J. W. Cohen Tervaert

Predominance of IgG1 and IgG4 subclasses of anti-neutrophil cytoplasmic autoantibodies (ANCA) in patients with Wegener's granulomatosis and clinically related disorders.

In view of the supposed hypersensitivity, the elevated levels of IgE, and the occurrence of eosinophilia reported in Wegeners granulomatosis and related conditions, we studied the IgG subclass distribution of ANCA directed against a 29‐kD serine protease and myeloperoxidase (MPO) in 41 untreated ANCA‐positive patients with several forms of active vasculitis and/or glomerulonephritis. We found that both 29‐kD ANCA and MPO ANCA were predominantly of the IgG1 and IgG4 subclass in all groups of patients. The additional presence of IgG3 subclass was associated with renal involvement. We compared the subclass distribution of ANCA with that of total IgG subclass levels, and with the IgG subclass distribution of antibodies to cytomegalovirus (CMV) as a persistent endogenous antigen and antibodies to tetanus toxoid (TT) as an exogenous recall antigen. Total levels of IgG4 were elevated in the majority of the patients together with elevated IgG1 levels. Antibodies to CMV and TT, however, had the same subclass distribution as found in normals and did not show enhanced IgG4 expression. ANCA belong predominantly to the IgG1 and IgG4 subclass, which may suggest that the production of ANCA is related to recurrent exposition to the antigen(s) involved, possibly as part of a hypersensitivity reaction.

European therapeutic trials in ANCA-associated systemic vasculitis: disease scoring, consensus regimens and proposed clinical trials EUROPEAN COMMUNITY STUDY GROUP ON CLINICAL TRIALS IN SYSTEMIC VASCULITIS ECSYSVASTRIAL (BMHl-Cr93-1078)

In previous phases of this project, proteinase 3 (PR3) and myeloperoxidase (MPO), the main antigenic target molecules of antineutrophil cytopiasmic antibodies, were isolated and applied in standardized ELISAs. In this study, standardized ELISAs with three PR3 preparations (from Copenhagen (CO), Raisdorf (RS) and Leiden (LF)) and one MPO preparation (from Copenhagen), were evaluated in a large retro-and prospective clitiical study. New patients (n=174) with primary systemic vasculitis (Wegeners granulomatosis, microscopic polyangiitis and idiopathic rapidly progressive glomerulonephritis, classical PAN and Churg-Strauss Syndrome) were included. Retrospectively, another 190 patients were evaluated. Furthermore control sera were obtained from patients with other forms of vasculitis, glomerulonephritis or granulomatous diseases (disease controls, n = 184) and healthy donors (healthy controls, n = 728). All patients were categorized by a system based on clinical and histoiogical data. Patients were followed up for at least 1 year after diagnosis in order to evaluate a possible correlation between ANCA levels and disease activity. The sensitivity of the anti-PR3 assays for histologically proven WG was between 59% and 69% in new patients, with a sensitivity of 22% for the anti-MPO assay. Similar figures were found for patients with clinically suspected WG. This was comparable with the results of the IIF test. In MPA and IRPGN a larger percentage of patients had antiMPO antibodies than in WG. Only a few patients with PAN and CSS were investigated, and most of these were negative in the ELISAs. The specificity ofthe assays for disease controls was 89-91% for the anti-PR3 assays and 95% for the anti-MPO assay. In the healthy controls the specificity was 98-99%. The specificity of the IIF test was 97% for a cANCA pattern and 81 % for a pANCA pattern in disease controls. The combination of cANCA with anti-PR3 and pANCA with anti-MPO both had a specificity of 99%. Further details will be presented during the meeting, in addition to the results of a follow-up study with correlation ofdisease activity and ANCA level. From this study we can conclude that ELISAs using purified PR3 or MPO are not more sensitive than the IIF test. However, the anti-MPO assay is more specific for systemic vascuitis as compared to disease controls with related diseases. Furthermore, the combination of the IIF test with antigen-specific ELISAs is very specific for the diagnosis Wegetiers granulomatosis, microscopic polyangiitis and idiopathic rapidly progressive gtomerulonephritis.

Regulatory B cells in ANCA-associated vasculitis

Objectives B cells have immunoregulatory function acting as antigen-presenting cells. A separate subset of interleukin (IL)-10 producing B cells (Breg) regulating T cell mediated immunity has been identified. In the present study, we investigated the role of Breg in antineutrophil cytoplasmic antibodies-associated vasculitis (AAV). Methods 17 healthy controls (HCs) and 41 patients with AAV were enrolled. 30 patients with AAV were in remission. Furthermore, 11 patients with AAV with active disease were studied. Breg were defined as IL-10+CD19+ B cells upon culture with cytosine-phosphate-guanosine oligodeoxynucleotide (CpG ODN) 2006. Next to Breg, CD4+CD127lowCD25hiCD39neg/CD39+ regulatory T-cells (Treg), interferon (IFN)γ+, IL-4+ and Il-17A+T helper cell subsets were determined via flow cytometry. Results Patients with active or quiescent disease showed a diminished fraction of Breg as compared with HCs. The frequency of IFNγ+ T helper cells was negatively associated with Breg in untreated AAV in remission but not in active vasculitis or in HCs. Interestingly, the total Treg population and the CD39+ Treg subpopulation correlated positively with Breg in inactive patients with AAV. Conclusions IL-10 producing B cells are diminished in AAV. Furthermore, Breg might regulate Th1 cells and are associated with Treg in quiescent AAV. Suppression of Th1 cells by Breg may be insufficient in active AAV.

