Functional Impact of Phosphodiesterase 4 (PDE4) Subtypes in Human Primary Immune Cells

Lade...
Vorschaubild
Dateien
Diss_Peter.pdf
Diss_Peter.pdfGröße: 6.41 MBDownloads: 1797
Datum
2006
Autor:innen
Peter, Daniel
Herausgeber:innen
Kontakt
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
DOI (zitierfähiger Link)
ArXiv-ID
Internationale Patentnummer
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Open Access Green
Sammlungen
Core Facility der Universität Konstanz
Gesperrt bis
Titel in einer weiteren Sprache
Funktionelle Bedeutung von Phosphodiesterase 4 (PDE4) Subtypen in Humanen Primären Immunzellen
Forschungsvorhaben
Organisationseinheiten
Zeitschriftenheft
Publikationstyp
Dissertation
Publikationsstatus
Published
Erschienen in
Zusammenfassung

As a second messenger, adenosine 3 -5 cyclic monophosphate (cAMP) plays a crucial role for intracellular signaling. In immune cells, cAMP has mainly inhibitory properties on cellular responses. Thus, type 4 phosphodiesterases (PDE4), which are capable to hydrolyze cAMP, are critical regulators of inflammatory cell signals by attenuating the negative constraint of cAMP. The PDE4 isoenzyme family consists of four distinct subtypes, each encoded by one gene. Because PDE4 subtypes are widely expressed in inflammatory cells, the PDE4 family became an attractive drug target for the treatment of chronic inflammatory diseases. In fact, PDE4 inhibitors have been shown to have anti-inflammatory and immunomodulatory effects on a broad range of inflammatory and immunocompetent cells and have proved efficacy in clinical trials. However, it is largely unknown which PDE4 subtype(s) mediate(s) the inhibitory effects in a defined immune cell.
The objectives of the present study were i) to ascertain the expression patterns of PDE4 subtypes in different human primary immune cells via quantitative PCR, PDE activity assays, and immunodetection experiments; ii) to validate an antisense- and a siRNA-mediated, PDE4 subtype-specific knockdown technique in the cell line A549 and to transfer the superior knockdown strategy to human primary CD4+ T cells; and iii) to elucidate the functional impact of PDE4 subtypes in human primary CD4+ T cells.

In resting human primary CD4+ T cells, low levels of PDE4A were detected, whereas PDE4B and PDE4D mRNA expression and protein activity were substantially higher, with PDE4D being the predominant subtype. After anti-CD3/CD28 stimulation, PDE4A and PDE4D mRNA as well as protein activity were upregulated within five days, whereas PDE4B mRNA and activity showed a transient upregulation. Remarkably, the PDE4B and PDE4D short forms were found to contribute largely to the time-dependent upregulation of PDE4 subtypes in human primary CD4+ T cells. In resting human primary CD8+ T cells, PDE4B and PDE4D mRNA were predominantly expressed, whereas PDE4A mRNA had low expression levels. After anti-CD3/CD28 stimulation, the induction of PDE4 subtypes in CD8+ T cells resembled, also not equal in a quantitative manner, the induction of PDE4 subtypes in CD4+ T cells. In contrast to T cells, PDE4B mRNA was the predominantly expressed PDE4 subtype in freshly isolated human primary monocytes, followed by PDE4A and very low levels of PDE4D. During differentiation of monocytes to macrophages or to dendritic cells, PDE4B mRNA expression dramatically decreased, whereas PDE4A and PDE4D mRNA levels were less regulated. LPS stimulation of macrophages or dendritic cells strongly induced PDE4B, but not PDE4A and PDE4D mRNA.

With the intention to elucidate the impact of individual PDE4 subtypes on cell function and because of the lack of PDE4 subtype-specific inhibitors, two strategies were evaluated to specifically knock down PDE4 subtypes. Initially, several second generation antisense (AS2nd) constructs or pools of short interfering RNAs (siRNAs) targeting PDE4A, PDE4B, or PDE4D mRNA were transfected into the human lung adenocarcinoma epithelial cell line A549. AS2nd constructs as well as siRNAs were shown to induce substantial, PDE4 subtype-specific knockdown of mRNA and protein 24 h and 72 h after transfection. Because siRNAs were effective in lower concentrations, were well tolerated, and showed less variability in between experiments, the siRNA-mediated knockdown strategy was transferred to human primary CD4+ T cells. The transfection of individual siRNAs into CD4+ T cells with the nucleofector electroporation technique resulted in significant, specific, and well-tolerated knockdown of PDE4 subtypes on mRNA and protein level.

