Antje Müller

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

OBJECTIVE To 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. METHODS Competitive 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. RESULTS Patients 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. CONCLUSION Our data indicate that a Thl cytokine pattern predominates in the granulomatous inflammation in patients with WG.

Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum

The obligate intracellular pathogen Leishmania major survives and multiplies in professional phagocytes. The evasion strategy to circumvent killing by host phagocytes and establish a productive infection is poorly understood. Here we report that the virulent inoculum of Leishmania promastigotes contains a high ratio of annexin A5-binding apoptotic parasites. This subpopulation of parasites is characterized by a round body shape, a swollen kinetoplast, nuclear condensation, and a lack of multiplication and represents dying or already dead parasites. After depleting the apoptotic parasites from a virulent population, Leishmania do not survive in phagocytes in vitro and lose their disease-inducing ability in vivo. TGF-β induced by apoptotic parasites is likely to mediate the silencing of phagocytes and lead to survival of infectious Leishmania populations. The data demonstrate that apoptotic promastigotes, in an altruistic way, enable the intracellular survival of the viable parasites.

Elevated interleukin-4 and interleukin-13 production by T cell lines from patients with Churg-Strauss syndrome

OBJECTIVE To investigate cytokine production patterns of T cell lines (TCL) from patients with Churg-Strauss syndrome (CSS). METHODS Short-term polyclonal TCL were generated from peripheral blood of patients with CSS or Wegeners granulomatosis (WG) and healthy controls (HC). TCL were established in the presence of interleukin-2 (IL-2) and phytohemagglutinin and were phenotypically characterized by flow cytometry. Th1/ Th2 cytokine production by stimulated TCL (72 hours) was analyzed by enzyme-linked immunosorbent assay. RESULTS TCL that represented the progeny of in vivo-activated T cells from CSS patients displayed a heterogeneous immunophenotype, with a predominance of CD4+ T cells when compared with WG TCL, which were predominantly CD8+. All CSS TCL shared the ability to produce large amounts of interferon-gamma (IFNgamma), IL-4, and IL-13 compared with HC (P = 0.014 for all 3). Production of IL-4 and IL-13 was higher in CSS TCL than in WG TCL (P = 0.014 for both). IL-5 production was up-regulated in WG TCL compared with CSS TCL (P = 0.014). Compared with HC, WG TCL showed increased production of IFNgamma (P = 0.021), IL-5 (P = 0.043), and IL-13 (P = 0.021). CONCLUSION Our results indicate that, while there is evidence for both a type 1 and a type 2 response in CSS, type 2 cytokine production pattern appears to predominate in this disease when compared with WG and HC.

Localized Wegener's granulomatosis: predominance of CD26 and IFN‐γ expression

The immune response in Wegeners granulomatosis (WG) has been characterized as a predominant, potentially pathogenic Th1‐like reaction by blood T cells and T‐cell clones from diseased tissues. To elucidate further the immunopathogenic mechanisms, this study analysed the phenotypes of inflammatory infiltrates in frozen nasal biopsies with involvement of the upper respiratory tract only (localized or ‘initial phase’ WG) and with multi‐organ involvement, including systemic vasculitis (generalized WG). The expression and production of Th1 and Th2 cytokines were examined in tissue specimens and peripheral blood mononuclear cells (PBMCs) of localized and generalized WG. The number of CD3+ T cells in inflammatory infiltrates ranged from 50 to 70%, together with approximately 30% CD14+ monocytes/macrophages. An average of 40% of T cells expressed CD26 in nasal biopsies of localized WG, compared with about 16% in specimens of generalized WG. In parallel, a higher number of interferon‐γ (IFN‐γ)‐positive cells were detected in nasal tissue of localized than in generalized WG. PBMCs from localized WG similarly exhibited higher spontaneous IFN‐γ production in contrast to generalized WG (207 vs. 3 pg/ml, p<0.05). Interleukin‐4 (IL‐4) mRNA was found in higher amounts in generalized than in localized WG. IL‐4 production was negligible in both disease and controls. In addition, both IL‐10 mRNA and IL‐10 protein levels of activated PBMCs from localized WG were elevated when compared with generalized disease (574 vs. 154 pg/ml, p<0.05) or healthy controls (574 vs. 246 pg/ml, p<0.05). It is conluded that in nasal tissues, mainly CD4+/CD26+ T cells as well as IFN‐γ‐positive cells may support a polarized Th1‐like immune response. Furthermore, the data suggest that this in situ immune response is already initiated and established in localized WG, accompanied by increased peripheral IFN‐γ and IL‐10 production. Copyright

