Soluble cytokine receptors are frequently found in human serum, most of them possessing antagonistic properties. The Interleukin 6 receptor (IL-6R) is found as a transmembrane protein on hepatocytes ...and subsets of leukocytes, but soluble isoforms of the IL-6R (sIL-6R) are generated by alternative splicing or by limited proteolysis of the
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etalloproteinases (ADAM) gene family members ADAM10 and ADAM17. Importantly, the sIL-6R in complex with its ligand Interleukin 6 (IL-6) has agonistic functions and requires cells expressing the signal transducing ß-receptor gp130 but not the membrane-bound IL-6R. We have called this process IL-6 trans-signaling. Naturally occurring isoforms of soluble gp130 (sgp130), which are generated by alternative splicing, are natural inhibitors of IL-6 trans-signaling, leaving IL-6 classic signaling via the membrane-bound IL-6R unaffected. We used recombinant sgp130Fc protein and recently generated transgenic mice expressing high levels of sgp130Fc to discriminate between classic and trans-signaling
in vivo, and demonstrated that IL-6 trans-signaling is critically involved in generation and maintenance of several inflammatory and autoimmune diseases including chronic inflammatory bowel disease, rheumatoid arthritis, peritonitis and asthma, as well as inflammation-induced colon cancer.
Cytokine control of the synovial infiltrate is a central process in the development of inflammatory arthritis. In this study, we combine genetic approaches and intervention strategies to describe a ...fundamental requirement for IL-6-mediated STAT3 signaling in orchestrating the inflammatory infiltrate in monoarticular and systemic models of experimental arthritis. STAT3 activation via the common gp130 signal-transducing receptor for all IL-6-related cytokines led to increased retention of neutrophils and T cells within the inflamed synovium, which included STAT3-regulated IL-17A-secreting T cells. Control of leukocyte infiltration was reliant upon IL-6 signaling via its soluble receptor (termed IL-6 trans signaling), as evidenced by selective blockade of this alternative IL-6 signaling pathway using an engineered variant of soluble gp130 (sgp130Fc). This therapeutic intervention led to substantial clinical improvement in mice with emerging or established incidence of systemic arthritis. These data illustrate that IL-6 control of STAT3 is critical for regulating the synovial infiltrate in inflammatory arthritis, and suggest that selective inhibition of IL-6 trans signaling may provide a more refined intervention strategy for blocking IL-6-driven proarthritic activities.
Cytokines of the IL-12 family show structural similarities but have distinct functions in the immune system. Prominent members of this cytokine family are the pro-inflammatory cytokines IL-12 and ...IL-23. These two cytokines share cytokine subunits and receptor chains but have different functions in autoimmune diseases, cancer and infections. Accordingly, structural knowledge about receptor complex formation is essential for the development of new therapeutic strategies preventing and/or inhibiting cytokine:receptor interaction. In addition, intracellular signaling cascades can be targeted to inhibit cytokine-mediated effects. Single nucleotide polymorphisms can lead to alteration in the amino acid sequence and thereby influencing protein functions or protein–protein interactions. To understand the biology of IL-12 and IL-23 and to establish efficient targeting strategies structural knowledge about cytokines and respective receptors is crucial. A highly efficient therapy might be a combination of different drugs targeting extracellular cytokine:receptor assembly and intracellular signaling pathways.
Activation of the IL-6/Stat3 via IL-6 trans-signaling plays an important role in the pathogenesis of inflammatory bowel disease. Colitis-associated cancer (CAC) is a large bowel cancer and occurs ...with long-standing inflammatory bowel disease. The role of the IL-6/Stat3 in the development of CAC has not been fully understood. We investigate whether IL-6 trans-signaling contributes to the development of CAC using a mouse colitis-associated premalignant cancer (CApC) model. Chronic colitis (CC) was induced in BALB/c mice using dextran sodium sulfate. CApC was induced by dextran sodium sulfate treatment to CC-affected mice. IL-6 expression was determined by quantitative RT-PCR and immunofluorescence staining in colon. Phospho-Stat3 expression was examined by Western blotting and immunofluorescence analysis. The expression of IL-6 receptors (i.e., the IL-6R alpha-chain and gp130) and tumor necrosis factor-alpha converting enzyme in the colon was examined by laser-capture microdissection and immunofluorescence staining. Soluble IL-6R alpha (sIL-6R alpha) was examined by Western blotting of epithelial cell-depleted colonic tissues. We also investigated whether a soluble gp130-Fc fusion protein could prevent CApC. IL-6 expression was increased in the colon of CC- and CApC-affected mice and was restricted to lamina propria-macrophages. The expression of IL-6R alpha and tumor necrosis factor-alpha converting enzyme was increased in the lamina propria CD11b-macrophages of CC-affected mice. sIL-6R alpha expression was also increased in these tissues. Reduced levels of IL-6R alpha generation were observed in the colonic epithelial cells of CC- and CApC-affected mice and were associated with the increased expression of gp130 and phospho-Stat3. Treatment with soluble gp130Fc significantly reduced the CApC. IL-6 trans-signaling in epithelial cells induced by macrophage-derived IL-6/sIL-6R alpha plays a crucial role in the development of CAC.
