Sebastian Krause

Grafting of thrombopoietin‐mimetic peptides into cystine knot miniproteins yields high‐affinity thrombopoietin antagonists and agonists

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high‐affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin‐mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c‐Mpl receptor agonists with EC50 values in the low nanomolar or picomolar range. One selected miniprotein‐derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand‐induced dimerization.

Signal transduction by the atopy-associated human thymic stromal lymphopoietin (TSLP) receptor depends on Janus kinase function.

Abstract Thymic stromal lymphopoietin (TSLP) is an interleukin-(IL)-7-like cytokine with emerging pathological importance for the development of atopic diseases such as allergic asthma bronchiale. The TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor, shares the IL-7R α-subunit with the IL-7 receptor system. The specific TSLPR α-chain shows similarities with the γc receptor chain, but has some unusual features within the receptor family in both its ligand-binding and cytoplasmic domain. The murine TSLPR signals via the signal transducers and activators of transcription STAT5 and STAT3, but is unique among cytokine receptors in that it activates STATs without the involvement of Janus (JAK) tyrosine kinases, but instead utilizes the Src type kinase Tec. Here, we show by Western blotting and reporter gene experiments in combination with the application of a specific JAK inhibitor that the human TSLP receptor, in contrast, requires the function of JAK1 and JAK2 for STAT activation. Moreover, we demonstrate that the human TSLPR mediates gene regulation not only through STAT5 and STAT3 but has also the potential to mediate transcription via STAT1. Our work should help to understand more thoroughly how TSLP triggers inflammatory responses in the course of atopic diseases.

Interleukin‐13 acts as an apoptotic effector on lung epithelial cells and induces pro‐fibrotic gene expression in lung fibroblasts

Background IL‐13 promotes acute allergic asthma and is discussed to play a role in late asthmatic features such as fibrotic processes and airway remodelling. The contributions of IL‐13‐mediated mechanisms to subepithelial events related to fibrosis are not yet settled.

Expression of interleukin-13 receptor α 1-subunit on peripheral blood eosinophils is regulated by cytokines

Interleukin‐13 (IL‐13) is critical for the development of allergic asthma and is involved in the activation of eosinophils within the airways. IL‐13 exerts its activity on target cells via the dimeric IL‐13 receptor (IL‐13R), which comprises the IL‐13 receptor α1‐chain (IL‐13Rα1) as a specific component. The aim of this study was to investigate the expression of the IL‐13Rα1‐chain on primary human eosinophilic granulocytes. Furthermore, it addresses the regulatory influence of cytokines on the level of surface abundance of this receptor subunit. Expression of IL‐13‐ and IL‐4‐receptor subunits in purified primary human eosinophils was monitored at the messenger RNA level by reverse transcription polymerase chain reaction and at the protein level by flow cytometry. For the analysis of IL‐13Rα1 surface expression, a new monoclonal antibody, which was generated using genetic immunization, was employed. Different cytokines with established activity on eosinophils were studied with regard to their influence on IL‐13Rα1 in vitro by flow cytometry. Whereas IL‐13 and IL‐4 had inhibitory effects on IL‐13Rα1 expression on eosinophils, interferon‐γ, tumour necrosis factor‐α, and, to the largest extent, transforming growth factor‐β, enhanced the expression of this receptor subunit. A positive regulatory response evoked by transforming growth factor‐β and interferon‐γ does not prevent inhibitory effects caused by IL‐13. These findings suggest a regulatory cytokine network influencing the reactivity of eosinophils to IL‐13.

Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range.

Specific inhibition of interleukin-13 activity by a recombinant human single-chain immunoglobulin domain directed against the IL-13 receptor alpha1 chain.

Abstract Interleukin-13 (IL-13) is a T-cell-derived pleiotropic cytokine of particular medical importance because of its critical role in the development of allergic asthma. The effects of IL-13 on its target cells are mediated through a dimeric transmembrane receptor (IL-13R), which shares the IL-4Rα subunit with the IL-4R system, but contains as a specific component the IL-13Rα1 chain. We have generated a set of single-chain Fv fragments with specific binding capacity to the extracellular domain of the human IL-13Rα1 receptor. Bacteriophage clones displaying receptor-binding antibody domains were selected from both naive and synthetic libraries by repetitive panning on recombinant and cell surface-expressed recombinant IL-13Rα1. Their specific reactivity with native human IL-13Rα1 expressed on the surface of transfected cells was demonstrated by flow cytometry. One binder that specifically interfered with cell activation by IL-13 was extensively characterized. This scFv inhibited IL-13-driven gene transcription and cell proliferation in test cell lines, as well as IL-13-induced activation of primary human monocytes in a dose-dependent manner, with an IC50 below 300 nM. This novel reagent thus constitutes a valuable tool for the further elucidation of IL-13 function in disease and offers potential therapeutic perspectives.