Differences in CCR5 expression on peripheral blood CD4+CD28− T-cells and in granulomatous lesions between localized and generalized Wegener’s granulomatosis

Wegeners granulomatosis (WG) is an autoimmune disease characterized by granulomatous lesions and a necrotizing vasculitis. Th1-type-cells lacking CD28 are expanded independent of age and immunosuppressive therapy in WG. To address their migratory properties of CD4(+)CD28(-) T-cells we studied the expression of the inducible inflammatory Th1-type chemokine receptor CCR5 in localized WG and generalized WG. Expansion of CD4(+)CD28(-) T-cells was more prominent in generalized WG compared to localized WG. In localized WG a larger fraction of CD4(+)CD28(-) T-cells displayed CCR5 expression compared to generalized WG. CCR5 expression was also higher in granulomatous lesions in localized WG. Higher levels of CCR5 expression on CD4(+)CD28(-) T-cells in localized WG may favor stronger CCR5-mediated recruitment of this T-cell subset into granulomatous lesions in localized WG. Expansion of Th-1-type CD4(+)CD28(-)CCR5(+) effector memory T-cells might contribute to disease progression and autoreactivity, either directly, by maintaining the inflammatory response, or as a result of bystander activation.

Is PR3‐ANCA Formation Initiated in Wegener's Granulomatosis Lesions? Granulomas as Potential Lymphoid Tissue Maintaining Autoantibody Production

Abstract: In Wegeners granulomatosis (WG), antiproteinase 3 (PR3) autoantibodies (PR3‐ANCA) are crucial in the development of generalized vasculitis. Wegeners pathognomonic lesion, a granulomatous inflammation of the upper and lower respiratory tract, contains abundant lymphocytes and macrophages. Lymphocyte clusters in germinal center‐like formation within the granulomatous lesion are frequently observed, which suggests antigen‐driven B cell maturation. Wegeners autoantigen PR3, the target for autoreactive B and T cells, is expressed in granulomatous lesions. Disease progression in WG is accompanied by a profound generalized alteration of T cell differentiation with an increase of effector memory T cells (CD4+CD28−). The cytokine profile suggests an aberrant Th1‐type response either to an environmental trigger and/or the autoantigen PR3 itself. Staphylococcus aureus, a risk factor for disease exacerbation, is widely present in the upper airways in WG. The Ig gene repertoire from WG lesions indicates a predominance of VH3+ B cells with affinity to PR3 as well as to the S. aureus B cell superantigen SPA. Hence, within the WG lesion, S. aureus might support the maturation of PR3‐affinity B cells that enter a germinal center reaction in contact with PR3 and T cells and expand, leading to PR3‐ANCA production. Thus, granulomatous lesions could represent a potential lymphoid tissue‐maintaining autoantibody production rather than a simple, random leukocyte accumulation in WG.

Wegener’s granuloma harbors B lymphocytes with specificities against a proinflammatory transmembrane protein and a tetraspanin

Wegeners granulomatosis (WG) is a severe autoimmune disorder ranging from localized granulomatous disease to generalised anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. A previous analysis of immunoglobulin heavy chain genes derived from tissue, i.e. Wegeners granuloma indicated selection and affinity maturation towards local antigen(s). The current study focused on determining the specificity of immunoglobulins from distinct B lymphocytes out of Wegeners granuloma. Four pairs of variable region immunoglobulin light and heavy chain genes, isolated before, were recombinantly expressed using the baculovirus/insect cell system. These immunoglobulins were then analysed for their antigenic target employing a protein macroarray based upon a human fetal brain tissue cDNA expression library. The lysosomal transmembrane protein 9B, a key regulator for TNFα activation, was identified as the putative antigenic target of two immunoglobulins and a tetraspanin, which might play a role in leukocyte activation and motility, was identified as the putative antigenic target of another one. Recombinant monoclonal antibodies out of Wegeners granuloma represent a new tool aiding in elucidation of its and WG immunopathogenesis.