Autoimmunity to lysosomal enzymes: new clues to vasculitis and glomerulonephritis?

The demonstration of autoantibodies to neutrophil cytoplasmic antigens (ANCA) in (1) systemic vasculitides such as Wegeners granulomatosis, classic polyarteritis nodosa and the Churg Strauss syndrome and (2) idiopathic crescentic glomerulonephritis has placed these disorders within the spectrum of autoimmune diseases. The target antigens have been identified as myeloid lysosomal enzymes that are involved in tissue destruction by neutrophils. Recent data suggest a pathogenetic role for these autoimmune responses. The autoantibodies may activate neutrophils, resulting in extensive tissue necrosis. Alternatively, T-cell-mediated immunity to these positively-charged enzymes that localize to polyanionic basement membranes may, in concert with the presence of autoantibodies, result in glomerulonephritis and vasculitis.

Secondary prevention with fluvastatin decreases levels of adhesion molecules, neopterin and C-reactive protein

BACKGROUND Treatment of hypercholesterolaemia with HMG-CoA reductase inhibitors results in an earlier reduction of morbidity and mortality than expected from trials using conventional cholesterol-lowering therapies. Possible explanations for this effect include stimulation of angiogenesis, improvement of endothelial function, plaque stabilisation, inhibition of coagulation and/or thrombocyte aggregation and inhibition of the inflammatory response associated with atherosclerosis. METHODS We investigated whether statins exert their effects by inhibition of endothelial activation, inflammation and/or monocyte/macrophage activation by measuring plasma levels of soluble cell adhesion molecules, neopterin and C-reactive protein upon treatment with fluvastatin for a period of 12 months in patients with established atherosclerosis and hypercholesterolaemia. RESULTS Blood samples were taken at baseline and at 3 and 12 months after starting treatment with fluvastatin 80 mg daily. Upon treatment, a reduction of s-ICAM-1 (956.3+/-123.6 vs. 745.4+/-127.4 vs. 674.9+/-70.8 ng/ml, P<0.05) and s-E-selectin (58.6+/-6.7 vs. 47.0+/-6.1 vs. 44.9+/-3.2 ng/ml, P<0.01) was observed. In addition, levels of neopterin decreased, albeit transiently (7.1+/-0.7 vs. 6.0+/-0.5 vs. 6.5+/-0.8 nmol/l, P=0.02), suggesting a reduction in monocyte/macrophage activity. Moreover, we found a decrease in levels of C-reactive protein during follow-up (5.21+/-2.0 vs. 3.18+/-0.7 vs. 1.95+/-0.3 mg/l, P<0.05), compatible with a reduction in inflammatory activity. CONCLUSION We conclude that statins have a combined beneficial effect on monocyte/macrophage activity, endothelial function and systemic inflammatory activity.

Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener’s granulomatosis?

The majority of patients with Wegener’s granulomatosis (WG) are chronic nasal carriers of Staphylococcus aureus. Chronic nasal carriage of S. aureus is associated with an increased risk of developing a relapse of the disease. The mechanism by which this occurs is still unknown. We hypothesized that a cationic protein of S. aureus, staphylococcal acid phosphatase (SAcP), acts as a planted antigen and initiates glomerulonephritis and vasculitis in patients with WG. In order to test the hypothesis that SAcP can act as a planted antigen in WG, we studied the ability of SAcP to bind to human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells. We also studied whether this binding can be prevented by preincubation with an anionic protein, and whether binding of SAcP activates endothelial cells. We also evaluated whether antibodies in sera of patients with WG are able to bind to endothelial cell‐bound SAcP. The results show that SAcP can act as a planted antigen by binding to both types of endothelial cells in a concentration‐dependent manner. Binding of concentrations as low as 4 μg/ml can be detected on HUVEC within 5 min of incubation. Binding of SAcP to endothelial cells was charge‐dependent but did not activate endothelial cells. Finally, endothelial cell‐bound SAcP was recognized by sera of patients with WG. The data suggest a possible pathogenic role for SAcP by acting as a planted antigen thereby initiating glomerulonephritis and vasculitis in patients with WG.

