P. van Paassen

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.

Evaluation of a new fluorescent-enzyme immuno-assay for diagnosis and follow-up of ANCA-associated vasculitis

In this study we have evaluated a new, fully automated fluorescent-enzyme immuno-assay (FEIA) for detection and quantification of anti-PR3 and anti-MPO ANCA in diagnosis and follow-up of ANCA-associated small vessel vasculitis (AAV). PR3- and MPO-ANCA were determined by FEIA technology in (1) sera of 87 consecutive patients with biopsy-proven, pauci-immune necrotizing crescentic glomerulonephritis (NCGN) and 72 controls; (2) 120 sera (60 patients with Wegener’s granulomatosis and 60 controls) that were previously used in a multicentre comparison of direct and capture ELISAs for PR3-ANCA; (3) in samples preceding relapse in 23 PR3-AAV patients with and 23 matched PR3-AAV patients without relapse for prediction of relapses. PR3- and/or MPO-ANCA detection in pauci-immune NCGN by FEIA revealed an overall sensitivity of 82.8%. The FEIA specificity was 96% and 100% for PR3- and MPO-ANCA, respectively. The overall sensitivity of MPO- and PR3-ANCA could be increased to 88.5% by lowering the cut-off values without affecting the specificity (ROC-curve analysis), which is similar to a multistep ANCA procedure that combines indirect immunofluorescence with direct and capture ELISAs. The sensitivity for Wegener’s granulomatosis (WG) of the PR3-ANCA FEIA (60%) was more comparable to direct ELISAs (64%) than to capture ELISAs (74%). A rise of 100% in ANCA level as measured by FEIA appeared optimal (ROC-curve) for prediction of relapses and such a rise was observed in 26 patients. In 18 of these 26 patients the rise was followed by a relapse (PPV 69%), whereas in 15 of the 20 patients without a rise no relapse was observed (NPV 75%). In conclusion, detection of PR3- and MPO-ANCA by FEIA has excellent performance in terms of diagnosis of AAV patients. Furthermore, detection of rises in PR3-ANCA by FEIA for prediction of relapses gives results comparable to other techniques.

Evaluation of the FIDIS vasculitis multiplex immunoassay for diagnosis and follow-up of ANCA-associated vasculitis and Goodpasture's disease

Abstract:  We have evaluated a new‐multiplex immunoassay (FIDIS Vasculitis) for simultaneous detection and quantification of anti‐MPO, ‐PR3, and ‐glomerular basement membrane (GBM) antibodies in diagnosis and follow‐up of ANCA‐associated vasculitides (AAV) and Goodpastures disease. ANCA were determined in sera of (a) 87 consecutive patients with biopsy‐proven pauci‐immune NCGN and 72 controls; (b) 9 patients with Goodpastures disease; and (c) 60 WG patients and 60 controls, previously used in a multicenter comparison of direct and capture ELISA for PR3‐ANCA. Finally, for prediction of relapses, PR3‐ANCA was measured in samples preceding relapse in 23 PR3‐AAV patients and in 23 matched PR3‐AAV patients without relapse. The relative sensitivity of the FIDIS Vasculitis assay was 97.4% for MPO‐ANCA and 92.3% for PR3‐ANCA; specificity was 100% and 97.2%, respectively. Evaluation of the anti‐GBM antibody detection revealed a sensitivity of 100% and a specificity of 99.6%. The sensitivity for WG of the PR3‐ANCA detection (71.6%) approached the performance of capture ELISA (74%), although at the cost of specificity (96.7% versus 100%). For prediction of relapses a rise of 50% in ANCA level by FIDIS Vasculitis appeared optimal (ROC curve) for prediction of relapses. However, as compared to capture ELISA, both positive (63% versus 76%) and negative (68% versus 72%) predictive values were reduced. In conclusion, simultaneous detection of anti‐MPO, ‐PR3, and ‐GBM antibodies in the multiplex FIDIS Vasculitis assay has excellent performance in terms of diagnosis of patients with AAV or Goodpastures disease. However, detection of rises in PR3‐ANCA for prediction of relapses gives less optimal results when compared to capture ELISA.

Type II cryoglobulinemia is not associated with hepatitis C infection: the Dutch experience.

