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ABSTRACT: The cytokine IL-6 plays a protective role in immune responses against bacterial infections. However, the mechanisms of IL-6-mediated protection are only partially understood. IL-6 can signal via the IL-6R complex composed of membrane-bound IL-6Rα (mIL-6Rα) and gp130. Owing to the restricted expression of mIL-6Rα, classical IL-6 signaling occurs only in a limited number of cells such as hepatocytes and certain leukocyte subsets. IL-6 also interacts with soluble IL-6Rα proteins and these IL-6/soluble IL-6Rα complexes can subsequently bind to membrane-bound gp130 proteins and induce signaling. Because gp130 is ubiquitously expressed, this IL-6 trans-signaling substantially increases the spectrum of cells responding to IL-6. In this study, we analyze the role of classical IL-6 signaling and IL-6 trans-signaling in the innate immune response of mice against Listeria monocytogenes infection. We demonstrate that L. monocytogenes infection causes profound systemic IL-6 production and rapid loss of IL-6Rα surface expression on neutrophils, inflammatory monocytes, and different lymphocyte subsets. IL-6-deficient mice or mice treated with neutralizing anti-IL-6 mAb displayed impaired control of L. monocytogenes infection accompanied by alterations in the expression of inflammatory cytokines and chemokines, as well as in the recruitment of inflammatory cells. In contrast, restricted blockade of IL-6 trans-signaling by application or transgenic expression of a soluble gp130 protein did not restrain the control of infection. In summary, our results demonstrate that IL-6Rα surface expression is highly dynamic during the innate response against L. monocytogenes and that the protective IL-6 function is dependent on classical IL-6 signaling via mIL-6Rα.
The Journal of Immunology 12/2012; · 5.79 Impact Factor
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ABSTRACT: Hypomorphic ADAM17ex/ex mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6
receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17ex/ex mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued
in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of
human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced
IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible
for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory
shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an
as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer
of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity
of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences
for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.
Journal of Biological Chemistry 04/2011; 286(17):14804-14811. · 4.77 Impact Factor
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ABSTRACT: Hypomorphic ADAM17ex/ex mice showed defects in mucosal regeneration due to inefficient EGFR-shedding. ADAM17 is the main sheddase
of Interleukin-6 receptor (IL-6R) to induce IL-6 transsignaling. However, serum levels of soluble murine IL-6R were not reduced
in ADAM17ex/ex mice and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17
was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular
domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis
induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 was also identified as protease
responsible for Ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10 deficient murine embryonic fibroblasts
compensatory shedding of human IL-6R was mediated by ADAM17 but loss of ADAM10-mediated shedding of murine IL-6R was compensated
by a yet unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer
of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity
of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and men, which might have consequences
for the interpretation of phenotypes from ADAM17 and ADAM10 deficient mice.
Journal of Biological Chemistry 03/2011; · 4.77 Impact Factor
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ABSTRACT: Neutrophil depleted mice are protected from concanavalin A-mediated hepatitis, showing that neutrophils are critical for cellular liver damage. Interleukin-6 has pro- and anti-inflammatory properties and mediates neutrophil recruitment in diseases such as rheumatoid arthritis. In classic signaling, interleukin-6 binds to the membrane-bound interleukin-6-receptor and initiates signaling via gp130. In interleukin-6 trans-signaling, the agonistic soluble interleukin-6-receptor can form a soluble interleukin-6/interleukin-6-receptor complex and stimulate cells which only express gp130 but no interleukin-6-receptor. Interleukin-6 trans-signaling was shown to be important for liver regeneration and development of liver adenomas. Here, we show that blocking classic interleukin-6 signaling but not interleukin-6 trans-signaling reduced concanavalin A-induced liver damage in mice, with reduced liver STAT3 phosphorylation and liver neutrophil accumulation. However, the level of neutrophil-attracting chemokine KC is only reduced by inhibition of interleukin-6 trans-signaling. Analysis of circulating neutrophils after concanavalin A challenge revealed that classic interleukin-6 signaling is required for the mobilization of blood neutrophils. Reduced neutrophil infiltration was accompanied by increased levels of hepatoprotective monocyte chemoattractant protein-1 and reduced level of hepatodestructive interleukin-4. Abrogated classic interleukin-6 signaling in concanavalin A-mediated hepatitis exhibited liver-protective effects indicating that interleukin-6 classic but not interleukin-6 trans-signaling is responsible for liver damage. Classic interleukin-6 signaling is required to mount an efficient neutrophilia during concanavalin A-induced immune response, which might have clinical implications in the regard that blocking global interleukin-6 signaling pathways is a treatment option in different chronic inflammatory diseases.
Biochimica et Biophysica Acta 03/2011; 1812(3):290-301. · 4.66 Impact Factor
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ABSTRACT: Hypomorphic ADAM17(ex/ex) mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17(ex/ex) mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.
