and it is difficult to know to what extent the contribution of rituximab had in achieving remission (Keogh et al.). The lack of efficacy of rituximab was demonstrated in a series of eight patients, predominantly consisting of granulomatous disease, where rituximab was given in addition to the standard treatment (Aries et al.). Two prospective controlled trials RAVE and Rituxivas have been recently published. Rituxivas is a British trial. Their hypothesis was as rituximab being specific anti CD-20 MAb, it should result in less profound immunosuppression and thus fewer adverse events compared to cyclophosphamide. Forty-four patients were randomised (1:3) to receive cyclophosphamide or rituximab. However at 12 months there was no significant difference between remission rates and adverse events in either groups (Jones et al.). RAVE trial compared efficacy of daily oral cyclophosphamide to rituximab in 176 patients. Rituximab was not inferior to cyclophosphamide but authors concluded that it be superior in patients with grumbling disease (Stone et al.). Disappointingly these large RCTs suggests rather limited role of rituximab in the management of AASV. Another interesting trial (CYCLOWVAS) is in progress which combines these two drugs to achieve a prolonged remission with limited toxicity. Worryingly rituximab has also been linked with development of serious complications like progressive multifocal leukoencephalopathy, a progressive fatal condition for which no treatment available to date (FDA warning 2007 Mar-Apr;41(2):3). Another problem with 61

62 rituximab is the timing of retreatment as there is poor correlation between (peripheral) circulating CD-20+ve B cells, ANCA positivity and relapse. Development of human antichimeric antibodies (HACA) against rituximab has been reported. Patients with strongly positive HACA may not respond to rituximab and may have severe infusion reaction (Smith et al.). There is no doubt that rituximab will be an important addition to the limited armamentarium against AASV. Newer B-cell depleting therapies such as ocrelizumab (humanized anti CD20), ofatumumab (human anti-cd20) and epratuzumab (humanized anti-cd22) have been developed. Some of these agents have already been used in systemic lupus erythematosus and rheumatoid arthritis while no data have yet been reported in vasculitis. Another potential target of therapy is the B lymphocyte stimulator (BAFF/BLYS), a member of the TNF superfamily that is critical to B-cell development (Moore et al.). The crucial role of BAFF/BLYS in lupus is only just emerging (Cancro, D'Cruz, and Khamashta). TNF can increase the release of BAFF/BLYS from neutrophils (Assi et al.) and BAFF/BLYS serum levels have been found to be increased in patients with vasculitis. Therapeutic manipulation is already available through Belimumab (humanized anti-baff/blys). It remains to be seen whether BAFF/BLYS could become a therapeutic target in vasculitis. T cell directed therapies: The production of ANCA is dependent on T cells. T cells are highly activated with increased expression of HLA-DR and CD25 and predominantly show a T H 1 cytokine phenotype. They are found in affected tissues, in granulomas and vasculitic lesions. Effector memory T cells are increased and possibly not adequately controlled by regulatory T cells, leading to 62

63 chronic inflammation in AASV (Abdulahad et al.). Many of the established drug treatments, such as Azathioprine or MMF, affect T lymphocytes albeit in a nonspecific manner. 15- Deoxyspergualin (Gusperimus) is a novel T cell directed drug (Flossmann et al.). It inhibits the interleukin-2-stimulated maturation of T cells and the polarization of T cells into IFN-gamma-secreting Th1 effector T cells resulting in growth of activated naive CD4 T cells. Deoxyspergualin also inhibits cytokine and monocyte activation. It has been effective in a small group of patients resistant or intolerant to Cyclophosphamide (Birck et al.). Leucopenia is a significant side effect, but seems to be manageable. Another issue is that the drug is administered by subcutaneous self-injection, which may not be feasible in each and every patient. Unfortunately, disease flares were frequent after the drug was withdrawn. Another unspecific T cell directed drug is antithymocyte globulin, which represents a collection of polycloncal antibodies directed against the surface antigens of activated T cells. In a small study in 15 patients with GPA (WG) resistant to cyclophosphamide it has been shown to be effective (Frampton and Wagstaff). The monoclonal anti-cd52 antibody alemtuzumab is a more selective T cell drug (Walsh, Chaudhry, and Jayne). Alemtuzumab has been used earlier to treat patients with refractory or relapsing AASV (Mathieson et al.) (Lockwood et al.). In the largest available study conducted in 71 patients, 65% went into clinical remission and 20% had a significant improvement. Disease relapse and infectious complications, however, were quite common (Walsh, Chaudhry, and Jayne). T cell activation is thought to be due to a "two-signal model" in CD4+ T cells. Activation of CD4-positive T cells occurs through engagement of the T cell receptor and costimulation of CD28 on the T cell surface by the Major 63