Bactericidal/Permeability-Increasing Protein Is Expressed by Human Dermal Fibroblasts and Upregulated by Interleukin 4

ABSTRACT The bactericidal/permeability-increasing protein (BPI) is an antibiotic- and endotoxin-neutralizing protein of granulocytes and epithelial cells. Constitutive expression of BPI, which increases upon interleukin 4 stimulation, by human dermal fibroblast was demonstrated, suggesting an important role of BPI in gram-negative bacterial clearance and a dampened response to endotoxin in the skin.

Human proteinase 3 (PR3) and its binding molecules: implications for inflammatory and PR3-related autoimmune responses.

Abstract:  Human proteinase 3 (PR3) is a multifunctional serine protease, mainly located in the azurophilic granules and on the cell surface of polymorphonuclear leukocytes (PMN). Cumulated data indicate that PR3, which is the main target autoantigen of antineutrophilic cytoplasmic antibodies (ANCA), interacts with several surface receptors and participates in the local inflammatory response. Herein, we summarize the efforts made to elucidate ANCA‐binding epitopes of PR3, extended by data derived from the use of a random peptide library. The inserts for 107 peptides were obtained by panning of a random peptide library with PR3‐ANCA+ immunoglobulins. Analysis of the amino acid sequences of the inserted peptides derived from isolated positive clones suggested that they do not belong to linear epitopes of PR03 and possess a high proportion of positively charged amino acids. Furthermore, this article focuses on immune functions of PR3 with respect to PR3 modulation of cell activation via cleavage of protease‐activated receptor‐2 (PAR‐2) and as binding protein for the proinflammatory cytokine IL‐32α. Altogether, there are a number of (auto)molecules that bind to PR3, some of them even competitive and each binding interaction seems to have specific implications.

Environmental factor and inflammation-driven alteration of the total peripheral T-cell compartment in granulomatosis with polyangiitis.

Autoimmune diseases are initiated by a combination of predisposing genetic and environmental factors resulting in self-perpetuating chronic inflammation and tissue damage. Autoantibody production and an imbalance of effector and regulatory T-cells are hallmarks of autoimmune dysregulation. While expansion of circulating effector memory T-cells is linked to disease pathogenesis and progression, the causes driving alterations of the peripheral T-cell compartment have remained poorly understood so far. In granulomatosis with polyangiitis (GPA), a prototypical autoimmune disorder of unknown aetiology, we performed for the first time a combined approach using phenotyping, transcriptome and functional analyses of T-cell populations to evaluate triggers of memory T-cell expansion. In more detail, we found increased percentages of circulating CD4+CD28-, CD8+CD28- and CD4+CD161+ single-positive and CD4+CD8+ double-positive T-cells in GPA. Transcriptomic profiling of sorted T-cell populations showed major differences between GPA and healthy controls reflecting antigen- (bacteria, viruses, fungi) and cytokine-driven impact on T-cell populations in GPA. Concomitant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) - positivity was associated with a significant increase in the percentage of CD28- T-cells in GPA-patients compared to sole CMV- or EBV-positivity or CMV- and EBV-negativity. T-cells specific for other viruses (influenza A virus, metapneumovirus, respiratory syncytial virus) and the autoantigen proteinase 3 (PR3) were infrequently detected in GPA. Antigen-specific T-cells were not specifically enriched in any of the T-cell subsets. Altogether, on a genetic and cellular basis, here we show that alterations of the peripheral T-cell compartment are driven by inflammation and various environmental factors including concomitant CMV and EBV infection. Our study provides novel insights into mechanisms driving autoimmune disease and on potential therapeutic targets.