Thesis (Ph. D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 152 pages. Includes Vita. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18157号 / 医博第3877号 / 新制||医||1003(附属図書館) / 31015 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 清水 章, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

Cyclosporine is a potent immunosuppressive agent used to treat a wide range of canine inflammatory diseases. Unfortunately, optimal dosing protocols for achieving immunosuppression with cyclosporine in dogs remain unclear, and standard methods that objectively monitor effectiveness of immunosuppression have not been established. We evaluated an already established panel of biomarkers of immunosuppression in vivo with two oral dosages of cyclosporine in seven normal dogs, a high dosage known to induce immunosuppression and a lower dosage used to treat atopy, with a washout period between the two dosages. The biomarker panel included the flow cytometric evaluation of T-lymphocyte cytokine expression (IL-2, IL-4, and IFN-gamma). High dosage cyclosporine resulted in significant decreases in IL-2 and INF-gamma expression, but not IL-4 expression. Low dosage cyclosporine was associated with a significant decrease in INF-gamma expression, while IL-2 expression was not affected. The results demonstrated suppression of biomarkers in a dose-dependent manner.

Cardiovascular disease (CVD) including atherosclerosis is the leading cause of illness and death in the United States. The American Heart Association indicated that an estimated 81,100,000 American adults have one or more types of CVD and the estimated direct and indirect cost of CVD for 2010 is $503.2 billion, which is $27.9 billion more than last year. Although the exact cause of this disease remains unsolved, previous studies have demonstrated that pro-oxidative and pro-inflammatory pathways in vascular endothelium play a critical role in early pathological events of atherosclerosis. However, the detailed molecular signaling mechanisms underlying this process are not yet completely understood. Recently, the role of interleukin-4 (IL-4) in atherogenesis became controversial and gained attention. IL-4 is a pleiotropic immunomodulatory cytokine secreted by T-helper 2 (Th2) lymphocytes, eosinophils, and mast cells. It was traditionally believed to be an anti-inflammatory cytokine. Increasing evidence, however, has suggested that IL-4 contributes to the initiation and progression of atherosclerosis by oxidative stress-mediated up-regulation of pro-inflammatory mediators such as vascular cell adhesion moledule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in vascular endothelium. 5-Lipoxygenase (5-LOX) is one of the key sources that generate reactive oxygen species (ROS) via metabolic pathways of arachidonic acid. Although 5-LOX has been implicated in the development of atherosclerosis, it remains unclear whether 5-LOX-mediated ROS generation is associated with IL-4-induced MCP-1 expression in vascular endothelium. The present study was focused on the oxidative mechanisms by which IL-4 induces vascular inflammation as well as how 5-LOX is involved in this process. The results showed that IL-4 significantly up-regulates mRNA and protein expression of MCP-1 in vascular endothelium. In addition, DHE and DCF fluorescence staining demonstrated that IL-4 increases ROS production in human vascular endothelial cells. We have also provided the first novel evidence that 5-LOX, one of the enzymes associated with arachidonic acid metabolism, is responsible for the IL-4-induced ROS generation and MCP-1 expression in human vascular endothelial cells. / Master of Science

No / Inflammatory changes, characterized by an increase in pro-inflammatory cytokine production and up-regulation of the corresponding signaling pathways, have been described in the brains of aged rats and rats treated with the potent immune modulatory molecule lipopolysaccharide (LPS). These changes have been coupled with a deficit in long-term potentiation (LTP) in hippocampus. The evidence suggests that anti-inflammatory agents, which attenuate the LPS-induced and age-associated increase in hippocampal interleukin-1ß (IL-1ß) concentration, lead to restoration of LTP. Here we report that atorvastatin, a member of the family of agents that act as inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, exerts powerful anti-inflammatory effects in brain and that these effects are mediated by IL-4 and independent of its cholesterol-lowering actions. Treatment of rats with atorvastatin increased IL-4 concentration in hippocampal tissue prepared from LPS-treated and aged rats and abrogated the age-related and LPS-induced increases in pro-inflammatory cytokines, interferon-¿ (IFN¿) and IL-1ß, and the accompanying deficit in LTP. The effect of atorvastatin on the LPS-induced increases in IFN¿ and IL-1ß was absent in tissue prepared from IL-4¿/¿ mice. The increase in IL-1ß in LPS-treated and aged rats is associated with increased microglial activation, assessed by analysis of major histocompatibility complex II expression, and the evidence suggests that IFN¿ may trigger this activation. We propose that the primary effect of atorvastatin is to increase IL-4, which antagonizes the effects of IFN¿, the associated increase in microglial activation, and the subsequent cascade of events.

