Genetics of toll like receptor 9 in ANCA associated vasculitides.

OBJECTIVES: To investigate the contribution of genetic polymorphisms of toll like receptor (TLR) 9 and related genes on the susceptibility and clinical manifestation of anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV). METHODS: Four single nucleotide polymorphisms (SNPs) in TLR9 were genotyped in 863 German AAV cases and 1344 healthy controls. Significant results were replicated in a cohort of 426 Dutch and British AAV cases. 11 polymorphisms in TLR9 related genes were studied concomitantly. RESULTS: A strong association of TLR9 genotypes and haplotypes with granulomatosis with polyangiitis was observed as well as a contrariwise association with microscopic polyangiitis. The association was confirmed when cases were compared according to ANCA status rather than to clinical entity. This was partly replicated in the second cohort leading to a striking overall difference in TLR9 allele/haplotype frequencies between proteinase 3 (PR3) ANCA+ and myeloperoxidase (MPO) ANCA+ cases (p=0.00000398, pc=0.000016, OR 1.68 (95% CI 1.35 to 2.1) for rs352140; p=0.000011, pc=0.000044, OR 1.64 (95% CI 1.31 to 2.04) for a 3-SNP haplotype). No significant association or epistatic effect was detected for TLR9 related genes: interleukin 6, interleukin 23 receptor, myeloid differentiation primary response 88, TNF receptor-associated factor 6, interleukin-1 receptor-associated kinase 4, discs large homolog 5 and nucleotide-binding oligomerisation domain containing 2. CONCLUSIONS: We provide further evidence that PR3-ANCA+ AAV differs genetically from MPO-ANCA+ AAV. TLR9 signalling may be involved in disease pathology, favouring models of infectious agents triggering AAV development.

[Genetic risk factors for vasculitis]. / Genetische Risikofaktoren von Vaskulitiden.

Among the vasculitides, genome-wide association studies (GWAS) have so far been performed for Behçet's disease, Kawasaki disease, granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). These studies delivered valuable information with respect to the pathogenesis and therapeutic targets: Apart from HLA-B51 and HLA-A26, distinct polymorphisms in cytokine (IL-10) or cytokine receptor (IL-12R/IL-23R) genes, transcription factors (STAT4) and genes encoding for proteins involved in antigen presentation (ERAP-1) have been identified as risk factors for Behçet's disease. The results of two GWAS performed for antineutrophil cytoplasmic antibody (ANCA) associated vasculitis GPA and MPA in Europe and the USA confirmed that the HLA-DP locus is the most relevant risk factor for GPA. Furthermore, the European GWAS confirmed SERPINA-1, a deficiency allele of the α-1-antitrypsin gene, as a genetic risk factor in GPA and identified a polymorphism in the proteinase 3 gene (PR3), one of the target antigens of ANCA, as a risk factor for GPA and PR3-ANCA-associated vasculitis.

Changes in proteinase 3 anti-neutrophil cytoplasm autoantibody levels in early systemic granulomatosis with polyangiitis (Wegener's) may reflect treatment rather than disease activity.

OBJECTIVES: To investigate the nature of the relationship between proteinase 3 anti-neutrophil cytoplasm autoantibody (PR3-ANCA) and relapse in patients with early systemic granulomatosis with polyangiitis (Wegener's) (GPA). METHODS: Clinical data from 16 relapsing and 12 non-relapsing patients with early systemic GPA from a randomised clinical trial were correlated to monthly PR3-ANCA values over 18 months. Each sample was examined using 9 different enzyme-linked immunosorbent assays (ELISAs) to ensure reliability of ANCA results. PR3-ANCA peaks were identified by the highest sum of logarithmic transformation values from all assays in samples after remission. RESULTS: A PR3-ANCA peak was identified in all relapsing and non-relapsing patients and coincided with relapse in all 14 evaluable relapsing patients. The monthly increment before the peak, however, was similar in relapsing and non-relapsing patients in all assays. Increments from remission to peak were higher in relapsing patients in 2/9 assays. PR3-ANCA values at entry and peak PR3-ANCA values were higher in relapsing patients in 3/9 and 2/9 assays, respectively. However, large overlaps of PR3-ANCA values prevented a distinction between relapsing and non-relapsing patients. The median time to reach peak values was 14 months in relapsing and 12 months in non-relapsing patients with scheduled termination of treatment at 12 months. CONCLUSIONS: The predictive value for relapses of PR3-ANCA determinations confirm and extend previous reports. Although all relapses were related to PR3-ANCA increases, reduction or withdrawal of immunosuppression without relapse was also related to increases and may explain the lack of predictive value of sequential PR3-ANCA determinations.

