The alternative activation of macrophage populations by Interleukin-4 (IL-4) is well characterised. Alternatively activated macrophages (AAM) express high levels of the arginine converting enzyme arginase-1, and express a plethora of IL-4 driven molecules including the resistin like molecule alpha (RELMα) and the chitinase like molecule Ym1/2. Dendritic cells (DCs) are the professional antigen presenting cells (APC) of the immune system, responsible for the detection of invading pathogens, secretion of cytokines and the subsequent activation of T-cells. This thesis addresses whether IL-4 is able to ‘alternatively activate’ DCs both in vitro and in vivo, in a manner similar to that of AAM. The impact of IL-4 on DC and macrophage activation was compared and contrasted, and it was confirmed for the first time that IL-4 can alternatively activate DCs, inducing high level expression of a range of alternative activation associated markers including RELMα, Ym1/2, CCL24 and dectin-1, with the exception of arginase. DCs were significantly more capable at the in vivo priming of T-cell responses in the context of both Th1 and Th2 polarising antigens than similarly exposed macrophages, confirming their superior capacity as APC. The requirements for DC IL-4Rα expression were assessed, and IL-4 responsiveness was found to be required for the optimal induction of Th1 responses. Conversely, selective loss of only one facet of the IL-4 response, namely RELMα expression, limited the ability of IL-4 exposed DCs to induce the regulatory cytokine IL-10 both in vitro and in vivo. Furthermore, alternatively activated DCs (AADCs) were found in the spleen following 8 weeks of infection with the parasitic trematode Schistosoma mansoni, highlighting a role for DC alternative activation in a disease setting. IL-4 was shown to induce expression of the vitamin A converting enzyme aldehyde dehydrogenase, and the product of such activity, retinoic acid (RA), was found to promote the expression of RELMα in IL-4 exposed DCs. Aldehyde dehydrogenase activity was found to inversely correlate with DC expression of Ym1/2 and inhibition of RA signalling limited IL-4 driven RELMα and promoted Ym1/2.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, June 2001. / Includes bibliographical references. Also available on the Internet.

Cytokines play an important regulatory role during immune responses against pathogens. The outcome of an induced cytokine pattern is determined by many factors. It strongly depends on the nature of the pathogen and the host’s ability to control the quality and strength of cytokine signals. In pulmonary infection with Cryptococcus neoformans T helper (Th) 1 and Th17 cell subsets and their associated cytokines confer protection, whereas a Th2-biased response with production of interleukin (IL) -4 confers susceptibility. Since inappropriate Th responses often lead to death in immunosuppressed human patients, especially HIV-1 infected patients, this work aimed to elucidate mechanisms of Th2 induction and regulation by assessing the Th2 hallmark cytokine IL-4 in an experimental model of cryptococcosis. Therefore, a kinetic study of IL-4 expression during 70 days after intranasal infection was performed in susceptible mice. The analyses included characterization of pulmonary leukocytes and Th cell cytokine profiling. IL-4 profiling revealed Cryptococcus-specific IL-4 production not before six weeks after infection. This unexpected finding was further validated by equal results observed in a kinetic study done in IL-4 reporter mice. These mice express a green fluorescent protein simultaneously to IL-4 expression in the same cell and this protein can be detected by flow cytometry. Two cellular sources of IL-4 were identified: Th2 cells were found as expected, but also, as shown for the first time, eosinophilic granulocytes could be demonstrated to secrete IL-4. Next, the influence of eosinophils on pulmonary inflammation and disease development was investigated using ΔdblGATA-1 mice constitutively devoid of eosinophilic granulocytes. Experiments with infected ΔdblGATA-1 mice revealed novel regulatory functions of eosinophils in cryptococcosis. In the absence of eosinophils pulmonary Th cell recruitment was significantly diminished. In addition, Th2 polarization was reduced in ΔdblGATA-1 mice as shown by reduced numbers of Th2 cells expressing the Th2-related surface marker T1/ST2 and reduced albeit not absent IL-4 production by Th cells. In addition to reduced IL-4 production, in the absence of eosinophils Th cells with enhanced interferon-γ and IL-17 production were observed. However, control of pulmonary fungal growth was only slightly enhanced in the absence of eosinophils and dissemination of cryptococci to the brain was unaltered. This may be related to the shared IL-4 production by not only eosinophils but also Th2 cells. Blocking more than one cellular source of IL-4 could be required to prevent immunopathology. To test the hypothesis of gradual IL-4-dependent immunopathology, experiments were conducted using mice expressing only one allele of the IL-4receptor (R) alpha (α) chain (+/-) instead of two (+/+). Indeed, mono-allelic expression of the IL-4Rα resulted in an intermediate expression of the IL-4R on the surface of myeloid and lymphoid cells indicating a gene-dosage effect for IL-4R expression. Infected IL-4Rα+/- mice displayed reduced susceptibility as compared with IL-4Rα+/+ mice, and IL-4Rα-/- mice completely lacking IL-4R expression were found to be protected with survival for the complete time period of the experiment (i.e. up to 275 days). Reduced susceptibility found in infected IL-4Rα+/- mice was associated with decreased serum levels of immunoglobulin E, reduced mucus production by airway epithelia, attenuation of airway hyper-reactivity, and reduced formation of alternatively activated macrophages in lung parenchyma – pathophysiological features, which are typically found in experimental models of asthma but also in asthma of humans and animals. Since no up-regulation of IL-4R by the infection with Cryptococcus neoformans was found, the experimental pulmonary infection model used appears to be a very sensitive low-level IL-4 system. This work highlights the outstanding role of IL-4 and its different cellular sources as well as its receptor in cryptococcosis and provides novel insights into pathogenesis. Moreover, a cellular (i.e. eosinophils) and a molecular (i.e. IL-4R) target for treatment of this mycosis and possibly of asthma is provided.