Bacteria Displaying Interleukin‐4 Mutants Stimulate Mammalian Cells and Reflect the Biological Activities of Variant Soluble Cytokines

We describe a novel procedure that allows the rapid determination of cytokine activity on cells that express their cognate receptor. The four‐helix bundle cytokine interleukin‐4 (IL‐4) was inducibly expressed as a fusion with the E. coli outer‐membrane protein intimin, such that IL‐4 was presented on the surfaces of the bacteria. Expression and accessibility of the cytokine on the cell exteriors were monitored by Western blotting and fluorescence microscopy, making use of two epitopes flanking the IL‐4 component of the fusion protein. To demonstrate the biological activity of the immobilized cytokine, a Ba/F3‐derived cell line stably transfected with both the bipartite human IL‐4 receptor and an IL‐4‐specific luciferase reporter gene construct was employed. Bacterial cells displaying interleukin‐4 elicited a specific, dose‐dependent response in the reporter cells. Two variants of IL‐4 with previously characterized (partial) antagonistic properties were also expressed as membrane‐bound fusion proteins and were tested for their activity in the immobilized state. In comparison with bacteria displaying wild‐type IL‐4, E. coli clones presenting variants IL‐4 Y124G and Y124D showed diminished or abolished activity, respectively, on murine reporter cells. The relative signaling potencies of the immobilized IL‐4 variants thus closely mirror the agonistic properties of the corresponding soluble cytokines. This approach should be generally applicable for the mutational analysis of numerous signal mediators that trigger cellular responses through dimerization of transmembrane receptors.

A versatile platform for activity determination of cytokines and growth factors based on the human TSLP (thymic stromal lymphopoietin) receptor

&NA; Cytokines and growth factors are signaling proteins involved in communication processes between cells. They are involved in the control of numerous essential physiological processes such as cell proliferation, gene transcription and differentiation; therefore being in the focus of basic and applied research. Many of them are also of relevance for human diseases. When observed as potential targets for pharmacological intervention and objects of structure/function studies, it is important to measure their biological activities, optionally along with potential inhibitors, in a convenient and rational manner. Such tests are frequently laborious to set up and their establishment is complicated by the necessity to employ problematic cell types and sophisticated assays. Here we present a robust and modular activity assay system which can be adapted to virtually all ligands that signal through dimerization of membrane receptors from different families. The technique rests on fusing ligand‐binding domains of specific receptors to the transmembrane and intracellular components of the thymic stromal lymphopoietin (TSLP) receptor which translates signals into readily quantifiable luciferase expression in reporter cells. We show that the activation of various hematopoietic cytokine receptors, of receptor tyrosine kinases as well as of receptors bearing serine/threonine kinase domains by their respective ligands was faithfully reflected both upon transient and stable introduction of hybrid receptor and reporter gene constructs into the murine pro‐B cell line Ba/F3. Moreover, we demonstrate the suitability of this platform for the functional characterization of cytokine/growth factor receptor inhibitors.

Model cell systems representing expression regulation and signal transduction of thymic stromal lymphopoietin (TSLP) in the development of asthmatic symptoms

Thymic stromal lymphopoietin (TSLP) is a novel interleukin-7-like cytokine, which triggers dendritic cell-mediated inflammatory responses ultimately executed by Th2 helper cells. TSLP is a central player in the development of allergic, especially asthmatic symptoms, and has been suggested to represent a missing link in the information flow from antigen-exposed epithelial cells via dendritic cells (DCs) to T helper cells. We have challenged the lung epithelial cell line A549 with various allergens such as extracts from dust mites, pollen and fungi, and could show by real time PCR for the first time an allergen-dependent specific transcriptional upregulation of TSLP. To approach the as yet poorly defined TSLP-induced signal transduction in dendritic cells, we established a cellular model system on the basis of the murine pro-B cell line Ba/F3. The heterodimeric human TSLP receptor (hTSLPR) consisting of the novel TSLP receptor chain and the IL-7 receptor alpha chain was functionally reconstituted in factor-dependent Ba/F3 cells. We could demonstrate by phosphotyrosine protein analysis and by STAT type-specific reporter gene assays, that the ligand-stimulated TSLPR triggers activation of STAT3 and STAT5 as well as of STAT1. Employment of specific inhibitors proved the functional involvement of Janus kinases (JAKs) in TSLP-dependent, receptor-mediated cellular responses. These achievements provide tools for research towards a better molecular understandimg of atopic asthma and for the future development of new therapeutics interfering with TSLP function.