Vitamin D receptor activation modulates the allergic immune response [Elektronische Ressource] / Björn Hartmann. Betreuer: Roland Lauster

Björn Hartmann

Sujets

• Biologie

Informations

• Publié par: technische_universitat_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 58
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Vitamin D receptor activation modulates
the allergic immune response

vorgelegt von
Diplom-Biochemiker
Björn Hartmann

Von der Fakultät III – Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation

Promotionsausschuss:
Vorsitzender: Prof. Dr. rer. nat. Peter Neubauer
Gutachter: Prof. Dr. rer. nat. Roland Lauster
Gutachter: Prof. Dr. med. Margitta Worm
Gutachter: Prof. Dr. rer. nat. Leif-Alexander Garbe
Tag der wissenschaftlichen Aussprache: 22.02.2011

durchgeführt an der
Charité, Klinik für Dermatologie, Venerologie und Allergologie, Berlin



Berlin 2011
D 83


“A vitamin is a substance that makes you ill if you don’t eat it.”
(Albert Szent-Gyorgyi, Nobel Prize in Physiology or Medicine, 1937)



















Meinen Eltern
Table of Contents I
1. LIST OF ABBREVIATIONS................................................................................................................. 1
1. ABSTRACT............................................................................................................................................ 8
2. ZUSAMMENFASSUNG ........................................................................................................................ 9
3. INTRODUCTION................................................................................................................................ 11
3.1. VITAMIN D AND PHYSIOLOGY ......................................................................................................... 11
3.2. VITAMIN D RECEPTOR AND SIGNALING ............................................................................................ 14
3.3. VITAMIN D AND THE IMMUNE SYSTEM............................................................................................. 19
3.4. VITAMIN D IN ALLERGIC DISEASES AND ATOPIC DERMATITIS ............................................................ 21
3.4.1. Type I allergic immune response ............................................................................................ 21
3.4.2. Role of T 2 cells in the allergic immune response................................................................... 22 H
3.4.3. Mechanism of IgE class switch recombination........................................................................ 23
3.4.4. Vitamin D in the context of IgE and atopic dermatitis (AD)..................................................... 25
3.4.5. Atopic dermatitis (AD)........................................................................................................... 25
4. OBJECTIVES ...................................................................................................................................... 30
5. MATERIALS AND METHODS.......................................................................................................... 31
5.1. MATERIALS .................................................................................................................................... 31
5.1.1. Antibodies.............................................................................................................................. 31
5.1.2. Buffers and solutions.............................................................................................................. 32
5.1.3. Chemical and biological reagents .......................................................................................... 33
5.1.4. Labware ................................................................................................................................ 35
5.1.5. Technical equipment .............................................................................................................. 36
5.1.6. Software ................................................................................................................................ 37
5.2. METHODS....................................................................................................................................... 38
5.2.1. Cellular methods.................................................................................................................... 38
5.2.1.1. Human cell isolation......................................................................................................................... 38
5.2.1.2. Murine splenocyte isolation .............................................................................................................. 38
5.2.1.3. Cell culture conditions...................................................................................................................... 39
5.2.2. Animal work .......................................................................................................................... 39
5.2.2.1. Mouse model of type I allergy .......................................................................................................... 39
5.2.2.2. Mouse model of allergen-induced eczema ......................................................................................... 40
5.2.2.3. Assessment of AD symptoms ........................................................................................................... 41
5.2.3. Immunological methods ......................................................................................................... 42
5.2.3.1. Enzyme-linked immunosorbent assay (ELISA) ................................................................................. 42
5.2.3.2. Human immunoglobulin ELISA ....................................................................................................... 42
5.2.3.3. Murine immunoglobulin ELISA ....................................................................................................... 43
5.2.3.4. Enzyme-linked immunospot (ELISPOT)........................................................................................... 43
5.2.3.5. Principles of flow cytometry............................................................................................................. 44
5.2.3.6. Flow cytometric analysis .................................................................................................................. 45
5.2.3.7. 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) dilution analysis ............................... 45
5.2.3.8. Flow cytometric analysis of STAT6 phosphorylation......................................................................... 45
Table of Contents II
5.2.3.9. Flow cytometric analysis of IκBα degradation................................................................................... 46
5.2.4. Immunohistochemistry ........................................................................................................... 46
+ +5.2.4.1. Staining of CD4 and CD8 T cell infiltrates ..................................................................................... 46
+5.2.4.2. Staining of Foxp3 cells.................................................................................................................... 47
5.2.4.3. Histological Analyses....................................................................................................................... 47
5.2.5. Molecular biology methods.................................................................................................... 47
5.2.5.1. RNA isolation from murine skin ....................................................................................................... 47
5.2.5.2. RNA isolation from cultured cells..................................................................................................... 48
5.2.5.3. cDNA synthesis ............................................................................................................................... 48
5.2.5.4. Real-time PCR/quantitative PCR (qPCR).......................................................................................... 48
5.2.6. Statistical analyses................................................................................................................. 50
6. RESULTS ............................................................................................................................................. 51
6.1. THE VDR AGONIST MEDIATES VDR ACTIVATION IN B CELLS ........................................................... 51
6.2. VDR ACTIVATION BY THE VDR AGONIST INHIBITS IGE PRODUCTION IN B CELLS .............................. 52
6.3. STAT6 PHOSPHORYLATION AND IκBα DEGRADATION IS NOT MODULATED ....................................... 54
6.4. REDUCTION OF AICDA EXPRESSION BY CALCITRIOL AND THE VDR AGONIST ...................................... 56
+ HIGH +6.5. REDUCTION OF THE CD19 CD27 CD38 B CELL POPULATION BY ACTIVATED VDRS ................... 57
6.6. VDR ACTIVATION REDUCES THE IGE RESPONSE IN A TYPE I ALLERGY MOUSE MODEL WITHOUT
CALCEMIC SIDE EFFECTS.................................................................................................................. 59
6.7. VDR AGONIST TREATMENT AMELIORATES ALLERGEN-TRIGGERED SKIN ECZEMA IN MICE .................. 61
6.8. VDR ACTIVATION DOES NOT CHANGE THE NUMBERS OF INFILTRATING T CELLS IN LESIONAL SKIN..... 63