Cytokine receptors, which exist in membrane‐bound and soluble forms, bind their ligands with comparable affinity. Although most soluble receptors are antagonists and compete with their ...membrane‐associated counterparts for the ligands, certain soluble receptors are agonists. In these cases, complexes of ligand and soluble receptor bind on target cells to second receptor subunits and initiate intracellular signaling. The soluble receptors of the interleukin (IL)‐6 family of cytokines (sIL‐6R, sIL‐11R, soluble ciliary neurotrophic factor receptor) are agonists capable of transmitting signals through interaction with the universal signal‐transducing receptor for all IL‐6 family cytokines, gp130. In vivo, the IL‐6/sIL‐6R complex stimulates several types of cells, which are unresponsive to IL‐6 alone, as they do not express the membrane IL‐6R. We have named this process trans‐signaling. The generation of soluble cytokine receptors occurs via two distinct mechanisms—limited proteolysis and translation—from differentially spliced mRNA. We have demonstrated that a soluble form of the IL‐6 family signaling receptor subunit gp130, which is generated by differential splicing, is the natural inhibitor of IL‐6 trans‐signaling responses. We have shown that in many chronic inflammatory diseases, including chronic inflammatory bowel disease, peritonitis, rheumatoid arthritis, asthma, as well as colon cancer, IL‐6 trans‐signaling is critically involved in the maintenance of a disease state, by promoting transition from acute to chronic inflammation. Moreover, in all these models, the course of the disease can be disrupted by specifically interfering with IL‐6 trans‐signaling using the soluble gp130 protein. The pathophysiological mechanisms by which the IL‐6/sIL‐6R complex regulates the inflammatory state are discussed.
Interleukin‐6 (IL‐6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL‐6 trans‐signaling through the soluble IL‐6/IL‐6R complex is ...involved in this process. However, the long‐term contribution of IL‐6 trans‐signaling for liver regeneration after PHX is unknown. PHX‐induced generation of the soluble IL‐6R by ADAM (a disintegrin and metallo) proteases enables IL‐6 trans‐signaling, in which IL‐6 forms an agonistic complex with the soluble IL‐6 receptor (sIL‐6R) to activate all cells expressing the signal‐transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL‐6 in complex with membrane‐bound IL‐6R and gp130 activates classic signaling. Here, we describe the generation of IL‐6 trans‐signaling mice, which exhibit boosted IL‐6 trans‐signaling and abrogated classic signaling by genetic conversion of all membrane‐bound IL‐6R into sIL‐6R proteins phenocopying hyperactivation of ADAM‐mediated shedding of IL‐6R as single substrate. Importantly, although IL‐6R deficient mice were strongly affected by PHX, survival and regeneration of IL‐6 trans‐signaling mice was indistinguishable from control mice, demonstrating that IL‐6 trans‐signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long‐term consequences of global IL‐6 signaling inhibition versus IL‐6 trans‐signaling selective blockade after PHX by IL‐6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL‐6 blockade and selective inhibition of IL‐6 trans‐signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL‐6 trans‐signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL‐6 trans‐signaling, but not classic signaling, controls liver regeneration following PHX.