Finally, the validated siRNA-mediated knockdown technique was used to ascertain the functional relevance of PDE4 subtypes in human primary CD4+ T cells. SiRNA-mediated knockdown of either PDE4B or PDE4D inhibited anti-CD3/CD28 induced IL-2 release 24 h after stimulation to an extent overall similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast), pointing to an overlapping function of PDE4B and PDE4D for IL-2 synthesis. At 48 h after stimulation, efficacies of PDE4B- and PDE4D-siRNAs to suppress IL-2 release were slightly diminished. Considering the anti-CD3/CD28 induced generation of IFN-g and IL-5, PDE4D-siRNA showed a predominant inhibitory effect, suggesting nonredundant roles of PDE4 subtypes for T cell cytokine production. However, the inhibition of cytokines was most effective when PDE4 subtype-specific siRNAs were applied in combination, indicating complementary functions of PDE4 subtypes. Although the effect of PDE4 inhibition on T cell proliferation was small, the PDE4D-targeting siRNA alone had similar anti-proliferative effects than RP73401, whereas PDE4A- or PDE4B-siRNAs were hardly effective.

Zusammenfassung in einer weiteren Sprache

Als sekundärer Botenstoff spielt zyklisches Adenosinmonophosphat (cAMP) eine entscheidende Rolle für die intrazelluläre Signalweiterleitung. In Immunzellen hat cAMP hauptsächlich hemmende Eigenschaften. Da Typ 4 Phosphodiesterasen (PDE4) cAMP hydrolisieren und dadurch den hemmenden Einfluss von cAMP abschwächen können, sind sie kritische Regler von Entzündungssignalen. Die PDE4-Familie besteht aus vier Subtypen, die von jeweils einem Gen kodiert werden, und wurde ein attraktives Ziel für die Arzneimittelforschung für die Behandlung von chronisch entzündlichen Krankheiten. In der Tat haben PDE4-Inhibitoren entzündungshemmende und immunmodulatorische Effekte in einer Reihe von entzündlichen und immunkompetenten Zellen und bewiesen Wirksamkeit in klinischen Studien. Allerdings ist größtenteils unbekannt, welche PDE4-Subtypen in einer bestimmten Immunzelle die inhibitorischen Effekte vermitteln.
Die Ziele der vorliegenden Arbeit waren a) durch die Anwendung von quantitativen PCR-Experimenten, von PDE Aktivitätstests und von Immundetektionsanalysen das Expressionsmuster von PDE4-Subtypen in verschiedenen humanen primären Immunzellen herauszufinden; b) eine Antisense- und siRNA-vermittelte Technik zur spezifischen Herunterregulation ( Knockdown ) von PDE4-Subtypen in der Zelllinie A549 zu validieren und die besser geeignete Knockdownstrategie auf humane primäre CD4+ T-Zellen zu überführen; und c) die funktionelle Bedeutung von PDE4-Subtypen in humanen primären CD4+ T-Zellen herauszufinden.

In ruhenden humanen primären CD4+ T-Zellen wurde die geringste Expression und Enzymaktivität für PDE4A gemessen, während die PDE4B- und PDE4D-Expression und Proteinaktivität größer waren und PDE4D als der vorherrschende Subtyp vorlag. Nach Stimulation der Zellen mit anti-CD3/CD28 wurden sowohl die Expression als auch die Enzymaktivität von PDE4A und PDE4D innerhalb von fünf Tagen hochreguliert, während PDE4B nur vorübergehend hochreguliert war. In ruhenden humanen primären CD8+ T-Zellen wurde die geringste mRNA-Expression für PDE4A gemessen, während sie für PDE4B und PDE4D etwa gleich groß war. Nach Stimulation der Zellen wurde die Expression der PDE4-Subtypen nach ähnlichem Muster wie in den CD4+ T-Zellen zeitabhängig induziert, wenn auch nicht qualitativ im gleichen Ausmaß. Im Gegensatz zu T-Zellen war PDE4B der vorherrschend exprimierte Subtyp in frisch isolierten humanen primären Monozyten, gefolgt von PDE4A und einem sehr geringen Expressionsniveau von PDE4D. Während der Differenzierung von Monozyten zu Makrophagen oder Dendritischen Zellen nahm die PDE4B-Expression drastisch ab, während die PDE4A- und PDE4B-Expression weniger reguliert wurde. Stimulation mit LPS induzierte in diesen Makrophagen oder Dendritischen Zellen stark PDE4B mRNA, aber nicht PDE4A und PDE4D mRNA.

