Interleukin-13 (IL-13) has been implicated as a critical inducer of a number of features of allergy and asthma including the induction of nonspecific airway hyperresponsiveness (AHR), eosinophilic inflammatory response, eotaxin production, excess mucus formation, and fibrosis. Determining the mechanism(s) of AHR, a hallmark of asthma, is crucial to our understanding of both the pathogenesis and successful treatment of asthma. After carrying out initial experiments to determine the effect of IL-13-induced AHR on murine and rat tracheal rings, mice tissues were chosen for subsequent experiments due to their consistent results and the fact that the mouse genetic map was completed in 1996, which will enable subsequent gene therapy work. Human and mouse share a high percentage of their genes with an average of 85% homology. Numerous IL-13 signalling studies have concentrated on the JAK/STAT6 pathway. IL-13 also activates phosphoinositide 3-kinase (PI3K) and downstream effector molecules. In experiments presented in this thesis pharmacological and genetic approaches implicate the involvement of PI3K and its individual isoform PI3Kδ in IL-13 induced AHR in vitro and this involvement was confirmed using a small interference RNA (siRNA) technology approach. However, IL-13 induced an early activation of PI3K, whereas increased responsiveness was not observed until overnight incubation. Arginase I induction was demonstrated to be another PI3K-dependent potential mechanism of IL-13-induced hyperresponsiveness. The epithelium is also implicated in IL-13-induced hyperresponsiveness, however, the induction of arginase I was demonstrated in both intact and denuded epithelium tracheal rings. The siRNA approach was also employed in 9HTEo-, A549 and BEAS-2B cell lines using different transfecting agents. From these findings, it is concluded that class IA p110δ could be a useful target for the treatment of asthma by preventing IL-13-induced airway smooth muscle hyperresponsiveness and also that arginase I may be involved in IL-13-induced hyperresponsiveness through PI3K- and epithelial-dependent pathways.

Zsfassung in engl. Sprache. - Würzburg, Univ., Diss., 2008

Der Transkriptionsfaktor Stat6 vermittelt zentrale Wirkungen von IL-4 und IL-13, die in der Pathologie atopischer Erkrankungen eine Rolle spielen. Seine Spezifität für diese beiden allergieassoziierten Cytokine ist eine wesentliche Motivation ihn näher zu untersuchen. In dieser Arbeit sollte mehr über die Funktion von Stat6 herausgefunden werden. Außerdem wurden Möglichkeiten untersucht dieses Verhalten zu beinflussen. Einen Schwerpunkt der Arbeit bildete die Regulation des Eotaxin-1-Promotors. Eotaxin-1 ist einer der stärksten Rekrutierungsfaktoren für Eosinophile, die eine zentrale Rolle bei der Immunpathologie allergischer Erkrankungen spielen. Mit Hilfe der Daten konnte eine neue Hypothese zur Regulation des Eotaxin-1-Promotors entwickelt werden. Zum Vergleich wurde mit der Untersuchung des Promotors eines weiteren Chemokins, des MCP-4, begonnen. In Zusammenarbeit mit Dr. Sascha Stolzenberger wurde ein Weg untersucht den Stat6-Signalweg zu hemmen. Dabei wurden mit Hilfe des Antennapedia-Peptides Stat6-Bindepeptide in die Zelle transportiert, um dort über eine kompetitive Hemmung die Signaltransduktion zu unterbinden. Ergebnis dieser Arbeiten ist ein hochspezifischer, aber nur transient wirkender Stat6 Inhibitor. Die Stat6/DNA-Wechselwirkung wurde mit der Magnetobead-Technik untersucht. Dabei werden Promotorfragmente an Magnetkügelchen gekoppelt und unter Ausnutzung der Magnetisierung an die DNA bindende Proteine isoliert und über SDS-PAGE/Immunoblotanalyse untersucht. Mit dem Verfahren konnte die Stat6-Bindung an acht verschiedene Promotoren nachgewiesen werden. In Zusammenarbeit mit der Arbeitsgruppe Pallardy aus Paris wurde die Wechselwirkung von Stat6 mit dem Glucocorticoid-Rezeptor untersucht. Glucocorticoide kontrollieren Entzündungen und Interaktionen des aktivierten Rezeptors mit anderen Proteinen aus der Stat-Familie sind seit längerem bekannt. Wie in dieser Arbeit gezeigt wurde, interagiert Stat6 mit dem Glucocorticoidrezeptor unabhängig von einer Bindung an DNA. Zusätzlich wurde der Mucin-2-Promotor auf Stat6-Regulierung untersucht. Mucine sind wichtige Bestandteile des Schleimes. Verstärkte Schleim-Sekretion ist ein klinisches Symptom asthmatischer Erkrankungen und trägt zur Zerstörung der Lunge bei. Ein potentiell Stat6 reguliertes Fragment aus dem Mucinpromoter wurde mit Hilfe von PCR-Techniken isoliert und in Reportergenvektoren kloniert. / The transcriptionfactor Stat6 mediates central effects of the interleukins (IL)-4 and -13, that play important roles in the pathology of Allergy and Asthma. The specificity of these both Allergy-associated cytokines is a strong motivation to investigate the detailed functions of Stat6 and to search for possibilities to influence the behaviour of this transcriptionfactor. The