Abstract:  Mixed cryoglobulinemia (MC) are cryoprecipitable immunocomplexes. In type II MC, a combination of polyclonal and monoclonal immunoglobulins is found, whereas in type III a combination of polyclonal immunoglobulins is detected. MC is usually associated with hepatitis C (HCV) infection as has been found in studies that have been performed in countries with a high prevalence of HCV. Because HCV has an extremely low prevalence in the Netherlands (<0.1% of the population), we wondered whether HCV is also associated with MC in our regional referral center. To answer this question, we tested consecutive patients with type II MC for HCV antibodies and for HCV‐mRNA by polymerase chain reaction (PCR). Between January 2000 and June 2005, 22 patients tested positive for type II MC. Seven patients had essential MC, 2 patients had MC due to a lymphoproliferative disease, 10 patients had MC in the context of a systemic autoimmune disease, and 3 patients had MC without a clear diagnosis. HCV antibodies were not detected in any of the 22 patients. Also, all samples tested negative for HCV‐mRNA. During follow‐up none of these patients developed an HCV infection. In summary, the estimated occurrence of HCV in 60–90% of patients with MC is not found in our region where MC is only infrequently associated with HCV. In a substantial proportion of our patients a really “essential MC” is observed. A search for yet unknown etiological factors is clearly needed in these patients, who frequently have severe renal involvement warranting aggressive immunosuppressive therapy.

Prevalence and prognostic relevance of cardiac involvement in ANCA-associated vasculitis: Eosinophilic granulomatosis with polyangiitis and granulomatosis with polyangiitis

BACKGROUND To investigate the prevalence and prognostic relevance of cardiac involvement in an ANCA-associated vasculitis (AAV) population of eosinophilic granulomatosis with polyangiitis (EGPA) and granulomatosis with polyangiitis (GPA) patients. METHODS Prospective cohort study of fifty EGPA and forty-one GPA patients in sustained remission without previous in-depth cardiac screening attending our clinical immunology outpatient department. Cardiac screening included clinical evaluation, ECG, 24-hour Holter registration, echocardiography and cardiac magnetic resonance imaging (CMR) with coronary angiography and endomyocardial biopsy upon indication. Fifty age-, sex- and cardiovascular risk factor-matched control subjects were randomly selected from a population study. Long-term outcome was assessed using all-cause and cardiovascular mortality. RESULTS A total of 91 AAV-patients (age 60±11, range 63-87years) were compared to 50-matched control subjects (age 60±9years, range 46-78years). ECG and echocardiography demonstrated cardiac abnormalities in 62% EGPA and 46% GPA patients vs 20% controls (P<0.001 and P=0.014, respectively). A total of 69 AAV-patients underwent additional CMR, slightly increasing the prevalence of cardiac involvement to 66% in EGPA and 61% in GPA patients. After a mean follow-up of 53±18months, presence of cardiac involvement using ECG and echocardiography in AAV-patients showed increased all-cause and cardiovascular mortality (Log-rank P=0.015 and Log-rank P=0.021, respectively). CONCLUSION Cardiac involvement in EGPA and GPA patients with sustained remission is high, even if symptoms are absent and ECG is normal. Moreover, cardiac involvement is a strong predictor of (cardiovascular) mortality. Therefore, risk stratification using cardiac imaging is recommended in all AAV-patients, irrespective of symptoms or ECG abnormalities.

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 …

Functional implications of IgG anti-endothelial cell antibodies in pulmonary arterial hypertension

Abstract The objective of this study was to research the functionality of anti-endothelial cell antibodies (AECA) in pulmonary arterial hypertension (PAH) by assessing the effects of IgG from AECA-positive PAH patients on the induction of adhesion molecules on human umbilical vein endothelial cells (HUVECs) and on the production of pro-inflammatory cytokines and chemokines by HUVECs. To achieve this purified IgG from 28 PAH patients were included. IgG from systemic sclerosis (SSc) (n = 58) and systemic lupus erythematosus (SLE) (n = 16) patients without PAH were included as disease controls. Intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression on HUVECs, incubated with patient IgG, were quantified by flow cytometry. Production of interleukin (IL)-1β, -6, -8, and CC chemokine ligand 2 (CCL2) by HUVECs, incubated with patient IgG, were quantified by multiplex flow cytometry. Our results showed that IgG from AECA-positive PAH, SSc and SLE patients induced significantly higher expression of ICAM-1, VCAM-1, and E-selectin and production of IL-6, -8, and CCL2 compared to IgG from AECA-negative patients and IgG from healthy controls. Like in SLE and SSc, IgG from AECA-positive PAH patients can activate endothelial cells to a pro-adhesive and pro-inflammatory state. Therefore, IgG AECA could play a pathogenic role by inducing inflammatory injury of vascular endothelium which is considered a key player in the initiation and progression of PAH.