Mit dem Ziel, die Bedeutung von individuellen PDE4-Subtypen auf die Zellfunktion aufzuklären, wurden zwei Strategien evauliert, um spezifisch PDE4-Subtypen herunterzuregulieren. Zunächst wurden mehrere Antisense-Konstrukte oder Kombinationen von kurzen doppelsträngigen RNAs (siRNAs), die gegen PDE4-Subtypen gerichtet waren, in die humane Zelllinie A549 transfiziert. Es wurde gezeigt, dass sowohl Antisense-Konstrukte als auch siRNAs beträchtliche, spezifische Herunterregulationen von PDE4-Subtypen auf mRNA- und Proteinebene bewirken konnten. Da siRNAs in geringeren Konzentrationen wirksam und gut verträglich waren und geringere Variabilitäten in den Experimenten zeigten, wurde die siRNA-vermittelte Knockdownstrategie auf humane primäre CD4+ T-Zellen übertragen. Die Transfektion von einzelnen siRNAs in CD4+ T-Zellen resultierte in signifikanten, spezifischen, und gut verträglichen Herunterregulationen von mRNA und Protein der PDE4-Subtypen.

Letztendlich wurde die validierte siRNA-vermittelte Knockdowntechnik benutzt, um die funktionelle Relevanz von PDE4-Subtypen in humanen primären CD4+ T-Zellen zu ermitteln. Sowohl die siRNA-vermittelte Herunterregulation von PDE4B als auch von PDE4D hemmten die anti-CD3/CD28 induzierte IL-2 Freisetzung 24 Stunden nach Stimulation in ähnlichem Ausmaß wie der panPDE4-Inhibitor RP73401 (Piclamilast). Dieser Befund deutet auf eine überlappende Rolle von PDE4B und PDE4D für die IL-2 Synthese hin. Betrachtet man die anti-CD3/CD28 induzierte Bildung von IFN-g und IL-5, so zeigte die gegen PDE4D gerichtete siRNA einen vorwiegenden inhibitorischen Effekt auf diese Zytokine. Dies lässt vermuten, dass die PDE4-Subtypen keine redundanten Rollen für die Zytokinproduktion innehaben. Allerdings war die Hemmung der Zytokine dann am größten, wenn alle PDE4-Subtyp-spezifischen siRNAs zusammen angewendet wurden, was auf komplementäre Funktionen der PDE4-Subtypen hindeutet. Obwohl die Effekte der PDE4-Hemmung auf die T-Zellproliferation gering waren, hatte nur die gegen PDE4D gerichtete siRNA eine ähnliche anti-proliferative Wirkung wie RP73401, während PDE4A- oder PDE4B-siRNAs kaum effektiv waren.