main focus of this work was the regulation of the Eotaxin-1-promoter. The Eotaxin-1 chemokine is one of the most potent recruiting factors for eosinophils, that play a central role in the immunopathology of allergic diseases. On the basis of these data a new model for the regulation was created. In addition to this the investigation of another chemokine promoter, the MCP-4-promoter, was started. In another part of this work a specific Stat6-binding-peptide to inhibit the IL-4 signaltransduction pathway was established. Using the Antennapedia-carrier-peptide allowed to shuttle Stat6-binding peptides into cells where they prevented Stat6 mediated signalling by competitive inhibition. Thus the Stat6-binding-peptide came out to be a transient Stat6 inhibitor with high specificity. The Stat6/DNA-interaction was investigated by DNA-pull-down assays with magnetobeads. Fragments of different promoters are linked to magnetobeads and by using magnetic forces the DNA binding proteins are isolated. This application was used to show Stat6-binding to 8 different promoters. Another subject of this work was the interaction of Stat6 with the glucocorticoid receptor. It is well known that glucocorticoids control inflammation and that the activated receptor interacts with different proteins of the Stat-family. In collaboration with the group of Marc Pallardy in Paris we were able to show that Stat6 interacts with the glucocorticoid receptor independently of DNA binding. In association with Stat6 the regulation of the Mucin-2-promoter seemed to be an interesting aspect. Mucins are essential components of mucus (slime). Enhanced mucus-secretion is a symptom of asthmatic diseases and contributes to the destruction of the lung. A potentially Stat6 regulated fragment was isolated by PCR-techniques and cloned into reportergene vectors.

<p>Cytokines play important roles in bone metabolism, participating in the complex interplay necessary for normal bone formation and turnover. The aim of the present thesis was to investigate the effects of two anti-inflammatory cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13) on bone. </p><p>Influence of pro- and anti-inflammatory cytokines on interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs) was investigated. IL-4 and IL-13 as well as interleukin-1 (IL-1) and tumour necrosis factor alpha and beta (TNF-α/β) stimulated IL-6 secretion in hOBs. Also, IL-4 and IL-13 synergistically potentiated the effect of IL-1 and TNFs on IL-6 secretion. </p><p>Effects of IL-4 and IL-13 on markers of osteoblastic activity in hOBs were investigated. IL-4 and IL-13 induced a dose-dependent increase in the formation of alkaline phosphatase (ALP) and pro-collagen type I carboxy-peptide (PICP) together with enhanced mineralization rate in hOBs. Formation of osteocalcin (OC) was unaffected. </p><p>The mechanism behind inhibited proliferation by IL-4 and IL-13 in hOBs was investigated. IL-4 and IL-13 caused a dose-dependent increase in DNA-fragmentation together with escalating Caspase-3 activity in hOBs, reflecting induced apoptosis. Osteoblast apoptosis was also confirmed by TNF-α, dexamethasone and by serum starvation.</p><p>The skeletal phenotype of IL-13^-/-, IL-4^-/-IL-13^-/- and WT mice was compared. An altered cortical bone mass was detected in adult male IL-4^-/-IL-13^-/- mice. They displayed a reduction in cortical bone mineral content (BMC) secondary to reduced cortical thickness. Mechanical strength of the cortical bone was reduced in level with the reduction detected in BMC. Trabecular bone mineral density (tvBMD) was unaffected. </p><p>Callus formation in IL-4^-/-IL-13^-/- and WT male mice was compared. No differences were found concerning radiological healing, biomechanical properties, callus parameters or histology. Heterotopic bone formation in IL-4^-/-IL-13^-/- and WT mice was compared using DXBM implants. No differences were found concerning mineralization of implants. Immuno-histology showed inhibition of autonomic nerves and lack of implant vascularization in IL-4^-/-IL-13^-/- mice. </p><p>In summery, the two anti-inflammatory cytokines IL-4 and IL-13 influence osteoblast activity and apoptosis <i>in vitro</i>. They also selectively influence cortical bone formation <i>in vivo</i>. These findings suggest a role for IL-4 and IL-13 in osteoblast differentiation, in bone metabolism and in bone formation. </p>

