Henko Tadema

High Prevalence of Autoantibodies to hLAMP-2 in Anti–Neutrophil Cytoplasmic Antibody–Associated Vasculitis

The involvement of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is controversial because of the absence of confirmatory data subsequent to the initial reports of their high prevalence in this disease. We characterized three assays for anti-hLAMP-2 antibodies: ELISA and Western blotting assays using unglycosylated recombinant hLAMP-2 expressed in Escherichia coli, and an indirect immunofluorescence assay using stably transfected ldlD cells that expressed glycosylated full-length hLAMP-2 on the plasma membrane. The assays detected autoantibodies to hLAMP-2 in human sera reproducibly and with comparable sensitivity and the assays gave the same results in 80.5% of the test panel of 40 selected positive and negative sera. In untreated patients at presentation, the frequencies of autoantibodies to LAMP-2 were 89%, 91%, and 80%, respectively, among three groups of patients with ANCA-associated vasculitis from Vienna, Austria (n=19); Groningen, the Netherlands (n=50) and Cambridge, United Kingdom (n=53). Prevalence of LAMP-2 autoantibodies was similar in both those with myeloperoxidase-ANCA and proteinase 3-ANCA. Furthermore, we detected LAMP-2 autoantibodies in two ANCA-negative patients. LAMP-2 autoantibodies rapidly became undetectable after the initiation of immunosuppressive treatment and frequently became detectable again during clinical relapse. We conclude that when robust assays are used, circulating autoantibodies to hLAMP-2 can be detected in most European patients with ANCA-associated vasculitis. Large-scale prospective studies are now needed to determine whether they are pathogenic or merely an epiphenomenon.

Vasculitis and infections : Contribution to the issue of autoimmunity reviews devoted to "autoimmunity and infection"

Abstract Infections are associated with secondary forms of vasculitis. However, there is increasing evidence that microbial agents play a role also in primary systemic vasculitides. For a long time it has been noted that Hepatitis B virus (HBV) is involved in polyarteritis nodosa (PAN) although the incidence of HBV-associated PAN seems to decline. Cryoglobulinemic vasculitis has been shown to be strongly associated with Hepatitis C Virus (HCV) infection, but this is most striking in Southern Europe and less in Northern Europe. Different microbial agents have been suggested to influence disease expression in other primary vasculitides but no specific association has been established. In Wegener’s Granulomatosis (WG) chronic carriage of Staphylococcus aureus (S. aureus) is associated with a strongly increased risk for relapsing disease. Various pathogenic pathways for this association have been suggested by clinical and experimental observations. Recent studies even suggest that S. aureus derived peptides, amongst others, may induce proteinase 3-ANCA via idiotypic–anti-idiotypic interactions. Treatment with co-trimoxazole in WG localized to the upper airways may result in (temporary) remission of the disease.

Bacterial DNA motifs trigger ANCA production in ANCA-associated vasculitis in remission

OBJECTIVES CpG motifs, which are highly prevalent in bacterial DNA, have been shown to trigger the production of ANCA in vitro by B lymphocytes from patients with active ANCA-associated vasculitis (AAV). Staphylococcus aureus is associated with relapses in AAV, and CpG motifs from staphylococcal DNA may trigger ANCA production in AAV patients in remission. We investigated the presence of ANCA-producing B lymphocytes during quiescent disease and tested the capacity of these cells to produce ANCA in response to CpG. METHODS Expression of Toll-like receptor 9 (TLR9) by B lymphocytes from AAV patients and controls was assessed. Peripheral blood mononuclear cells were isolated from 23 PR3-ANCA and 15 MPO-ANCA patients (33 quiescent, 5 active disease) and 14 healthy controls, and cultured for 12 days in the presence of cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) and IL-2. B-lymphocyte activation, differentiation, immunoglobulin production and in vitro ANCA production were studied. RESULTS TLR9 expression by B lymphocytes was comparable in AAV patients and controls. B lymphocytes were activated and differentiated towards a plasma cell phenotype in response to CpG-ODN and IL-2. ANCA were produced in vitro by 13 out of 23 PR3-ANCA patients and 3 out of 15 MPO-ANCA patients. CONCLUSIONS We conclude that ANCA-producing B lymphocytes can be present in the peripheral blood of AAV patients during remission. These autoreactive B lymphocytes are triggered by CpG-ODN and IL-2 to produce ANCA in vitro. CpG motifs may trigger the production of ANCA in vivo, contributing to the development of relapses in AAV.

Increased Expression of Toll-Like Receptors by Monocytes and Natural Killer Cells in ANCA-Associated Vasculitis

Introduction Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo. Methods Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed. Results In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4+ monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC. Conclusions In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation.

Bacterial infections in Wegener's granulomatosis: mechanisms potentially involved in autoimmune pathogenesis

Purpose of reviewWegeners granulomatosis is associated with bacterial infection, in particular nasal carriage of Staphylococcus aureus. Infection may play a role in the induction of autoimmunity as well as in the effector phase of the disease. Here, the current hypotheses aiming to explain the link between infections and Wegeners granulomatosis immunopathogenesis are reviewed and discussed. Recent findingsIn recent years, studies suggested that molecular mimicry could play a role in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV), either via direct mimicry between human lysosome-associated membrane protein-2 and bacterial FimH or indirectly via the development of antibodies against a peptide complementary to proteinase 3 (cPr3). More recent work has focused on Toll-like receptors (TLRs), a family of receptors specialized in the recognition of pathogen-associated molecular patterns. In animal models, it has been shown that TLR ligands can aggravate anti-MPO antibody-mediated disease. Furthermore, it was shown that a TLR9 ligand can trigger the production of ANCA in vitro by peripheral blood-derived B lymphocytes from AAV patients. The newly described process of ANCA-mediated neutrophil extracellular trap formation may provide an endogenous TLR9 ligand. Finally, TLR2 signaling is involved in the development of a Th17-driven immune response, consistent with skewing towards a Th17 T cell phenotype that has been observed in Wegeners granulomatosis. SummaryAlthough Wegeners granulomatosis pathophysiology is becoming better understood, the specific events leading to autoimmunity are not clear. Recent studies show that several mechanisms may be involved in linking infection to autoimmunity. Molecular mimicry may be involved, and a role for TLR signaling is suggested.