[Hypereosinophilic syndrome]. / Hypereosinophile Syndrome.

Hypereosinophilic syndrome is a heterogeneous group of diseases characterized by blood hypereosinophilia and eosinophil-related organ damage. A comprehensive diagnostic work-up is necessary to identify underlying conditions and to detect organ involvement, which are important for prognosis. Involvement of the heart is related with a poorer outcome. Some underlying conditions can be treated with targeted therapies, e.g., tyrosine kinase inhibitors. However, glucocorticoids in combination with steroid-sparing drugs are generally used for treatment. Furthermore, the growing understanding of the molecular pathogenesis will lead to new therapies, e.g., the use of anti-cytokine antibodies.

Association of ferritin autoantibodies with giant cell arteritis/polymyalgia rheumatica.

OBJECTIVES: Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are relatively common inflammatory disorders. Establishing the diagnosis however may be difficult, since so far no specific biomarkers of the disorders are available. METHODS: As a screening procedure, the authors used protein arrays for the detection of new autoantigens in GCA and PMR. The results of the protein array were confirmed by different ELISAs detecting IgG antibodies against the human ferritin heavy chain, N-terminal 27 amino acids of the human ferritin heavy chain or the homologous peptide of Staphylococcus epidermidis. Sera of patients with only GCA (n=64), only PMR (n=47) and both PMR and GCA (n=31) were used. RESULTS: In the ELISA using the human ferritin peptide, the sensitivity of IgG antibodies against ferritin was 92% in 36 GCA and/or PMR patients before initiation of treatment, 22/32 (69%) in patients with disease flares and 64/117 (55%) in the total cohort including treated and inactive patients. In controls, the false positive rate was 11/38 (29%) in systemic lupus erythematosus, 1/36 (3%) in rheumatoid arthritis, 0/31 (0%) in late onset rheumatoid arthritis, 3/46 (6.5%) in B-non-Hodgkin's lymphoma and 1/100 (1%) in blood donors. In the ELISA using the ferritin peptide of S epidermidis, 89% of 27 patients with untreated GCA and PMR were positive. CONCLUSION: Antibodies against the ferritin peptide were present in up to 92% of untreated, active GCA and PMR patients. They can be useful as a diagnostic marker of PMR and GCA.

[Esophagitis during immunosuppression]. / Ösophagitis unter Immunsuppression.

Esophagitis due to cytomegalovirus (CMV) has mostly been described in patients with acquired immunodeficiency syndrome (AIDS). Distal and hemorrhagic ulcerations are characteristic. A CMA esophagitis can, however, also occur in patients with no human immunodeficiency virus (HIV) infection as a complication of immunosuppressive therapy. In the case example presented here the disease was due to an excessive dosage of prednisolone medication over a period of many years. In all published cases of CMV esophagitis with rheumatic diseases, there was also a high dosage of glucocorticoid medication. To avoid complications regular rheumatological screening controls and adjustment of immunosuppressive therapy are therefore important to maintain control of the disease with low dosage glucocorticoids.

[Update on granulomatosis with polyangitis (GPA, Wegener's granulomatosis)]. / Update Granulomatose mit Polyangiitis (GPA, Wegener-Granulomatose).

Granulomatosis with polyangitis (GPA, Wegener's granulomatosis) is characterized by a granulomatous inflammation of the respiratory tract and a necrotizing ANCA-associated small to medium-size vessel vasculitis with a predilection for the lungs (pulmonary capillaritis) and kidneys (necrotizing glomerulonephritis). The disease evolves stage-wise and typically starts as inflammation of the respiratory tract followed by development of systemic vasculitis manifestations. Today, treatment is evidence-based and adapted according to activity and disease stage which has resulted in a significant improvement in long-term outcome. Early mortality during the first year of treatment poses one of the main problems and is a result of infections under immunosuppressive treatment. Furthermore, treatment of refractory disease activity which is often represented by granulomatous manifestations is still a challenge and may result in significant organ damage if not treated successfully.