64 histocompatibility complex peptide and B7 family members on the antigen presenting cell. Cytotoxic-T-lymphocyte-associated antigen 4 (CTLA 4) can prevent delivery of the second co-stimulatory signal required for complete activation of T-cells. The gene encoding CTLA-4 has multiple polymorphisms and a positive association between GPA (WG) and longer alleles of (AT)n in the CTLA-4 gene has been demonstrated (Reynolds et al.). In addition, CTLA4-IG has prevented disease progression in an animal model of crescentic glomerulonephritis (Suntharalingam et al.). Unfortunately a trial investigating CTLA-4 IG as an adjunctive to MTX to induce and maintain remission in early systemic ANCA-associated vasculitis was terminated, apparently due to low recruitment and supply issues with the sponsoring manufacturer. Finally, it has to be said that the catastrophic experience with the anti-cd28 Monoclonal Antibody TGN has dampened the initial enthusiasm regarding therapeutic manipulation of co-stimulatory signals. Therapy directed at other targets: Intravenous immunoglobulin (IVIG) is an immune-modulatory drug with multiple effects. It is thought to interact with the inhibitory Fc receptor. A few open-label studies have shown that IVIG is effective in refractory ANCA-associated vasculitis and in early active disease (van Timmeren, Chen, and Heeringa). IVIG is a potential treatment alternative if standard care is not feasible, e.g. in the context of active infection or pregnancy. As pathogenesis of AASV involves complex interplay between B-cells, T-cells, variety of targets offer potential for treatment of AASV and is represented in Figure-4 (Reprinted by permission of Edizioni Minerva Medica from: Dhaygude et al, Minerva Urol Nefrol 2009 December; 61(4): ) 64

65 Figure 4: Complex interplay between B cells and T cells and potential targets for therapeutic intervention for AASV. Abbreviations: B cells as current and future targets for therapeutic intervention. BAFF denotes the BAFF/BLYS factor. BAFFR denotes BAFF/BLYS receptor. CTLA4 denotes Cytotoxic T Lymphocyte Antigen 4 and B7 its receptor. APRIL denotes a proliferation-inducing ligand. TACI denotes transmembrane activator and CAML interactor, the receptor for BAFF/BLYSS and APRIL. BCMA denotes B cell maturation antigen. TCR denotes T cell receptor. CD40 denotes cluster of differentiation 40 and CD40L its ligand. CD 20 denotes cluster of differentiation 20. Note that BAFF/BLYS and APRIL promote survival of B lymphocytes. BAFF/BLYS and APRIL work via TACI whereas APRIL can also activate via BCMA. Therapeutic manipulation of BCMA and TACI is not yet available. Anti-CD40 is also available (Dacetuzumab). 65

66 In summary, recent years saw the publication of a number of seminal studies for the treatment of AASV. We now have a much better understanding of the role of pulse intravenous cyclophosphamide and plasma exchange than ten or even five years ago. Further studies must now show whether plasma exchange is also beneficial for less severely ill patients with AASV. Finally, as ever, it is hoped that further progress in understanding the disease pathogenesis will also provide more tailored and less toxic therapies. 66