Journal of Biological Chemistry 03/2011; 286(17):14804-11. · 4.77 Impact Factor
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ABSTRACT: The purinoreceptor P2X7 is expressed on subsets of T cells and mediates responses of these cells to extracellular nucleotides such as ATP or NAD(+). We identified P2X7 as a molecule highly up-regulated on conventional CD8alphabeta(+) and unconventional CD8alphaalpha(+) T cells of the intestinal epithelium of mice. In contrast, CD8(+) T cells derived from spleen, mesenteric lymph nodes, and liver expressed only marginal levels of P2X7. However, P2X7 was highly up-regulated on CD8(+) T cells from spleen and lymph nodes when T cells were activated in the presence of retinoic acid. High P2X7 expression on intestinal CD8(+) T cells as well as on CD8(+) T cells incubated with retinoic acid resulted in enhanced sensitivity of cells to extracellular nucleotides. Both cell populations showed a high level of apoptosis following incubation with NAD(+) and the ATP derivative 2',3'-O-(benzoyl-4-benzoyl)-ATP, and injection of NAD(+) caused selective in vivo depletion of intestinal CD8(+) T cells. Following oral infection with Listeria monocytogenes, P2X7-deficient mice showed similar CD8(+) T cell responses in the spleen, but enhanced responses in the intestinal mucosa, when compared with similarly treated wild-type control mice. Overall, our observations define P2X7 as a new regulatory element in the control of CD8(+) T cell responses in the intestinal mucosa.
The Journal of Immunology 10/2008; 181(6):3861-9. · 5.79 Impact Factor
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ABSTRACT: The intestinal mucosa represents a challenging environment for CD8+ T cells, which must tolerate nutrient antigens and commensal microorganisms while responding efficiently to pathogens. Consequently, specific regulatory mechanisms apply for CD8+ T cells in the intestinal environment, which should also be reflected in a tissue-specific gene expression profile of these cells. This study investigates whether such tissue-specific gene expression can be observed in CD8+ T cells primed during bacterial infection. To identify intestine-specific gene expression in conventional CD8alphabeta+ T cells, mice were infected with Listeria monocytogenes expressing ovalbumin (LmOVA). Using OVA257-264 tetramers, specific CD8+ T cells were sorted from spleen, liver and the small intestinal mucosa, and RNA samples from these cells were compared using microarrays. This approach allowed the identification of differences in gene expression in a highly defined CD8+ T-cell population with identical antigen specificity generated during infection. One group of genes with reduced expression in the intestinal mucosa comprised members of the C-type lectin-like natural killer receptor (NKR) family. Fluorescence-activated cell sorting analysis was used to assess protein expression of NKR. NKR expression on CD8+ T cells from the intestinal mucosa was dependent on the route of listeria application and consequently on the site of T-cell priming. Retinoic acid influenced NKR expression consistent with an imprinting of the NKR expression profile in intestine-associated lymphoid tissues. In contrast, NKR expression was largely independent from intestinal flora. Our results demonstrate that in the intestinal mucosa, conventional CD8alphabeta+ T cells lack NKR expression and thereby lose responsiveness to NKR ligands, which otherwise could possibly cause adverse activation or inhibition of T cells in this environment.
Immunology 03/2008; 125(1):38-47. · 3.32 Impact Factor
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ABSTRACT: Activation of naive T cells is tightly controlled and depends on cognate interactions with professional antigen-presenting cells. We analyzed dependency on secondary lymphoid tissues for the activation of naive and memory CD4(+) and CD8(+) T cells following primary and secondary Listeria monocytogenes infection, respectively. In splenectomized lymphotoxin-beta receptor-deficient mice, lacking all secondary lymphoid tissues, oral infection with L. monocytogenes failed to induce bacteria-specific CD4(+) and CD8(+) T cell responses. Treatment of splenectomized wild-type mice with FTY720, a drug that prevents egress of T cells from lymph nodes, also reduced T cell responses after oral L. monocytogenes infection and blocked T cell responses after intravenous infection. FTY720-treated wild-type and lymphotoxin-beta receptor-deficient mice show only slightly impaired recall responses. However, T cell responses were profoundly inhibited when mice were splenectomized subsequently to recovery from primary infection. T cell transfer experiments demonstrated that the impaired secondary T cell response was not simply due to removal of a large fraction of memory T cells by splenectomy. Overall, these results indicate that not only primary T cell responses, but also secondary T cell responses, highly depend on the lymphoid environment for effective activation.
European Journal of Immunology 02/2008; 38(1):127-38. · 5.10 Impact Factor
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ABSTRACT: The complement activating venom component Cobra Venom Factor (CVF), a functional and structural homologue of the human complement component C3, forms a stable CVF-dependent C3 convertase complex, which, in contrast to C3-dependent convertase effects continuous activation of the complement and, thereby, decomplementation. In order to elucidate the mechanism underlying the enhanced activity of CVF compared to human C3, we generated two CVF/C3 chimeras and established different affinity-based assay systems for functional analysis of these constructs. To allow for convenient expression and subsequent functional characterisation, the CVF/C3 chimeras as well as CVF and C3 were transiently expressed in mammalian cells. Problems due to the low concentration of the recombinant proteins in the supernatants of transient expressions were circumvented by fusion to peptide tags enabling their efficient immobilisation onto suitable surfaces and subsequent characterisation. In an alternative approach monoclonal antibody fragments generated from a semisynthetic phage display scFv library were employed for concentrating the recombinant proteins by immunoprecipitation. Utilising both approaches all transiently expressed proteins could be characterised for their complement consumption activity. The data obtained with the CVF/C3 chimeras demonstrate that the increased stability of the CVFBb complex is independent of the domains in CVF corresponding to binding sites of factor B and H and the cleavage sites of factor I in the human C3 molecule.
Molecular Immunology 06/2004; 41(1):19-28. · 2.90 Impact Factor