Mucin1 expression is enriched in the human stem cell fraction of cord blood and is upregulated in majority of the AML cases

OBJECTIVE Mucin1 is a membrane glycoprotein that is overexpressed in a variety of human cancers. Here, we analyzed the role of Mucin1 in human hematopoietic stem/progenitor cells as well as in acute myeloid leukemia (AML) cells. MATERIALS AND METHODS Mucin1 expression was determined within the normal stem cell and progenitor compartment, as well as in the AML CD34+ and CD34- subfractions of patient samples. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays in limiting dilution and progenitor frequencies in colony-forming cell (CFC) assays in methylcellulose, and consequences of elevated Mucin1 expression were studied using retroviral overexpression systems in cord blood (CB) CD34+ cells. RESULTS Ten percent of CB and 5% of peripheral blood CD34+ cells expressed Mucin1. Retroviral overexpression of Mucin1 in CB CD34+ cells resulted in elevated stem cell and progenitor frequencies as determined in LTC-IC and CFC assays without affecting differentiation, which coincided with increased proliferation. Overexpression of intercellular adhesion molecule-1, a ligand for Mucin1, in MS5 stromal cells further increased LTC-IC frequencies. Mucin1 overexpression was associated with increased nuclear factor-kappaB p50 nuclear translocation, suggesting that Mucin1-induced phenotypes involve increased cell survival mechanisms. Finally, we observed increased Mucin1 expression in 70% of the AML cases (n=24), suggesting that elevated Mucin1 levels might be involved in regulating the proliferative potential of the immature leukemic compartment as well. CONCLUSIONS Our data indicate that hematopoietic stem cells as well as CD34+ AML subfractions are enriched for Mucin1 expression, and that overexpression of Mucin1 in CB cells is sufficient to increase both progenitor and LTC-IC frequencies.

Reactivity against Complementary Proteinase-3 Is Not Increased in Patients with PR3-ANCA-Associated Vasculitis

The etiology of anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) is unknown, but the association between infections and autoimmunity has been studied extensively. In 2004, a novel theory was proposed that could link infection and autoimmunity. This ‘theory of autoantigen complementarity’ was based on the serendipitous finding of antibodies against complementary-PR3 (cPR3) in patients with PR3-ANCA-associated vasculitis. cPR3 demonstrated homology to several bacterial proteins, and it was hypothesized that PR3-ANCA develop in response to anti-cPR3 antibodies, as a consequence of the anti-idiotypic network. These data have not been confirmed in other patient cohorts. We investigated the presence of anti-cPR3 antibodies in a Dutch cohort of PR3-ANCA-associated vasculitis patients. Anti-cPR3 reactivity was determined in serum using ELISA. Two separate batches of cPR3 were used to determine reactivity in two separate cohorts of PR3-ANCA-associated vasculitis patients. We found that anti-cPR3-reactivity was not increased in our PR3-ANCA-associated vasculitis patients, in comparison to control groups. Further research will be necessary to prove the concept of autoantigen complementarity in autoimmune diseases.

Increased frequency of circulating IL-21 producing Th-cells in patients with granulomatosis with polyangiitis (GPA)

IntroductionThe present study aimed to explore a possible role for IL-21 producing Th-cells in the immunopathogenesis of granulomatosis with polyangiitis (GPA).MethodsPeripheral blood from 42 GPA patients in remission and 29 age-matched healthy controls (HCs) were stimulated in vitro, and the frequencies of IL-21 producing Th-cells were determined by flow cytometry. Since Th17-cells produce a low level of IL-21, IL-17 was also included in the analysis. Given that IL-21 is a hallmark cytokine for T follicular helper cells (TFH), we next evaluated the expression of their key transcription factor BCL-6 by RT-PCR and flow cytometry. To investigate the effect of IL-21 on autoantibody-production, PBMCs from GPA patients were stimulated in vitro with BAFF/IL-21 and total IgG and ANCA levels were measured in supernatants. In addition, the expression of IL-21-receptor on B-cells was analyzed.ResultsPercentages of IL-21 producing Th-cells were significantly elevated in GPA-patients compared to HCs, and were restricted to ANCA-positive patients. The expression of BCL-6 was significantly higher in ANCA-positive GPA-patients, as compared with ANCA-negative patients and HCs. IL-21 enhanced the production of IgG and ANCA in vitro in stimulated PBMCs from GPA patients. No difference was found in the expression of the IL-21-receptor on B-cells between ANCA-negative patients, ANCA-positive patients, and HCs.ConclusionThe increased frequency of circulating IL-21 producing Th-cells in ANCA-positive GPA patients and the stimulating capacity of IL-21 on ANCA-production suggest a role for these cells in the immunopathogenesis of GPA. Blockade of IL-21 could constitute a new therapeutic strategy for GPA.