La protéine IL4I1 est une enzyme sécrétée dont l’activité L-amino acide oxydase vis-à-visde la phénylalanine inhibe la prolifération des lymphocytes T in vitro (Boulland et al, Blood 2007).Comme d’autres enzymes immunosuppressives, elle est exprimée dans les tumeurs au niveau descellules myéloïdes et/ou des cellules tumorales (Carbonnelle-Puscian et al, Leukemia 2009). Le but decette thèse a été de caractériser les conditions d’expression d’IL4I1 et de comprendre son rôle dans lecancer.Nous avons montré que les macrophages et les cellules dendritiques représentent la principalesource d’IL4I1 in vitro et dans des lésions inflammatoires chroniques. L’expression d’IL4I1 dans lesphagocytes mononucléés est induite par les interférons ou les ligands de TLR, activant respectivementSTAT1 et NF-kB, tandis que les lymphocytes B expriment des niveaux nettement plus faibles d’IL4I1sous le contrôle de la voie IL-4/STAT6 et de la voie CD40/NFkB. L’expression d’IL4I1 par des cellulesmonocytaires inhibe la production de cytokines Th1 et pourrait donc contribuer à la régulation del’inflammation Th1 in vivo.Dans un modèle murin de cancer, l’expression d’IL4I1 facilite le développement tumoral endiminuant la réponse T cytotoxique spécifique de la tumeur. Ceci est observé à des niveaux d’activitéIL4I1 proches de ceux mesurés dans des tumeurs humaines, suggérant qu’IL4I1 puisse contribuer àl’échappement des tumeurs au système immunitaire chez l’homme. Nous avons développé plusieursmutants d’IL4I1, afin d’évaluer l’impact de l’activité enzymatique versus celui de l’éventuelle liaison del’enzyme à un récepteur, dans l’effet protumoral observé. Un de ces mutants est actuellementdisponible pour une étude chez la souris.Nos résultats installent définitivement IL4I1 dans le panorama des enzymesimmunosuppressives associées au cancer et ouvrent la voie au développement d’inhibiteursspécifiques comme outils thérapeutiques / The IL4I1 protein is a secreted L-amino acid oxidase, which inhibits T cell proliferationthrough phenylalanine degradation in vitro (Boulland et al, Blood 2007). Similar to previously describedimmunosuppressive enzymes, IL4I1 is expressed in cancer by myeloid cells and/or tumor cells(Carbonnelle-Puscian et al, Leukemia 2009). The aim of this work was to characterize the cells andstimuli associated with IL4I1 expression and to decipher its role in cancer.We showed that macrophages and dendritic cells are the main source of IL4I1 in vitro and inchronic inflammatory lesions. IL4I1 expression in mononuclear phagocytes is induced by interferons orTLR ligands, which act through STAT1 and NFkB respectively. Conversely, B cells express dramaticallylower levels of IL4I1 under the control of IL-4/STAT6 and CD40/NFkB. IL4I1 expression by monocyticcells inhibits the production of Th1 cytokines and may thus contribute to Th1 inflammation control invivo.In a murine model of cancer, IL4I1 expression facilitates tumor development by depressing thetumor specific cytotoxic T cell response. This is observed for IL4I1 activity levels in the range of thosemeasured in human tumors, suggesting that IL4I1 may contribute to tumor immune escape in humans.We developed several IL4I1 mutants to discriminate the role of the enzymatic activity versus the bindingto a putative cell surface receptor in the protumor effect observed. One of these mutants is currentlyavailable for in vivo testing.Our results definitively establish IL4I1 in the family of immunosuppressive enzymes associatedwith cancer and pave the way for the development of specific inhibitors as therapeutic tools

