Frank Moosig

Cytokine profiles in Wegener's granulomatosis: Predominance of type 1 (Th1) in the granulomatous inflammation

OBJECTIVEnTo determine whether a specific cytokine pattern (type 1 [Th1] or type 2 [Th2]) predominates in Wegeners granulomatosis (WG), by evaluating interferon-gamma (IFNgamma) and interleukin-4 (IL-4) expression in different compartments of the body (i.e., biopsied nasal mucosal tissue [NBS], bronchoalveolar lavage [BAL] fluid, and peripheral blood [PB]) and comparing the findings with those in disease and healthy control subjects.nnnMETHODSnCompetitive reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to assess IFNgamma and IL-4 expression in T cell clones (TCC), T cell lines (TCL), and polyclonal CD4+ and CD8+ cells derived from NBS, BAL, and PB.nnnRESULTSnPatients with WG and chronic rhinitis were found to share in situ production of messenger RNA (mRNA) specific for IFNgamma (Th1). Only 2 patients with WG expressed IL-4, whereas IL-4 mRNA PCR products were found in inflamed tissues of the disease control patients. The granuloma-derived T cells of WG patients produced only IFNgamma, while TCC, TCL, and CD4+ and CD8+ T cells from BAL and PB produced mainly IFNgamma.nnnCONCLUSIONnOur data indicate that a Thl cytokine pattern predominates in the granulomatous inflammation in patients with WG.

Costimulatory molecules in Wegener's granulomatosis (WG): lack of expression of CD28 and preferential up-regulation of its ligands B7-1 (CD80) and B7-2 (CD86) on T cells

T cells are most likely to play an important role in the pathogenesis of WG, and recently a predominant Th1 pattern of immune response has been demonstrated in granulomatous inflammation. Since the expression of costimulatory molecules has a significant impact on the cytokine profile and proliferation response of T cells, the goal of this study was to characterize the expression of costimulatory molecules (CD28, CTLA‐4 (CD152), B7‐1 (CD80), B7‐2 (CD86)) on T cells, monocytes and B cells in WG, and to correlate the findings with clinical parameters such as disease activity, extent and therapy. WG patients (nu2003=u200324) and healthy controls (HC; nu2003=u200317) were examined for the expression of costimulatory molecules by fluorescence‐activated cell sorter analysis, both in whole peripheral blood and after in vitro activation of T cells and antigen‐presenting cells. Results were correlated with clinical data. The expression of CD28 on CD4+ and CD8+ cells was significantly lower in WG than in HC (CD28+u200381.4% in WG versus 97.9% of CD4+ cells (Pu2003<u20030.0001); CD28+u200344.6% in WG versus 68.5% of CD8+ cells (Pu2003<u20030.00001)), both in peripheral blood and after in vitro activation. A lower percentage of monocytes was B7‐2+ in WG than in HC in peripheral blood, whereas no significant differences in the expression of B7‐1 and B7‐2 were observed after in vitro stimulation of monocytes and B cells. After in vitro activation a significantly higher percentage of B7‐1+ andu2003B7‐2+ T cells was seen in WG. There was no significant difference in the CTLA‐4 expression pattern between WG and HC. The percentage of CD28+ lymphocytes correlated negatively with the Disease Extent Index cumulated over the course of disease (ru2003=u2003−0.46, Pu2003=u20030.03), indicating a more severe manifestation in patients with lower CD28 expression. Correlations with other clinical parameters such as activity or therapy were not seen. WG patients show a lack of CD28 expression on T cells and an unusual up‐regulation of its ligands B7‐1 and B7‐2 on T cells after in vitro activation as well as a lower expression of B7‐2 on freshly isolated monocytes compared with HC. These features might promote the Th1 cytokine pattern and thereby contribute to persistently high levels of immune activation in WG.

Functionally relevant variations of the interleukin‐10 gene associated with antineutrophil cytoplasmic antibody–negative Churg‐Strauss syndrome, but not with Wegener's granulomatosis