67 2.0 HYPOTHESIS, AIMS AND OBJECTIVES 67

68 2.0 HYPOTHESIS, AIMS AND OBJECTIVES 2.1 Hypothesis: There has been a perception of increased incidence of ANCA associated vasculitis amongst the clinicians at Greater Manchester. This relatively rare condition has two subtypes, canca positive disease and panca positive disease. Pauciimmune glomerulonephritis is a well defined diagnosis and upto 90% patients are ANCA positive. This condition is rapidly progressive and thus is associated with burden on patient and health care provider. What are differences in genetic background of AASV patients compared with normals which may account for a group vulnerable to AASV, at risk of serious illness with associated burden on the health-care community? As there are many similarities between the clinical presentations of canca and panca positive patients irrespective of the phenotypical diagnosis of Granulomatosis with polyangiitis (Wegener s Granulomatosis) and microscopic polyangiitis; as a group, AASV patients may share some genetic factors which are different compared to the healthy volunteers. As there are epidemiological, clinical, histological and outcome related differences between canca and panca positive patients, we addressed the hypothesis that there must be subtle genetic differences responsible for these observed differences between these two conditions. 68

69 2.2 Aims and Objectives: Aims and objectives of this study are- 1) To asses the disease burden of ANCA associated vasculitis by calculating incidence of biopsy proven pauciimmune glomerulonephritis in Greater Manchester. 2) To compare the frequencies of inflammation related gene polymorphisms in patients with ANCA associated vasculitis and healthy volunteers. 3) To compare the frequencies of inflammation related gene polymorphisms in canca and panca positive patients. 4) To examine the correlation between the genetic polymorphisms and circulating cytokine profile. 5) To correlate laboratory findings (genetic polymorphisms and circulating cytokine profiles) with the clinical outcomes like relapses and renal outcomes. 6) To study difference between metabolomics profile of canca and panca positive patients during active vasculitis and remission state. 7) To study the gene expression signatures of active and remission state of ANCA associated vasculitis. 69

70 3.0 PATIENTS, MATERIALS AND METHODS 70

71 3.0 PATIENTS, MATERIALS AND METHODS: 3.1 Patients: Patients for this study were recruited from five renal centres from the North-West of England. These centres included South Manchester Vasculitis Clinic, Salford Royal (Hope) Hospital Renal Clinic, Manchester Royal Infirmary Renal Clinic, Royal Preston Hospital Renal Clinic and Aintree University Hospital Renal Clinic. Informed consent was obtained from all the patients. Inclusion criteria included all the patients with diagnosis of AASV, above age of eighteen years and willing and able to consent. Although initially ANCA negative pauciimmune small vessel vasculitis patients were included, they were excluded from the analysis. In total 147 patients were analysed in this study. 3.2 Material And Methods: SNPs Genotyping: DNA Extraction, Measurement and Dilution: DNA was isolated from the cell pellet using the Nucleon kit no (Tepnel Life Sciences). The kit was used following the manufacturer s instructions. The cell pellets were thawed and made up to 4mls with phosphate buffered saline. The sample was added to a 15ml polypropylene centrifuge tube and 8mls of reagent A was added. The tubes were placed on a rotator mixer for 5 minutes at room temperature and then centrifuged at 4100rpm (3500g) for 5 minutes. The supernatant was decanted and discarded and a further 4mls of reagent A was added to the pellet. The pellet was vortexed for up to 1 minute to disperse the pellet followed by centrifugation at 4100rpm for 5 minutes. The supernatant was decanted and discarded. The pellet was vortexed, 500µl of reagent B was added and incubated at 37 C in a water bath for 10 minutes. One hundred and seventy five microlitres of reagent C was 71

72 added and mixed by inversion of the tube. One hundred and fifty microlitres of Nucleon resin was added drop wise followed by centrifugation at 4100 rpm for 4 minutes. The supernatant was transferred to a clean tube an equal volume of propan-2-ol was added. This was mixed gently by inversion to precipitate the DNA pellet followed by centrifugation at 4300 rpm (3800g) for 5 minutes. The supernatant was gently poured off, retaining the DNA pellet, and discarded. One ml of 70% ethanol was added and the DNA pellet transferred to a screw topped microfuge tube. The excess ethanol was aspirated and the pellet allowed to air dry for 10 minutes and the pellet was re-suspended in 400µl of sterile water for injection (B. Braun Melsungen AG). The pellets were then placed on a rotator mixer and left overnight at room temperature. They were then stored at 4 C until the DNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The concentrated DNA was stored at -80 C in screw capped tubes and labelled with the subject s study number and DNA preparation date. A working dilution of 50ng/µl was made by diluting the stock DNA using sterile water for injection (B. Braun Melsungen AG), this was stored at -20 C until use DNA Measurement and dilution: Nano drop: Measuring DNA concentration was necessary to maximize the efficiency of the molecular test to be performed. A spectrophotometer was used to measure the DNA concentration of each sample. Double stranded DNA has an excitation spectrum that peaks at 260 nm and the spectrometer emits light at 260 nm. The 260 nm measurement gives the DNA concentration and the 280 nm value indicates contaminating protein. The 260/280 ratio gives an indication of relative purity of the sample. 72