Cytokines play important roles in bone metabolism, participating in the complex interplay necessary for normal bone formation and turnover. The aim of the present thesis was to investigate the effects of two anti-inflammatory cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13) on bone. Influence of pro- and anti-inflammatory cytokines on interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs) was investigated. IL-4 and IL-13 as well as interleukin-1 (IL-1) and tumour necrosis factor alpha and beta (TNF-α/β) stimulated IL-6 secretion in hOBs. Also, IL-4 and IL-13 synergistically potentiated the effect of IL-1 and TNFs on IL-6 secretion. Effects of IL-4 and IL-13 on markers of osteoblastic activity in hOBs were investigated. IL-4 and IL-13 induced a dose-dependent increase in the formation of alkaline phosphatase (ALP) and pro-collagen type I carboxy-peptide (PICP) together with enhanced mineralization rate in hOBs. Formation of osteocalcin (OC) was unaffected. The mechanism behind inhibited proliferation by IL-4 and IL-13 in hOBs was investigated. IL-4 and IL-13 caused a dose-dependent increase in DNA-fragmentation together with escalating Caspase-3 activity in hOBs, reflecting induced apoptosis. Osteoblast apoptosis was also confirmed by TNF-α, dexamethasone and by serum starvation. The skeletal phenotype of IL-13-/-, IL-4-/-IL-13-/- and WT mice was compared. An altered cortical bone mass was detected in adult male IL-4-/-IL-13-/- mice. They displayed a reduction in cortical bone mineral content (BMC) secondary to reduced cortical thickness. Mechanical strength of the cortical bone was reduced in level with the reduction detected in BMC. Trabecular bone mineral density (tvBMD) was unaffected. Callus formation in IL-4-/-IL-13-/- and WT male mice was compared. No differences were found concerning radiological healing, biomechanical properties, callus parameters or histology. Heterotopic bone formation in IL-4-/-IL-13-/- and WT mice was compared using DXBM implants. No differences were found concerning mineralization of implants. Immuno-histology showed inhibition of autonomic nerves and lack of implant vascularization in IL-4-/-IL-13-/- mice. In summery, the two anti-inflammatory cytokines IL-4 and IL-13 influence osteoblast activity and apoptosis in vitro. They also selectively influence cortical bone formation in vivo. These findings suggest a role for IL-4 and IL-13 in osteoblast differentiation, in bone metabolism and in bone formation.

Cytokines activate cell surface receptors to control and regulate immunity and hematopoiesis. Despite its enormous potential, pharmaceutical use of cytokines is in most cases hampered by their pleiotropic functionality, which renders cytokine-based therapies exceptionally difficult to control. Although there is growing evidence that the functional plasticity of cytokine receptors is largely encoded in the spatiotemporal dynamics of receptor complexes, no mechanistic correlation has hitherto been achieved. Two related aspects, the spatiotemporal organization and the activation mechanism of cytokine receptors in the plasma membrane, have further remained a topic of intensive and controversial debate. To shed to light into the mechanistic principles responsible for functional selectivity, this thesis aimed to quantitatively explore the molecular and cellular determinants governing cytokine receptor assembly and signaling using the type II interleukin-4 (IL-4) receptor as model system. To this end, by taking advantage of IL-4 and interleukin 13 (IL-13) agonists binding the receptor subunits IL-4Rα and IL-13Rα1 with different affinities and rate constants, an in vitro kinetic characterization of the receptor system was combined with live cell microscopy on the single molecule level and flow cytometry as well as in silico modeling approaches. The quantification of kinetics by a dedicated solid-phase detection method with the extracellular receptor domains tethered onto artificial membranes confirmed that the affinity and stability of the two-dimensional molecular interactions determine receptor dimerization levels and dynamics. Single molecule localization microscopy at physiological cell surface expression levels, however, revealed efficient ligand-induced receptor dimerization, largely independent of the two-dimensional receptor binding affinities, in line with similar STAT6 activation potencies observed for different IL-4 variants. Detailed spatiotemporal analyses and single molecule co-tracking of receptor subunits and ligands in conjunction with spatial-stochastic modeling identified confinement by actin-dependent membrane micro-compartments as an important cellular determinant for sustaining transient receptor dimers. By correlating downstream cellular responses with various three-dimensional binding affinities and kinetics of engineered IL-13 variants, distinct roles of ligand association and dissociation kinetics were uncovered. Whereas the extent of membrane-proximal effector activation is dependent on the association rate by controlling the number of formed receptor complexes in the plasma membrane, the lifetime of receptor complexes determines the potency of a ligand for inducing more distal responses and is, due to accumulation of signaling complexes in endosomes, directly connected to the kinetics of early signaling events.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Vita. Thesis advisor: Habib Zaghouani. Includes bibliographical references.