Correlation of serum level of high mobility group box 1 with the burden of granulomatous inflammation in granulomatosis with polyangiitis (Wegener's).

OBJECTIVES: To investigate the correlation of serum levels of high mobility group box 1 (HMGB1) with the extent of granulomatous inflammation in granulomatosis with polyangiitis (GPA). METHODS: From 169 patients with GPA, 17 patients with granulomatous inflammation, without evidence of vasculitis were identified and 36 patients without measurable 'granuloma' formation. HMGB1 serum levels were determined and compared between the two groups, using a Mann-Whitney U test. Serum levels of 26 healthy individuals served as controls. In a further 21 patients with GPA with a pulmonary granulomatous manifestation from the study population, CT volumetry of 'granuloma' was performed. Volumes were compared with serum levels of HMGB1 (Spearman rank order test). RESULTS: Serum levels of HMGB1 were significantly higher in patients with predominant granulomatous disease than in patients without measurable 'granuloma' manifestations (6.44 ± 4.53 ng/ml vs 3.85 ± 2.88 ng/ml; p=0.0107). In both groups, levels of HMGB1 were significantly higher than in controls (2.34 ± 2.01 ng/ml; p<0.01). A positive correlation of HMGB1 serum levels with volumes of pulmonary 'granuloma' (r=0.761, p<0.0017) was seen. CONCLUSIONS: HMGB1 serum levels are significantly higher in GPA with predominant granulomatous manifestations and correlate with volumes of pulmonary 'granuloma'. HMGB1 may be used as a marker of the burden of granulomatous inflammation in GPA.

[The genetics of vasculitides]. / Genetischer Hintergrund der Vaskulitiden.

Genetic association studies have been of great value in the past by contributing to the understanding of pathophysiological mechanisms of chronic inflammatory and autoimmune diseases. Many genetic risk factors have been identified which confer susceptibility for one or several (autoimmune) disease(s). Using a candidate-gene approach, the first genetic risk factors and polymorphisms of vasculitides have been identified. Due to the rarity of autoimmune vasculitides often only small sample numbers have been generated and analysed, leading to inconsistent results. Furthermore, differences in ethnic background may complicate analysis. Only few of the detected risk factors have been reliably replicated in larger cohorts, such as the association of the PTPN22*620W allele with WG and MPA, the deficiency allele Pi*Z of the alpha1 antitrypsin gene and the HLA-DPB*04041 allele with WG and the HLA-DRB3/DRB4 with CSS. Genome-wide association studies (GWAS) offer the advantage of screening the whole genome for risk factors rather than relying on disease models postulated by the investigator; however, they require even larger sample sizes. Initial results from GWA studies are available for Behçet's disease and Kawasaki syndrome, which identified new genetic associations but require replication, especially since some of the identified risk factors could not be linked to pathophysiological pathways to date.

[Therapy of vasculitides: according to recommendations of the European League Against Rheumatism (EULAR) and European Vasculitis Study Group (EUVAS)]. / Therapiestrategien bei Vaskulitiden: Entsprechend den Empfehlungen der European League Against Rheumatism (EULAR) und der European Vasculitis Study Group (EUVAS).

The stringent definition of disease and activity stage as well as the performance of several controlled trials in the field of vasculitis in the past years now enables an evidence-based stage and activity adapted treatment, especially for ANCA-associated vasculitides. On the basis of available controlled trials, the European League Against Rheumatism (EULAR) and European Vasculitis Study Group (EUVAS) established and published recommendations for the management of vasculitides. This manuscript summarizes the treatment recommendations published in 2009 and highlights new studies which have been published since then.

Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme.

The target antigen of anti-neutrophil cytoplasm antibodies (ACPA; also known as ANCA) was isolated by affinity chromatography from supernatants of human neutrophils, stimulated with phorbol ester to induce degranulation. Sequence analysis of the antigen revealed 17 NH2-terminal amino acids (IVGGHEAQPHIRPIYMA), which have considerable sequence homology with known serine proteinases. Investigation of the enzymatic activity showed that the antigen is a neutral serine proteinase that is able to cleave elastin. Since the molecular weight of the antigen, its substrate specificity, and its inhibitor profile reported in this study are identical with those reported recently for proteinase 3, we conclude that ACPA are most probably directed against proteinase 3.