Das Cytokin Interleukin-4 (IL-4) ist ein essentieller Faktor bei der Entstehung von Sofort-Typ Allergien. Die Bindung von IL-4 an seinen Rezeptor und die anschließende Phosphorylierung des IL-4 aktivierten Transkriptionsfaktors Stat6 ist ein Schlüsselereignis bei der allergischen Immunantwort. In der vorliegenden Arbeit werden Ergebnisse zur Hemmung der Stat6 vermittelten Signaltransduktion des IL-4 Rezeptors vorgestellt. Dazu wurde ein Vektorsystem etabliert, bei dem ein von dem Drosophila-Transkriptionsfaktor Antennapedia abgeleitetes 16 AS langes Peptid benutzt wird. Dieses Antennapediapeptid kann Plasmamembranen lebender Zellen energie- und rezeptorunabhängig durchqueren und dabei andere hydrophile Moleküle mittransportieren. Stat6 bindet über eine SH2 Domäne an phosphorylierte Reste von IL4Ra und bildet, nachdem es selbst phosphoryliert ist, mit anderen Stat6-Molekülen aktive Dimere. Ein aus der Stat6-Bindestelle des IL-4Ra abgeleitetes phosphoryliertes Peptid (Stat6BP) wurde mit Hilfe des Antennapediapeptids in verschiedene humane und murine Zellinien transportiert. Für Stat6BP konnte mit Hilfe von spezifischer Immunpräzipitation und Western-Blot gezeigt werden, dass es IL-4 induzierte Phosphorylierung und Aktivierung von Stat6 transient hemmen kann. Durch zusätzliche Applikation des Tyrosinphosphataseinhibitors Natriumpervanadat gelang es, die hemmende Wirkung von Stat6BP zu verlängern. Unter gleichen Bedingungen konnte auch gezeigt werden, dass Stat6BP spezifisch die Aktivierung von Stat6 hemmt, da die durch IL-4 oder IL-3 induzierte Phosphorylierung des eng verwandten Stat5 völlig unbeeinträchtigt bleibt. Ferner wurde durch das Peptid die Expression eines Stat6 kontrollierten Reportergens gehemmt. Im Rahmen dieser Arbeit wurde außerdem die Rolle der Src-Typ Kinasen p56lck und p59fyn in der IL-4 Signaltransduktion in unterschiedlichen T-Zellinien untersucht. Es zeigte sich, dass die Aktivierung der beide Kinasen stark von der getesteten Zellinie abhängt. In einigen T-Zellinien aktiviert IL-4 eher p56lck, in anderen eher p59fyn. / Interleukin-4 (IL-4) is the major factor in the development of allergic diseases like hay fever or asthma. The most important cytoplasmic event following stimulation with IL-4 is the activation of the transcription factor Stat6 (signal transducer and activator of transcription 6). Stat6 binds via a single SH2 domain first to tyrosine-phosphorylated motifs in the IL-4Ra-chain, and then to another Stat6 molecule, which results in the formation of active dimers. Since Stat6 is exclusively used by the IL-4 receptor, it is a promising approach to specifically disrupt IL-4 signal- transduction by inhibiting Stat6 activation. A vector system was established for the delivery of hydrophilic agents into living cells. To this purpose, a 16 amino acid membrane-permeable peptide derived from the Drosophila transcription factor Antennapedia was used. The Antennapedia peptide has been shown to internalize into living cell in a receptor- and energy-independent manner. In this thesis it is shown that a peptide derived from the Stat6-binding region of IL-4Ra (Stat6BP) is an effective inhibitor when it is delivered into cells by coupling with the Antennapedia peptide. Stat6BP completely inhibited IL-4 dependent phosphorylation of Stat6 in different human and murine cell lines, while IL-3 and IL-4 dependent phosphorylation of Stat5 was not affected. The inhibitory effect of Stat6BP was transient, but could be prolonged by treating the cells with the phospatase inhibitor sodium pervanadate. Transcription from a reporter gene construct with a Stat6-dependent promoter was inhibited by Stat6BP as well, indicating that the peptide is a suitable inhibitor for cellular responses downstream from Stat6 phosphorylation. Another aim of this study was to investigate the role of the src-kinases p56lck and p59fyn in IL-4 signaltransduction. The results indicate, that the activation of both kinases is celline dependent. In some T-cellines p56lck was activated dominantly, in others p59fyn.