Atopische Erkrankungen wie Asthma, atopische Dermatitis und allergische Rhinitis sind komplexe, multifaktorielle Erkrankungen. Diese Arbeit untersuchte Gen-Gen- und Gen-Umweltinteraktionen von insgesamt acht Polymorphismen in fünf Kandidatengenen. In einer großen deutschen Geburtskohorte (Multizentrische Allergie Studie, MAS 90) mit longitudinalen klinischen und serologischen Daten wurde der Effekt von Polymorphismen in Genen, die für Interleukin-13 (IL-13), Interleukin-4 (IL-4) und dessen gemeinsamer Rezeptoruntereinheit IL4R-a kodieren, auf Atopie-assoziierte Merkmale untersucht. Zwei Polymorphismen im IL13-Gen (Arg130Gln, C-1055T) zeigten über den gesamten Beobachtungszeitraum von sieben Jahren eine signifikante Assoziation mit erhöhten Gesamt-IgE-Werten. Weiterhin konnte diese Untersuchung zeigen, dass mütterliches Rauchen diesen Effekt auf die IgE-Werte noch verstärkt. Ein Polymorphismus des Komplementfaktor-C5-Rezeptors, der im Mausmodell als Suszeptibilitätslokus für Asthma identifiziert wurde, wurde in der MAS-Kohorte wie auch in einer afro-karibischen Population auf Assoziation mit Atopie-relevanten Merkmalen untersucht. Eine Assoziation konnte zwar nicht gefunden werden, jedoch waren hochsignifikante Unterschiede in der Allelfrequenz in den zwei ethnischen Gruppen auffällig; ein Phänomen, welches auch bei anderen genetischen Varianten in proinflammatorischen Genen beobachtet wurde. Ein funktioneller Polymorphismus des histaminabbauenden Enzyms Histamin-N-Methyl-Transferase wurde auf eine Assoziation mit bronchialer Hyperreagibilität und Asthma in der MAS-Kohorte sowie in einer weiteren kaukasischen Population von asthmatischen Kindern überprüft. Weder eine Assoziation mit Asthma noch eine modulierende Wirkung auf den Schweregrad der bronchialen Hyperreagibilität konnte in den Studienpopulationen gefunden werden. Im Gegensatz zu Untersuchungen bei erwachsenen Asthmatikern scheint diese Variante bei kindlichem Asthma keine entscheidende Rolle zu spielen. / Atopic diseases like asthma, atopic dermatitis and allergic rhinitis are complex traits of multifactorial origin. This study aimed to reveal gene-gene- and gene-environmental interactions of eight polymorphisms in five candidate genes. We examined whether 6 genetic variants of the genes coding for Interleukin-4 (IL-4), Interleukin-13 (IL-13) and their common receptor unit IL4R-alpha had genotypic effects on atopy-related traits such as total serum IgE levels in a large German birth cohort study (Multicenter Atopy Study, MAS 90) with longitudinally well defined phenotypes. Two single nucelotide polymorphisms (SNP) in the IL-13 gene (Arg130Gln, C-1055T) showed a significant association with increased serum IgE levels over the whole period of seven years. In addition, exposure to maternal smoking appears to modify the above effects on total serum IgE levels. We tested the association of atopy-related traits and a SNP of the complement factor 5 receptor (-245T) in the MAS cohort and in an afro-caribbean population. Although we could not observe any association, there was a highly significant difference in the frequency of the -245T allele between the two ethnic populations, a phenomenon previously described for other genetic variants. Histamine N-Methyltransferase (HNMT) catalyses the major pathway of histamine metabolism in the human lung. We tested the functional polymorphism C314T of HNMT for association with asthma and bronchial hyperresponsiveness in two German pediatric populations (MAS, Astmatic children from Freiburg). No association of the T314 Allele with asthma, BHR and other asthma related phenotypes could be observed. We concluded that this SNP might not play a major role in the pathogenesis of asthma or BHR in German children.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / "May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala univeritet, 2008. / Härtill 5 uppsatser.