73 10 µl of DNA sample was added to 990 µl of DEPC distilled water in a 1.5 µl microfuge tube. 1 ml of DEPC distilled water (blank sample) was added in a cuvette to calibrate the meter. (zero against distilled water at 260 nm) and OD at 260 nm and at 280 nm was measured and the difference noted. I then measured each sample OD at each wavelength. Calculation 1 DNA concentration in ng/µl = 50X OD 260 nm X Dilution factor Therefore for 1 in 100 dilution concentration = OD 260 nm X 5000ng/µl Calculation 2- Relative purity = OD 260 nm/ corrected OD 280 (OD 280 blank OD 280). All the DNA samples were between 1.5 to 1.7 and worked well with Taqman PCR. Volume of DNA stock in ml= 5000 concentration in ng/µl i.e. a solution of 5000 ng/µl will need = 10 µl stock + 90 µl DEPC dist. Water Taqman PCR technique: This method was used for genotyping of TNFα, PECAM-1, IL-8, TGFβ, CCL5 IL-10, IL-18, 1L-8, genes. Principles: This protocol is designed for single nucleotide polymorphism (SNP) detection from genomic DNA. Taqman chemistry relies on some basic principles, which occur during PCR. (Fig. 5) 73

74 Figure 5: Amplification of Target Sequence. The Plateau effect: After cycle threshold the amount of PCR product produced enter a logarithmic phase, where there is a linear relationship between the amount of PCR product formed and the number of PCR cycles, allowing quantification of starting template. After this phase a plateau is reached to progressive inactivation of Taq polymerase, reduction in the efficiency of the denaturation step, reduction in the efficiency of primer-template complex formation (due to product reannealing) and Taq polymerase becomes limiting. Taqman Chemistry: The primers will bind to their complimentary sequences as they do in a standard PCR. Taq polymerase will aid in the addition of bases to the primer sequence, where polymerisation is associated with a 5 to 3 nuclease activity. As the addition of bases corresponds with where the probe has bound to 74

75 the DNA template, the probe is displaced separating the reporter dye from the quencher resulting in a fluorescent emission corresponding to the bound probe. Allelic discrimination: FAM labelled probe specific for allele 1 VIC labelled probe specific for allele 2 Probes specific for a given allele have a different Tm than the mismatched probe. This allows the mismatched probe to be destabilised. The probes will compete with each other for binding to the correct allele, because of the destabilisation effect; the matched probe will out compete the mismatched probe. Probe discrimination: During the PCR reaction the probe will be cleaved resulting in a fluorescent emission with an exponential pattern as the number of PCR cycles increases. In the multi-component view of the software this will produce certain patterns of fluorescence depending on the binding and cleavage of a probe to a given allele. Allelic discrimination: Genotypes dye signals can be separated to give clustering patterns. For example a FAM homozygote will be in a different wavelength of the spectrum to a VIC homozygote, with the being somewhere in the middle (Fig-6) 75

76 Figure 6: Allelelic discrimination showing homozygote for alleles X and Y and heterozygote (XY) PCR Methods: 1) Preparing the DNA : 50 ng/µl concentration of genomic DNA was diluted at 1:10 to make 5ng/µl solution. 2 µl of the 5 ng/µl DNA was added to the bottom of a Tqman 96 well plate. Centrifuge briefly at 1500 rpm and then place on PCR machine at 90 0 C for 15 minutes with lid off to dry down the DNA. 2) Preparing the PCR reaction: Prepare mastermix using 2X Taqman universal mastermix (2.5µl); 40 X Primer/Probe mixes (0.25µl) and sterile distilled water (2.25 µl). Total volume is 5 µl per reaction and 105 reactions were used for a 96 76