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Immunzellen, 3-5 cAMP Phosphodiesterase, PDE4 inhibition, Immune cells, 3-5-cAMP-phosphodiesterase, inflammation, PDE4 inhibition
Konferenz
Rezension
undefined / . - undefined, undefined
Zitieren
ISO 690PETER, Daniel, 2006. Functional Impact of Phosphodiesterase 4 (PDE4) Subtypes in Human Primary Immune Cells [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Peter2006Funct-7018,
  year={2006},
  title={Functional Impact of Phosphodiesterase 4 (PDE4) Subtypes in Human Primary Immune Cells},
  author={Peter, Daniel},
  address={Konstanz},
  school={Universität Konstanz}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/7018">
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:30:53Z</dcterms:available>
    <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/>
    <dcterms:title>Functional Impact of Phosphodiesterase 4 (PDE4) Subtypes in Human Primary Immune Cells</dcterms:title>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7018/1/Diss_Peter.pdf"/>
    <dc:format>application/pdf</dc:format>
    <dcterms:issued>2006</dcterms:issued>
    <dcterms:abstract xml:lang="eng">As a second messenger, adenosine 3 -5  cyclic monophosphate (cAMP) plays a crucial role for intracellular signaling. In immune cells, cAMP has mainly inhibitory properties on cellular responses. Thus, type 4 phosphodiesterases (PDE4), which are capable to hydrolyze cAMP, are critical regulators of inflammatory cell signals by attenuating the negative constraint of cAMP. The PDE4 isoenzyme family consists of four distinct subtypes, each encoded by one gene. Because PDE4 subtypes are widely expressed in inflammatory cells, the PDE4 family became an attractive drug target for the treatment of chronic inflammatory diseases. In fact, PDE4 inhibitors have been shown to have anti-inflammatory and immunomodulatory effects on a broad range of inflammatory and immunocompetent cells and have proved efficacy in clinical trials. However, it is largely unknown which PDE4 subtype(s) mediate(s) the inhibitory effects in a defined immune cell.&lt;br /&gt;The objectives of the present study were i) to ascertain the expression patterns of PDE4 subtypes in different human primary immune cells via quantitative PCR, PDE activity assays, and immunodetection experiments; ii) to validate an antisense- and a siRNA-mediated, PDE4 subtype-specific knockdown technique in the cell line A549 and to transfer the superior knockdown strategy to human primary CD4+ T cells; and iii) to elucidate the functional impact of PDE4 subtypes in human primary CD4+ T cells.&lt;br /&gt;&lt;br /&gt;In resting human primary CD4+ T cells, low levels of PDE4A were detected, whereas PDE4B and PDE4D mRNA expression and protein activity were substantially higher, with PDE4D being the predominant subtype. After anti-CD3/CD28 stimulation, PDE4A and PDE4D mRNA as well as protein activity were upregulated within five days, whereas PDE4B mRNA and activity showed a transient upregulation. Remarkably, the PDE4B and PDE4D short forms were found to contribute largely to the time-dependent upregulation of PDE4 subtypes in human primary CD4+ T cells. In resting human primary CD8+ T cells, PDE4B and PDE4D mRNA were predominantly expressed, whereas PDE4A mRNA had low expression levels. After anti-CD3/CD28 stimulation, the induction of PDE4 subtypes in CD8+ T cells resembled, also not equal in a quantitative manner, the induction of PDE4 subtypes in CD4+ T cells. In contrast to T cells, PDE4B mRNA was the predominantly expressed PDE4 subtype in freshly isolated human primary monocytes, followed by PDE4A and very low levels of PDE4D. During differentiation of monocytes to macrophages or to dendritic cells, PDE4B mRNA expression dramatically decreased, whereas PDE4A and PDE4D mRNA levels were less regulated. LPS stimulation of macrophages or dendritic cells strongly induced PDE4B, but not PDE4A and PDE4D mRNA.&lt;br /&gt;&lt;br /&gt;With the intention to elucidate the impact of individual PDE4 subtypes on cell function and because of the lack of PDE4 subtype-specific inhibitors, two strategies were evaluated to specifically knock down PDE4 subtypes. Initially, several second generation antisense (AS2nd) constructs or pools of short interfering RNAs (siRNAs) targeting PDE4A, PDE4B, or PDE4D mRNA were transfected into the human lung adenocarcinoma epithelial cell line A549. AS2nd constructs as well as siRNAs were shown to induce substantial, PDE4 subtype-specific knockdown of mRNA and protein 24 h and 72 h after transfection. Because siRNAs were effective in lower concentrations, were well tolerated, and showed less variability in between experiments, the siRNA-mediated knockdown strategy was transferred to human primary CD4+ T cells. The transfection of individual siRNAs into CD4+ T cells with the nucleofector electroporation technique resulted in significant, specific, and well-tolerated knockdown of PDE4 subtypes on mRNA and protein level.&lt;br /&gt;&lt;br /&gt;Finally, the validated siRNA-mediated knockdown technique was used to ascertain the functional relevance of PDE4 subtypes in human primary CD4+ T cells. SiRNA-mediated knockdown of either PDE4B or PDE4D inhibited anti-CD3/CD28 induced IL-2 release 24 h after stimulation to an extent overall similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast), pointing to an overlapping function of PDE4B and PDE4D for IL-2 synthesis. At 48 h after stimulation, efficacies of PDE4B- and PDE4D-siRNAs to suppress IL-2 release were slightly diminished. Considering the anti-CD3/CD28 induced generation of IFN-g and IL-5, PDE4D-siRNA showed a predominant inhibitory effect, suggesting nonredundant roles of PDE4 subtypes for T cell cytokine production. However, the inhibition of cytokines was most effective when PDE4 subtype-specific siRNAs were applied in combination, indicating complementary functions of PDE4 subtypes. Although the effect of PDE4 inhibition on T cell proliferation was small, the PDE4D-targeting siRNA alone had similar anti-proliferative effects than RP73401, whereas PDE4A- or PDE4B-siRNAs were hardly effective.</dcterms:abstract>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dc:rights>terms-of-use</dc:rights>
    <dc:contributor>Peter, Daniel</dc:contributor>
    <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/7018"/>
    <dc:language>eng</dc:language>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:30:53Z</dc:date>
    <dc:creator>Peter, Daniel</dc:creator>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7018/1/Diss_Peter.pdf"/>
    <dcterms:alternative>Funktionelle Bedeutung von Phosphodiesterase 4 (PDE4) Subtypen in Humanen Primären Immunzellen</dcterms:alternative>
  </rdf:Description>
</rdf:RDF>
Interner Vermerk
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Kontakt
URL der Originalveröffentl.
Prüfdatum der URL
Prüfungsdatum der Dissertation
December 12, 2006
Finanzierungsart
Kommentar zur Publikation
Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Begutachtet
Diese Publikation teilen