OBJECTIVEnWegeners granulomatosis (WG) and Churg-Strauss syndrome (CSS) belong to the heterogeneous group of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Current understanding of their pathogenesis and genetic background is limited. Expression levels of interleukin-10 (IL-10), a potent and pleiotropic cytokine, are largely determined by variations in the gene encoding the IL-10 precursor. This study was undertaken to determine the impact of IL10 polymorphisms on the pathogenesis of both WG and CSS in large cohorts.nnnMETHODSnThree single-nucleotide polymorphisms (SNPs) tagging the promoter haplotypes of the IL10 gene (IL10 -3575, IL10 -1082, and IL10 -592) were analyzed in 403 patients with WG and 103 patients with CSS as well as 507 matched control subjects from Germany. In addition, 3 informative SNPs in other parts of IL10 were genotyped.nnnRESULTSnNone of the markers or their haplotypes was associated with WG or any of its subgroups classified according to ANCA status, sex, or presence of further WG genetic risk factors. In contrast, the IL10 -3575/-1082/-592 TAC haplotype, part of the extended ancient haplotype IL10.2, was highly significantly associated with ANCA-negative CSS (chi2 = 19.14, P = 0.000012, corrected P = 0.0003, odds ratio 2.16, 95% confidence interval 1.52-3.06).nnnCONCLUSIONnThese findings challenge those from previous studies of IL10 in WG and provide further evidence that CSS and WG have distinct genetic backgrounds. Because the IL10.2 haplotype has been correlated reproducibly with increased IL10 expression, the possible role of IL-10 in the pathogenesis of ANCA-negative CSS needs to be further elucidated.

Opsonization of apoptotic neutrophils by anti-neutrophil cytoplasmic antibodies (ANCA) leads to enhanced uptake by macrophages and increased release of tumour necrosis factor-alpha (TNF-α)

Since proteinase 3 (PR3)‐ANCA interact with PR3 on the surface of apoptotic polymorphonuclear neutrophils (PMN) and ingestion of apoptotic PMN is known to modulate macrophage inflammatory reactions, we raised the question whether PR3‐ANCA‐opsonized apoptotic PMN influence the uptake by macrophages and their state of activation. We therefor analysed the effects of PR3‐ANCA‐opsonized apoptotic PMN on the uptake process by enzymatic assay. We further investigated the production of TNF‐α, IL‐10, IL‐12 and the secretion of lipid inflammatory mediators (TxB2, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2)) by human monocyte‐derived macrophages using FACS and ELISA methods. We show that PMN‐opsonization by PR3‐ANCA substantially enhances phagocytosis by macrophages and thereby triggers the production of TNF‐α and TxB2. These in vitro findings indicate that PR3‐ANCA opsonization of apoptotic PMN might be an important mechanism in the pathogenesis of Wegeners granulomatosis (WG), prompting macrophages to produce proinflammatory mediators. These mediators, mainly TNF‐α, might prime further PMN leading to perpetuation of the known priming‐dependent mechanisms of ANCA action.

Increased Expression Of Ctla-4 (Cd152) By T And B Lymphocytes In Wegener'S Granulomatosis

CTLA‐4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA‐4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Furthermore, CTLA‐4 has a role in inducing a Th1 response and suppressing Th2 cytokines, an effect which is antagonized by CD28. Many autoimmune diseases are characterized by an overwhelming production of Th1 cytokines. Recently, the predominance of the Th1 cytokine pattern has been directly observed in the granulomatous inflammation of patients with Wegeners granulomatosis. The balance between CD28 and CTLA‐4 expression by T lymphocytes could be a factor in the pathogenesis of autoimmune diseases. Down regulation of CD28 predominantly on CD8+u2003T cells has been described in Wegners granulomatosis; however, analysis of CTLA‐4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA‐4 on PBMC in patients with Wegeners granulomatosis (nu2003=u200325) in comparison with healthy controls (nu2003=u200319). Expression levels of CTLA‐4 were significantly increased selectively on CD4+ and possibly also on CD4−/CD8− T cells in Wegeners granulomatosis. High CTLA‐4 expression by T lymphocytes was associated with more severe disease. In contrast, after stimulation with the mitogen PHA, CTLA‐4 levels were strongly increased on T cells from controls but in T cells from Wegeners granulomatosis patients this response was severely impaired. Interestingly, while CTLA‐4 was seen exclusively on T cells in control individuals, about half of the Wegeners patients showed CTLA‐4 expression by a fraction of peripheral B lymphocytes. CTLA‐4 positive B cells in the periphery were associated with less acute disease.

Immunological and clinical follow up of hepatitis C virus associated cryoglobulinaemic vasculitis