Interleukin-6 (IL-6)-type cytokines not only have key immunomodulatory functions that affect the pathogenesis of diseases such as autoimmune diseases, chronic inflammatory conditions, and cancer, but ...also fulfill important homeostatic tasks. Even though the pro-inflammatory arm has hindered the development of therapeutics based on natural-like IL-6-type cytokines to date, current synthetic trends might pave the way to overcome these limitations and eventually lead to immune-inert designer cytokines to aid type 2 diabetes and brain injuries. Those synthetic biology approaches include mutations, fusion proteins, and inter-cytokine swapping, and resulted in IL-6-type cytokines with altered receptor affinities, extended target cell profiles, and targeting of non-natural cytokine receptor complexes. Here, we survey synthetic cytokine developments within the IL-6-type cytokine family and discuss potential clinical applications.
OBJECTIVE—Interleukin (IL)-17A is regarded as an important cytokine to drive psoriasis, an inflammatory skin disease marked by increased cardiovascular mortality. We aimed to test the hypothesis that ...overproduction of IL-17A in the skin leading to dermal inflammation may systemically cause vascular dysfunction in psoriasis-like skin disease.
APPROACH AND RESULTS—Conditional overexpression of IL-17A in keratinocytes caused severe psoriasis-like skin inflammation in mice (K14-IL-17A mice), associated with increased reactive oxygen species formation and circulating CD11b inflammatory leukocytes in blood, with endothelial dysfunction, increased systolic blood pressure, left ventricular hypertrophy, and reduced survival compared with controls. In K14-IL-17A mice, immunohistochemistry and flow cytometry revealed increased vascular production of the nitric oxide/superoxide reaction product peroxynitrite and infiltration of the vasculature with myeloperoxidaseCD11bGR1F4/80 cells accompanied by increased expression of the inducible nitric oxide synthase and the nicotinamide dinucleotide phosphate (NADPH) oxidase, nox2. Neutrophil depletion by anti-GR-1 antibody injections reduced oxidative stress in blood and vessels. Neutralization of tumor necrosis factor-α and IL-6 (both downstream of IL-17A) reduced skin lesions, attenuated oxidative stress in heart and blood, and partially improved endothelial dysfunction in K14-IL-17A mice.
CONCLUSIONS—Dermal overexpression of IL-17A induces systemic endothelial dysfunction, vascular oxidative stress, arterial hypertension, and increases mortality mainly driven by myeloperoxidaseCD11bGR1F4/80 inflammatory cells. Depletion of the GR-1 immune cells or neutralization of IL-17A downstream cytokines by biologicals attenuates the vascular phenotype in K14-IL-17A mice.
Cytokine signaling is transmitted by cell surface receptors which act as natural biological switches to control cellular functions such as immune reactions. Recently, we have designed synthetic ...cytokine receptors (SyCyRs) consisting of green fluorescent protein (GFP)- and mCherry-nanobodies fused to the transmembrane and intracellular domains of cytokine receptors. Following stimulation with homo- and heterodimeric GFP-mCherry fusion proteins, the resulting receptors phenocopied signaling induced by physiologically occurring cytokines. GFP and mCherry fusion proteins were produced in E. coli or CHO-K1 cells, but the overall yield and stability was low. Therefore, we applied two alternative multimerization strategies and achieved immunoglobulin Fc-mediated dimeric and coiled-coil GCN4pII-mediated trimeric assemblies. GFP- and/or mCherry-Fc homodimers activated synthetic gp130 cytokine receptors, which naturally respond to Interleukin 6 family cytokines. Activation of these synthetic gp130 receptors resulted in STAT3 and ERK phosphorylation and subsequent proliferation of Ba/F3-gp130 cells. Half-maximal effective concentrations (EC50) of 8.1 ng/ml and 0.64 ng/ml were determined for dimeric GFP-Fc and mCherry-Fc, respectively. This is well within the expected EC50 range of the native cytokines. Moreover, we generated tetrameric and hexameric GFP-mCherry-Fc fusion proteins, which were also biologically active. This highlighted the importance of close juxtaposition of two cytokine receptors for efficient receptor activation. Finally, we used a trimeric GCN4pII motif to generate homo-trimeric GFP and mCherry complexes. These synthetic cytokines showed improved EC50 values (GFP3: 0.58 ng/ml; mCherrry3: 0.37 ng/ml), over dimeric Fc fused variants. In conclusion, we successfully generated highly effective and stable multimeric synthetic cytokine receptor ligands for activation of synthetic cytokine receptors.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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