No abstract available

Graduation date: 2002

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

Zugl.: Berlin, Freie Univ., Diss., 2008

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 177-212).

Cytokines play an important regulatory role during immune responses against pathogens. The outcome of an induced cytokine pattern is determined by many factors. It strongly depends on the nature of the pathogen and the host’s ability to control the quality and strength of cytokine signals. In pulmonary infection with Cryptococcus neoformans T helper (Th) 1 and Th17 cell subsets and their associated cytokines confer protection, whereas a Th2-biased response with production of interleukin (IL) -4 confers susceptibility. Since inappropriate Th responses often lead to death in immunosuppressed human patients, especially HIV-1 infected patients, this work aimed to elucidate mechanisms of Th2 induction and regulation by assessing the Th2 hallmark cytokine IL-4 in an experimental model of cryptococcosis. Therefore, a kinetic study of IL-4 expression during 70 days after intranasal infection was performed in susceptible mice. The analyses included characterization of pulmonary leukocytes and Th cell cytokine profiling. IL-4 profiling revealed Cryptococcus-specific IL-4 production not before six weeks after infection. This unexpected finding was further validated by equal results observed in a kinetic study done in IL-4 reporter mice. These mice express a green fluorescent protein simultaneously to IL-4 expression in the same cell and this protein can be detected by flow cytometry. Two cellular sources of IL-4 were identified: Th2 cells were found as expected, but also, as shown for the first time, eosinophilic granulocytes could be demonstrated to secrete IL-4. Next, the influence of eosinophils on pulmonary inflammation and disease development was investigated using ΔdblGATA-1 mice constitutively devoid of eosinophilic granulocytes. Experiments with infected ΔdblGATA-1 mice revealed novel regulatory functions of eosinophils in cryptococcosis. In the absence of eosinophils pulmonary Th cell recruitment was significantly diminished. In addition, Th2 polarization was reduced in ΔdblGATA-1 mice as shown by reduced numbers of Th2 cells expressing the Th2-related surface marker T1/ST2 and reduced albeit not absent IL-4 production by Th cells. In addition to reduced IL-4 production, in the absence of eosinophils Th cells with enhanced interferon-γ and IL-17 production were observed. However, control of pulmonary fungal growth was only slightly enhanced in the absence of eosinophils and dissemination of cryptococci to the brain was unaltered. This may be related to the shared IL-4 production by not only eosinophils but also Th2 cells. Blocking more than one cellular source of IL-4 could be required to prevent immunopathology. To test the hypothesis of gradual IL-4-dependent immunopathology, experiments were conducted using mice expressing only one allele of the IL-4receptor (R) alpha (α) chain (+/-) instead of two (+/+). Indeed, mono-allelic expression of the IL-4Rα resulted in an intermediate expression of the IL-4R on the surface of myeloid and lymphoid cells indicating a gene-dosage effect for IL-4R expression. Infected IL-4Rα+/- mice displayed reduced susceptibility as compared with IL-4Rα+/+ mice, and IL-4Rα-/- mice completely lacking IL-4R expression were found to be protected with survival for the complete time period of the experiment (i.e. up to 275 days). Reduced susceptibility found in infected IL-4Rα+/- mice was associated with decreased serum levels of immunoglobulin E, reduced mucus production by airway epithelia, attenuation of airway hyper-reactivity, and reduced formation of alternatively activated macrophages in lung parenchyma – pathophysiological features, which are typically found in experimental models of asthma but also in asthma of humans and animals. Since no up-regulation of IL-4R by the infection with Cryptococcus neoformans was found, the experimental pulmonary infection model used appears to be a very sensitive low-level IL-4 system. This work highlights the outstanding role of IL-4 and its different cellular sources as well as its receptor in cryptococcosis and provides novel insights into pathogenesis. Moreover, a cellular (i.e. eosinophils) and a molecular (i.e. IL-4R) target for treatment of this mycosis and possibly of asthma is provided.