Leukocyte activation and cytokine production during experimental human endotoxemia

Background: At present, it is unclear whether in experimental endotoxemia, the pro-inflammatory response observed in healthy volunteers is followed by an anti-inflammatory response, as observed in patients with sepsis. We studied the evolution of a number of inflammatory parameters during a prolonged period (24 h) after infusion of endotoxin in healthy subjects. Methods: Six healthy male subjects received an infusion of endotoxin (4 ng/kg body weight). Blood was drawn before, and at various intervals up to and including 24 h after, endotoxin infusion. Circulating cytokine levels, leukocyte activation surface markers, plasma lactoferrin, and neopterin levels were measured, and clinical signs and symptoms were noted during a 24-h period. Monocyte and neutrophil activation after endotoxin infusion is investigated in relation to the inflammatory response. The extent of neutrophil and monocyte activation was correlated to clinical markers and blood levels of inflammatory mediators and cytokines. Results: Tumor necrosis factor-alpha appeared 30 min after infusion in the circulation, peaking (5665+/-1910 pg/ml) at 2 h. Interleukin-10 appeared 60 min after infusion, peaking (427+/-348 pg/ml) at 3 h. The expression of leukocyte activation markers increased significantly after infusion. Expression of HLA-DR on monocytes decreased significantly after 3 h (P=0.03). There was a correlation between the TNF-alpha:IL-10 ratio and the CD11b:HLA-DR ratio (P=0.03). Conclusions: During experimental human endotoxemia, an initial pro-inflammatory response is successfully compensated by an anti-inflammatory response, leading to homeostasis. This is in contrast to what happens in septic patients with compensatory anti-inflammatory response syndrome. The inflammatory balance, expressed as the cytokine pro:anti-inflammatory ratio, is reflected at a cellular level.

Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis

Anti‐neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegeners granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3‐ANCA do not always correspond to clinical disease activity.

Prevalence of anti-endothelial cell antibodies in idiopathic pulmonary arterial hypertension

To the Editors: Pulmonary arterial hypertension (PAH) is a rare disease often resulting in right-sided heart failure and premature death 1. PAH is idiopathic (IPAH), heritable, or related to conditions such as connective tissue diseases (CTD) 2. IPAH prognosis remains poor despite improved patient survival with current treatment options. Therefore, elucidating the pathophysiology of IPAH is important for the discovery of novel therapeutic approaches. Inflammation and immune reactivity have been implicated in the pathophysiology of IPAH. In 2005, Tamby et al. 3 described the presence of anti-endothelial cell antibodies (AECA) in IPAH, pointing at the involvement of humoral immunity. AECA are a heterogeneous family of auto-antibodies capable of reacting with different endothelial cell (EC) structures 4. In PAH, pulmonary EC dysfunction is considered a key player in the initiation and progression of the disease 5. Systemic sclerosis (SSc) is a paradigm of autoimmune PAH, as 10–15% of these patients develop PAH 6. Interestingly, in SSc, immunoglobuliln (Ig)G AECA targeting cell-surface antigens have indeed been shown to induce EC dysfunction 7, 8. Thus, IgG AECA-induced endothelial dysfunction may be the initiating event in IPAH. Whereas IgG auto-antibodies are considered pathogenic in various autoimmune diseases, IgM auto-antibodies have been proposed to be protective in these diseases 9. Regarding a pathophysiological role of AECA, it is important to identify whether AECA are reactive with EC-surface antigens. To corroborate and further investigate the role of the humoral immune system in IPAH, we aimed to study the prevalence of IgG, as well as IgM AECA targeting cell-surface EC antigens using a cell-based ELISA with viable human umbilical vein EC (HUVEC). We screened five study cohorts for the presence of AECA. 1) 29 IPAH patients; mean pulmonary artery …

Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener's granulomatosis

Elevated soluble thrombomodulin (sTM) levels are an accepted marker of endothelial damage. The physiological significance of plasma endothelial protein C receptor (sEPCR) levels is not known. To assess the relevance of this plasma protein in Wegener’s granulomatosis (WG), sEPCR levels were measured in sera obtained from WG patients and related to disease activity, sTM levels, and other known markers of disease activity. In total, 129 sera (37 at active disease, 92 during follow‐up) from 31 WG patients were tested. During active disease, eight (22%) and 17 (46%) out of 37 active sera had elevated levels of sEPCR and sTM, respectively (NS); sEPCR (r = 0·39; P = 0·02) and sTM (r = 0·53; P < 0·01) levels correlated with disease activity (Birmingham Vasculitis Activity Score). Analysis of longitudinal sera revealed a significant increase in sEPCR (P = 0·01) and sTM (P = 0·04) levels prior to the moment of a relapse. Corrected for renal function, the increase in sEPCR remained significant (P = 0·04) whereas sTM did not (NS). Levels of sEPCR correlated with sTM levels (r = 0·32; P < 0·001). Plasma levels of sEPCR respond to changes in the disease in patients with WG.