Neutrophil activation by anti-proteinase 3 antibodies in Wegener's granulomatosis: role of exogenous arachidonic acid and leukotriene B4 generation.

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA-positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody-related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA (10 microM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha-priming and AA incubation was ineffective. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-lipoxygenase pathway was demasked, with the appearance of excessive quantities of leukotriene (LT)B4, LTA4, and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-lipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4-related autocrine loop of cell activation. In contrast, the increased synthesis of platelet-activating factor in response to TNF-alpha-priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5-lipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.

Wegener's granulomatosis: anti-proteinase 3 antibodies are potent inductors of human endothelial cell signaling and leakage response.

Anti-neutrophil cytoplasmic antibodies (ANCAs) targeting proteinase 3 (PR3) have a high specifity for Wegener's granulomatosis (WG), and their role in activating leukocytes is well appreciated. In this study, we investigated the influence of PR3-ANCA and murine monoclonal antibodies on human umbilical vascular endothelial cells (HUVECs). Priming of HUVECs with tumor necrosis factor alpha induced endothelial upregulation of PR3 message and surface expression of this antigen, as measured by Cyto-ELISA, with a maximum occurrence after 2 h. Primed cells responded to low concentrations of both antibodies (25 ng-2.5 microg/ml), but not to control immunoglobulins, with pronounced, dose-dependent phosphoinositide hydrolysis, as assessed by accumulation of inositol phosphates. The signaling response peaked after 20 min, in parallel with the appearance of marked prostacyclin and platelet-activating factor synthesis. The F(ab)2 fragment of ANCA was equally potent as ANCA itself. Disrupture of the endothelial F-actin content by botulinum C2 toxin to avoid antigen-antibody internalization did not affect the response. In addition to the metabolic events, anti-PR3 challenge, in the absence of plasma components, provoked delayed, dose-dependent increase in transendothelial protein leakage. We conclude that anti-PR3 antibodies are potent inductors of the preformed phosphoinositide hydrolysis-related signal tranduction pathway in human endothelial cells. Associated metabolic events and the loss of endothelial barrier properties suggest that anti-PR3-induced activation of endothelial cells may contribute to the pathogenetic sequelae of autoimmune vasculitis characterizing WG.

[Classification and therapy of vasculitis according to recommendations of the European League Against Rheumatism (EULAR)]. / Klassifikation und leitliniengerechte Empfehlungen zur Diagnose und Therapie von Vaskulitiden.

Vasculitis is still being classified according the criteria of the American College of Rheumatology and the Chapel Hill Consensus Conference Definitions. Diagnostic criteria are currently being established. The classification criteria are based on the size of the inflamed blood vessel (e.g. large vessel vasculitis with inflammation of the aorta and its branches), clinical symptoms and findings (such as cephalalgia in giant cell arteritis) and histological findings. In recent years a definition of disease stages and activity has been established and a number of controlled trials have been carried out in order to provide evidence-based stage and activity adapted therapy regimens. Recommendations for the management of vasculitis have been published in 2009 by EULAR (European League Against Rheumatism). This article gives a review of the classification of vasculitis and summarizes the current European guidelines on management.

[The significance of health-related quality of life in systemic vasculitides]. / Bedeutung der gesundheitsbezogenen Lebensqualität bei systemischen Vaskulitiden.

Systemic vasculitides are a heterogenous group of chronic relapsing diseases. Initial data show that health-related quality of life (HRQoL) from the patient's perspective is reduced on all levels (physical, social and emotional). These studies suggest that generic questionnaires do not encompass all relevant and important levels of HRQoL in vasculitis. Moreover, data indicate that tiredness (fatigue) and reduced energy levels play a significant role. This article presents an overview of the existing literature.

Novel association of the CD226 (DNAM-1) Gly307Ser polymorphism in Wegener's granulomatosis and confirmation for multiple sclerosis in German patients.