Anti‐oxidized low‐density lipoprotein antibodies in myeloperoxidase–positive vasculitis patients preferentially recognize hypochlorite‐modified low density lipoproteins

Many patients surviving vasculitis are prone to accelerated atherosclerosis and often have enhanced levels of antibodies to oxidized low‐density lipoprotein (oxLDL). To measure anti‐oxLDL antibodies, oxidation of LDL is achieved with copper (Cu) or malondialdehyde (MDA). Because, in vivo, LDL may be oxidized with myeloperoxidase (MPO) or its product hypochlorite, we measured anti‐hypochlorite LDL antibodies in patients with vasculitis, haemodialysis patients and healthy controls. A newly developed enzyme‐linked immunosorbent assay (ELISA) was used to detect antibodies to oxLDL as modified by hypochlorite. Results are compared with data obtained by standard LDL oxidation using MDA–LDL or Cu–LDL as substrate. Results were compared between anti‐neutrophil cytoplasmic antibodies (ANCA)‐associated vasculitis (AAV) patients (n = 93), haemodialysis (HD) patients (n = 59) and healthy controls (HC; n = 43). Furthermore, patients with MPO–ANCA‐associated vasculitis (n = 47) were compared with patients with proteinase 3 (PR3)–ANCA associated vasculitis (n = 46). Optimal cut‐off points were determined by receiver operator characteristic (ROC) curve analysis. Anti‐oxLDL antibodies are enhanced in AAV patients (MDA–LDL and hypochlorite–LDL) and in HD patients (hypochlorite–LDL), when compared to HC. Furthermore, patients with MPO–ANCA‐associated vasculitis had higher levels of antibodies to hypochlorite–LDL than patients with PR3–ANCA‐associated vasculitis. Our newly developed assay, in which hypochlorite–LDL is used as substrate, seems a more sensitive assay than traditional assays to measure oxLDL antibodies. Furthermore, our results suggest that enhanced MPO‐mediated LDL oxidation occurs in patients with MPO–ANCA.

Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension?

Endothelial cell (EC) apoptosis seems to play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). We aimed to test the hypothesis that circulating anti‐endothelial cell antibodies (AECA) of PAH patients induce EC apoptosis. Immunoglobulin (Ig)G was purified from sera of PAH patients (n = 26), patients with systemic lupus erythematosus (SLE) nephritis without PAH (n = 16), patients with systemic sclerosis (SSc) without PAH (n = 58) and healthy controls (n = 14). Human umbilical vein endothelial cells (HUVECs) were incubated with patient or healthy control IgG for 24 h. Thereafter, apoptosis was quantified by annexin A5 binding and hypoploid cell enumeration by flow cytometry. Furthermore, real‐time cell electronic sensing (RT–CES™) technology was used to monitor the effects of purified IgG from patient and healthy control IgG on HUVECs. As demonstrated previously, IgG of AECA‐positive SLE nephritis patients (n = 7) induced a higher percentage of apoptosis of HUVECs compared to IgG of AECA‐negative SLE nephritis patients and healthy controls. Furthermore, IgG of AECA‐positive SLE nephritis patients induced a marked decrease in cell index as assessed by RT–CES™ technology. IgG of AECA‐positive PAH patients (n = 12) and SSc patients (n = 13) did not alter the percentage of HUVEC apoptosis or cell index compared to IgG of AECA‐negative PAH and SSc patients and healthy controls. AECA‐positive PAH patients, in contrast to SLE nephritis patients, do not have circulating IgG AECA that enhances apoptosis of HUVECs in vitro. Further studies should focus on other mechanisms by which AECA may enhance EC apoptosis in PAH, such as antibody‐dependent cell‐mediated cytotoxicity.

ANCA-Associated Vasculitis: Microscopic Polyangiitis, Eosinophilic Granulomatosis With Polyangiitis (Churg–Strauss Syndrome) and Granulomatosis With Polyangiitis (Wegener’s Granulomatosis)

Antineutrophil cytoplasmic antibodies (ANCA) associated systemic vasculitis (AAV) is a grouping of systemic small-vessel vasculitides, autoimmune diseases wherein patients have pathogenic autoantibodies reacting to myeloperoxidase (MPO) or proteinase 3 (PR3). AAV comprises three disease types: microscopic polyangiiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg–Strauss Syndrome), and granulomatosis with polyangiitis (GPA; formerly known as Wegeners granulomatosis). With current therapy, AAV has become a chronic relapsing rather than a fatal condition. With this prolonged survival, patients may experience long-term sequelae as a result of vasculitis or its treatment. Large-scale evidence shows that patients with AAV have an increased mortality rate as a consequence of cardiovascular disease. Moreover, several studies have shown that cardiac involvement in these patients is an independent predictor of mortality. More importantly, both symptomatic and asymptomatic patients have demonstrated cardiac involvement that was associated with worse outcome. Despite this overwhelming evidence, current guidelines do not recommend cardiac screening in AAV patients. Finally, the impact of glucocorticoids and/or additional immunosuppressive regimens is poorly defined and it remains largely unknown whether these treatment strategies can prevent or reverse cardiac damage. Herein, we review all published data on cardiac involvement and its prognostic relevance in AAV and the current therapeutic evidence to prevent or treat these abnormalities.