Alternatives mRNA-Splicing ist ein häufig beobachtetes Phänomen, das es der Zelle ermöglicht, unterschiedliche Proteine aus einem Gen zu generieren. In den letzten Jahren wurden immer mehr alternativ gespleißte Transkripte entdeckt, und einigen der daraus resultierenden Protein-Isoformen konnten geänderte biologische Funktionen zugeordnet werden. In dieser Arbeit ist erstmals ein alternativ gespleißtes Transkript der Interleukin-4 Rezeptor alpha (IL-4R-alpha) Kette beschrieben. Dieser mRNA Splice-Variante, genannt IL-4R-alpha-IT, fehlt im membranproximalen Bereich der zytoplasmatischen Domäne ein komplettes Exon. Dies führt zur Verschiebung des Leserasters und so zur Entstehung eines vorzeiten Stop-Codons. Der resultierenden Protein-Isoform fehlt der größte Teil der intrazellulären Kette mit den dort enthaltenen, für die Signaltransduktion essentiellen Domänen. Die Untersuchung der biologischen Funktion der Rezeptor-Varianten in einem geeigneten Zellsystem der Maus zeigte, daß die Splice-Variante IL-4R-alpha-IT keine Proliferation der Zellen vermitteln und auch den Übergang der Zellen in die Apoptose nicht verhindern kann. Bei der Quantifizierung der Expression von IL-4R-alpha-IT-mRNA in Relation zum IL-4R-alpha voller Länge mit einer kompetitiven RT-PCR an Knochenmark und peripheren Blutlymphozyten von Kindern mit ALL zeigte sich zunächst ein irreführender Unterschied zwischen Proben von Kindern mit ALL-Ersterkrankung und Rezidiv. Weitere Untersuchungen ergaben jedoch, daß der Zeitraum zwischen Abnahme und Aufarbeitung des Untersuchungsmaterials für diesen scheinbaren Zusammenhang verantwortlich war. Während direkt nach Abnahme aufgearbeitetes Untersuchungsmaterial eine nur niedrige relative Expression der Splice-Variante zeigte, nahm diese bei verzögerter Aufarbeitung drastisch zu. Diese Beobachtung wurde experimentell an Proben gesunder Probanden wiederholt bestätigt. Interessanterweise konnte derselbe Effekt in unterschiedlicher Ausprägung auch bei Splice-Variante anderer Zytokine und -Rezeptoren wie IL-7, IL-7R und beta-C beobachtet werden. mRNA-Stabilitäts-Assays und die Bestimmung der einzelnen Transkripte mit einer semiquantitativen RT-PCR zeigten, daß es tatsächlich zu einer absoluten Hochregulation der IL-4R-alpha-IT-mRNA in den verzögerte aufgearbeiteten Proben kommt. Wurden die Zellen wieder in Kultur genommen, war dies innerhalb weniger Stunden reversibel. Desweiteren scheinen auch unterschiedlichen mRNA-Stabilitäten eine Rolle zu spielen. / Alternative pre-mRNA splicing is a widespread mechanism contributing to the diversity of gene expression. The number of newly detected alternatively spliced transcripts has continuously risen, and distinct biological functions have been attributed to some protein isoforms resulting from these mRNA variants. We report on the detection of a novel alternatively spliced transcript of the human interleukin-4 receptor alpha (IL-4R-alpha) chain, which has been called IL-4R-alpha-IT mRNA. A premature stop codon due to omission of one exon in the membrane-proximal region of the cytoplasmic domain leads to an mRNA variant, which encodes an intracellular truncated receptor protein lacking domains which are essential for signal transduction. The investigation of the biological function of the IL-4Ra splice variants in a suitable mouse cell system showed, that the truncated receptor variant is not able to mediate cell proliferation or prevention of apoptosis. Bone marrow and peripheral blood samples from children with acute lymphoblastic leukemia were analyzed for the expression of IL-4R-alpha-IT mRNA relative to the full-length receptor transcript by competitive RT-PCR. Initially, there was found a difference of IL-4R-alpha-IT mRNA expression in patients with initial ALL versus relapsed ALL. However, this difference turned out to be due to the time interval between collection and preparation of samples. While freshly isolated material was associated with low levels of IL-4R-alpha-IT mRNA, samples with a longer period until cell preparation exhibited a drastic increase of IL-4R-alpha-IT mRNA levels. The same results were obtained for peripheral blood samples from healthy donors by imitating a prolonged time of transport until cell preparation. Interestingly, a similar effect could be demonstrated for splice variants of other cytokine receptors and cytokines (beta-C, IL-7R, and IL-7), although to different extents. mRNA stability assays and semiquantitative RT-PCR specific for IL-4Ra or IL-4R-alpha-IT, respectively, indicated that the expression of IL-4R-alpha-IT mRNA increases absolutely in these samples, although mRNA degradation may be of importance as well.

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.

Intensive study of interleukin-4 for more than three decades has revealed multiple functions of this cytokine in diverse processes. Nevertheless, the wide distribution of Interleukin-4 suggests the possibility of unexplored roles. Indeed, in here we present a novel role of IL-4 for the maintenance of different populations of stromal cells in peripheral lymph nodes at homeostasis and describe a role of IL-4 in the expansion of these stromal populations following antigen challenge. In consequence, IL-4 is fundamental for mounting an appropriate humoral response to a primary immunization, and absence of this cytokine is detrimental for the development of a Type 2 response. Furthermore, we describe the role of IL-4 in the immune responses of offspring antenatally exposed to <i>Schistosoma mansoni</i>antigens. Diminished IL-4 production is linked to reduced cellular T and B cells responses in offspring derived from infected mothers, which is of critical relevance to understand vaccination failure. Finally, we describe the protective role of Schistosomiasis infection in atherosclerosis and propose possible mechanism that helps explain the athero-protection. This will contribute to the discovery of novel pathways inducing protection from cardiovascular disease and help to identify possible targets for novel treatments