Recently, there has been increasing evidence that a non-synonymous exchange (Gly307Ser) in the gene for CD226 is linked to several autoimmune diseases including type 1 diabetes, multiple sclerosis (MS), rheumatoid arthritis and Grave's disease. Here we present evidence that this polymorphism also predisposes to Wegener's granulomatosis (WG), an autoimmune condition belonging to the group of ANCA (antineutrophil cytoplasmic autoantibody)-associated vasculitides. We found a significant association of the 307Ser allele in separate panels of 520 Northern German (P=0.016, odds ratio (OR)=1.20) and 122 Southern German (P=0.020, OR=1.37) WG cases compared with 1226 healthy controls. The importance of this single-nucleotide polymorphism in the etiopathology of ANCA-associated vasculitides is supported by similar effect sizes that we found in British WG cases (n=105) and German patients with Churg-Strauss syndrome (n=119), which, however, miss significance level because of the relatively small cohorts available for these rare disorders. Finally, we confirm the association with MS in a cohort of 422 German patients (P=0.011, OR=1.23).

Association of UCP2 -866 G/A polymorphism with chronic inflammatory diseases.

We reported earlier that two mitochondrial gene polymorphisms, UCP2 -866 G/A (rs659366) and mtDNA nt13708 G/A (rs28359178), are associated with multiple sclerosis (MS). Here we aim to investigate whether these functional polymorphisms contribute to other eight chronic inflammatory diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Wegener' granulomatosis (WG), Churg-Strauss syndrome (CSS), Crohn's disease (CD), ulcerative colitis (UC), primary sclerosing cholangitis (PSC) and psoriasis. Compared with individual control panels, the UCP2 -866 G/A polymorphism was associated with RA and SLE, and the mtDNA nt13708 G/A polymorphism with RA. Compared with combined controls, the UCP2 -866 G/A polymorphism was associated with SLE, WG, CD and UC. When all eight disease panels and the original MS panel were combined in a meta-analysis, the UCP2 was associated with chronic inflammatory diseases in terms of either alleles (odds ratio (OR)=0.91, 95% confidence interval (95% CI): 0.86-0.96), P=0.0003) or genotypes (OR=0.88, (95% CI: 0.82-0.95), P=0.0008), with the -866A allele associated with a decreased risk to diseases. As the -866A allele increases gene expression, our findings suggest a protective role of the UCP2 protein in chronic inflammatory diseases.

Severe impaired respiratory ciliary function in Wegener granulomatosis.

OBJECTIVE: The pathogenesis of granulomatous inflammation in the respiratory tract and autoimmunity in Wegener granulomatosis (WG) are poorly understood. Since mucociliar clearance represents the first major line of defence in the respiratory tract and its breakdown facilitates chronic inflammation, we investigated ciliary beat frequency (CBF) in WG. METHODS: Nasal epithelial cells were obtained from 30 patients with WG with involvement of the upper respiratory tract, 12 patients with other inflammatory rheumatic disease and 10 healthy controls. CBF was measured at 5 and 24 h after collection. RESULTS: were correlated with clinical data. Results: CBF was significantly reduced in WG compared to disease and healthy controls after 5 and 24 h. In WG, CBF almost stagnated after 24 h. Reduction of CBF correlated with the cumulative number of immunosuppressive agents in WG, but not in disease controls. No correlation was found between CBF impairment and cyclophosphamide levels, disease extent, disease activity, disease duration, serological and microbiological findings, or inflammation markers. CONCLUSION: CBF is severely impaired in WG, potentially influenced by immunosuppressive treatment. To what extent CBF impairment and subsequent barrier dysfunction are caused by other factors still has to be elucidated. Supportive measures to improve mucociliary clearance should be discussed in patients with WG.

ANCA-associated vasculitides: pathogenetic aspects and current evidence-based therapy.

A lot of progress has been made regarding the therapy of ANCA-associated vasculitides in the past decades. Cyclophosphamide still is standard therapy for remission induction in generalized disease, however, duration and cumulative dose of cyclophosphamide have been curtailed successfully to reduce toxicity. MTX is a safe alternative for remission induction in early systemic disease and medications available for remission maintenance have been extended. Rituximab and TNFalpha-antagonists represent promising options for refractory disease. The current evidence of therapy of ANCA-associated vasculitides is summarized in this review.