OBJECTIVE To study immunological markers and compare these markers with standard measures for the clinical and immunological follow up of vasculitis activity in hepatitis C virus (HCV) associated cryoglobulinaemic vasculitis (CV). METHODS Serial serum samples from eight patients with newly diagnosed HCV associated CV were followed during interferon α treatment induced remission of the CV. Vasculitis activity and disease extent were evaluated with the Birmingham vasculitis activity score (BVAS) and disease extent index (DEI). Cryoglobulinaemia, complement levels (C3c, C4, and CH50), rheumatoid factor (RF), autoantibodies such as antinuclear antibodies, soluble interleukin 2 receptor (sIL2r), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble CD30 (sCD30) were determined. RESULTS All patients achieved either complete or partial remission of their CV during interferon α treatment. There was a significant reduction in vasculitis activity and disease extent (BVAS, DEI), cryoglobulinaemia, RF, sIL2r, sICAM-1, and sCD30. Complement C3c levels increased significantly during this period. Erythrocyte sedimentation rate and levels of complement C4 and CH50 did not change significantly. Both clinical measures (BVAS and DEI) correlated significantly only with C3c and sCD30. CONCLUSIONS Although this study was of only a small group of patients, it shows that BVAS and DEI as clinical measures and C3c and sCD30 as immunological markers may be useful in the follow up of disease activity of HCV associated CV. The data indicate that activity of the humoral (cryoglobulinaemia, RF, autoantibodies) and cellular (sIL2r, sICAM-1, sCD30) immune response and endothelial damage (sICAM-1) are found in HCV associated CV.

Wegener’s Granulomatosis: The Current View

In the last few years, substantial progress has been achieved in elucidating the pathogenesis of Wegener’s granulomatosis. Several genetic risk factors have been described. The structure of the granuloma and its possible function as ectopic lymphoid tissue have been defined. Furthermore, the consecutive immunopathological reactions leading to induction of PR3-antineutrophil cytoplasmic antibodies (ANCA) and the role of ANCA itself is getting clearer. However, the initial events leading to granuloma formation are still widely unknown. Concerning therapy, the significance of the so-called biological agents (TNF-α-blockers, Rituximab) still has to be defined.

Successful use of bortezomib in a patient with systemic lupus erythematosus and multiple myeloma

Bortezomib belongs to the family of proteasome inhibitors and is a well established first line drug in multiple myeloma (MM).1 In mouse models of lupus nephritis, bortezomib ameliorated glomerulonephritis, prolonged survival and reduced autoantibody production.2nnWe report the case of a 63-year-old woman who presented with systemic lupus erythematosus (SLE) fulfilling the American College of Rheumatology (ACR) criteria and requiring treatment for autoimmune thrombocytopoenia, Coombs positive autoimmune haemolytic anaemia, polyarthritis and hair loss. Anti-nuclear antibody (ANA), extractable nuclear antigen (ENA) (U1 riboucleoprotein (U1RNP)) and anti-double-stranded (ds)DNA antibody tests were positive, and serum complement C3 and C4 levels were decreased (European Consensus …

Serum HMGB1 levels are increased in active Wegener's granulomatosis and differentiate between active forms of ANCA-associated vasculitis

High mobility group box 1 (HMGB1) is a non-histone nuclear protein released during cell necrosis and late apoptosis. It contributes to the activation of monocytes/macrophages and induces the secretion of proinflammatory cytokines.1,–,4 HMGB1 serum concentrations are increased in conditions associated with severe tissue damage such as haemorrhagic shock.5nnThe hypothesis behind this study was that HMGB1 may be released from damaged tissue in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and serum levels may correlate with disease activity. Furthermore, with respect to the known sources of HMGB1, high levels were expected in particular in Wegeners granulomatosis (WG) which is characterised by granulomatous inflammation.nnSerum samples were obtained from 46 patients with WG, 44 with …

Identification of the PTPN22 functional variant R620W as susceptibility genetic factor for giant cell arteritis

Objective To analyse the role of the PTPN22 and CSK genes, previously associated with autoimmunity, in the predisposition and clinical phenotypes of giant cell arteritis (GCA). Methods Our study population was composed of 911 patients diagnosed with biopsy-proven GCA and 8136 unaffected controls from a Spanish discovery cohort and three additional independent replication cohorts from Germany, Norway and the UK. Two functional PTPN22 polymorphisms (rs2476601/R620W and rs33996649/R263Q) and two variants of the CSK gene (rs1378942 and rs34933034) were genotyped using predesigned TaqMan assays. Results The analysis of the discovery cohort provided evidence of association of PTPN22 rs2476601/R620W with GCA (PFDR=1.06E-04, OR=1.62, CI 95% 1.29 to 2.04). The association did not appear to follow a specific GCA subphenotype. No statistically significant differences between allele frequencies for the other PTPN22 and CSK genetic variants were evident either in the case/control or in stratified case analysis. To confirm the detected PTPN22 association, three replication cohorts were genotyped, and a consistent association between the PTPN22 rs2476601/R620W variant and GCA was evident in the overall meta-analysis (PMH=2.00E-06, OR=1.51, CI 95% 1.28 to 1.79). Conclusions Our results suggest that the PTPN22 polymorphism rs2476601/R620W plays an important role in the genetic risk to GCA.