DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Annette Hering :
Analyses of IFN-gamma kinetics of induction of human naive Th-cells
Analysen zur IFN-gamma Induktionskinetik von humanen naiven Th-Zellen

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Abstract

Rheumatoid arthritis (RA) is an autoimmune diseas characterized by chronic inflammation of synovial tissue of multiple joints. CD4+T-lymphocytes are described as the dominant cell population infiltrating the synovial membrane in RA patients. It has been discussed wether the predominance of inflammatory cytokines (Th1-cytokines) produced by CD4+Memory-cells triggers and maintains the disease process. This work demonstrates for the first time that naive CD4+cells isolated from peripheral blood of RA patients produce elevatet amounts of the Th1 cytokin IFN-gamma after stimulation. An isolation protocoll was established to purify naive CD31+ and CD31- CD45RA+CD45RO-CD4+T-cells from peripheral blood mononuclear cells (PBMC) by using magnetic cell sorting systems. PBMC were obtained from RA patients and healthy donors. Both cell populations were cultured and stimulated for 5 days with anti CD28 and anti CD3 in the presence of increasing concentrations of IL-12. We show that IL-12 and anti IL-4 induce Th1 differentation in CD31+ and CD31- CD45RA+CD45RO-CD4+T-cells. We saw an increase in the number of IFN-gamma producing cells due to the rising concentration of IL-12. The separate analysis of CD31+ and CD31- T-cells was necessary. CD31+T-cells of different healthy donors responded homogeneously to IL-12. In contrast the response of CD31-T-cells to IL-12 varied significantly between different donors. The naive T-cells of RA patients are characterized by an enhanced IL-12 sensibility and even unpolarised cells produce larger amounts of IFN-gamma than cells of healthy donors. These data support the hypothesis of a genetic background of RA, because we could showed a detectable level disturbance in the cytokine balance in naive T-cells of RA patients.

Table of Contents

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0. Titelblatt und Inhaltsverzeichnis
1. Einleitung 1
2. Immunologische Grundlagen 3
2.1 Rheumatoide Arthritis 3
2.2 Funktion des Immunsystems 4
2.3 T-Helferzellen 5
2.4 Naive und antigenerfahrene Zellen 7
2.5 Die zentrale Rolle der CD4+Th-Zellen in der Pathogenese der RA 9
3. Material und Methoden 13
3.1 Zellkulturmedien und -bedingungen 13
3.2 Methoden der Zelltrennung 13
3.3 Antikörper 19
3.4 Kopplung von Haptenen und Flurochromen an Antikörper 21
3.5 Polyklonale in vitro Th-Zell-Stimulation 23
3.6 Coaten von Zellkulturplatten mit immobilisierten Antikörpern 24
3.7 Immunfluoreszenzfärbungen 24
3.8 Durchflußzytometrie 25
3.9 Quantifizierung der IFN-gamma Konzentration in Zellkulturüberständen durch fluoreszente Beads 26
4. Ergebnisse 27
4.1 Isolierung humaner naiver Th-Zellen 27
4.2 Analyse der Effektor-Zytokinproduktion der CD45RA+CD4+Th-Zellen 33
4.3 CD31+ und CD31- CD45RA+CD45RO-CD4+Th-Zellsubpopulationen 42
4.4 CD31+ und CD31-Th-Zellisolierung mittels FACS-Sorter 51
4.5 IFN-gamma Induktionskinetik von gesunden Spendern und Patienten 60
5. Diskussion 75
5.1 Die magnetische Mehrparameter Zellsortierung 75
5.2 CD45RA+CD45RO-CD4+Th-Zellkulturen 76
5.3 CD45RA+CD45RO-CD4+Th-Zellen: eine homogene Population? 78
5.4 CD31+und CD31-Th-Zellen 80
5.5 Analyse des Zytokinmusters von Th1 polarisierten CD31+ und CD31- Th-Zellen gesunder Spender 82
5.6 Analyse der Patienten 84
5.7 MACS und FACS-Sortierung im Vergleich 85
5.8 Weiterführende Experimente 87
6. Zusammenfassung 89
Anhang 91
Literaturverzeichnis 91
Abkürzungsverzeichnis 101
Danksagung 103

More Information:

Online available: http://www.diss.fu-berlin.de/2002/170/indexe.html
Language of PhDThesis: german
Keywords: Rheumatoid artrithis, naive T-cells, CD31-cells
DNB-Sachgruppe: 33 Medizin
Date of disputation: 23-Jul-2002
PhDThesis from: Fachbereich Humanmedizin, Freie Universität Berlin
First Referee: Prof. Dr. med. Jürgen Braun
Second Referee: Prof. Dr. med. Jochen Sieper
Contact (Author): kollonitsch@hmi.de
Contact (Advisor): thiel@drfz.de
Date created:29-Aug-2002
Date available:30-Aug-2002

 

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