Hyporesponsive synovial fluid T-cells in rheumatoid arthritis [Elektronische Ressource] / by Jan Thomas Barth

Thomas Barth

Sujets

• Biologie

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2003
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

From the Clinical Research Unit for Rheumatology
and Department of Rheumatology and Clinical Immunology
of the University Hospital Freiburg i. Breisgau
Hyporesponsive Synovial Fluid T cells
in Rheumatoid Arthritis
Inaugural-Dissertation
Submitted for the Degree of
Doctor of Natural Science
from the Faculty of Biology
of the Albert-Ludwigs-University
Freiburg i. Breisgau
by
Jan Thomas Barth
2003Supervised by: PD Dr. Hermann Eibel
Second censor: PD Dr. Ursula Klingmüller
Submitted: 25. May 2003Index
Index page
1 Introduction
1.1 Pathogenesis of rheumatoid arthritis (RA) 7
1.2 Cells involved in the pathogenesis RA 7
1.2.1 Fibroblast like synovial lining cells (synoviocytes) 7
1.2.2 Macrophages and monocytes 8
1.2.3 Neutrolphilic granulocytes 8
1.2.4 Dendritic cells (DC) 8
1.2.5 Osteoclasts & chondrocytes 10
1.2.6 B cells. 10
1.3 T cells 11
1.3.1 T cells in general. 11
1.3.2 T cell activation 11
1.3.3 TCR-mediated signal transduction. 12
1.3.4 Signaling of co-stimulatory receptor CD28 15
1.3.5 Regulation of T cell proliferation upon TCR and CD28 stimulation 17
1.3.6 Further T cell activation-modifying proteins 19
+ +
1.3.7 Generation of CD4 T cells and CD8 CTL of effector and memory phenotype 19H
+ +
1.3.8 Induction of hyporesponsiveness in CD4 T cells and CD8 CTL 20H
1.3.8.1 T cell anergy 20
1.3.8.2 Regulatory suppressor T cells. 21
1.3.9 T 1 and T 2 cytokines 22H H
1.3.9.1 IFN-γ and IL-2 23
1.3.9.2 IL-13 24
1.3.10 T cells in rheumatoid arthritis 24
+ +
1.4 Survival and apoptosis in CD4 T cells and CD8 CTL 26H
1.4.1 CD95-mediated apoptosis in T cells 26
1.4.2 CD95-mediated signaling. 27
1.4.3 Survival of activated T cells is mediated by anti-apoptotic cellular factors 28
1.5 Chemokine directed T cell migration within the body 31
1.5.1 Chemokines 31
1.5.2 Chemokine Receptors 32
1.5.2.1 CCR7 32
1.5.2.2 CXCR6 33
1.5.3 Chemokines in RA 33
1.6 MAFA and KLRG1 34
1.7 Other forms of chronic arthritis 35
1.8 Aims of the thesis 36
2Index
2.1 Material
2.1.1 Apparatus 38
2.1.2 Disposable hardware 39
2.1.3 Chemicals 39
2.1.4 Reagents and Kits 40
2.1.5 Antibodies used 40
2.1.5.1 Antibodies used for immunoblotting experiments 40
2.1.5.2 Antibodies used for in vitro activation assays of T lymphocytes 40
2.1.5.3 Antibodies and dyes used in flow cytometric analysis 41
2.1.5.4 Antibodies used for intracellular determination of cytokines 41
2.1.6 Solutions and buffers used in experiments 42
2.1.6.1 Common buffers 42
2.1.6.2 Flow cytometric analysis 42
2.1.6.3 Immunoblotting 42
2.1.6.4 Cell culture 43
2.1.6.5 Cultivation of bacteria 43
2.1.7 Marker and standards 43
2.1.8 Eukaryotic cell lines and bacterial strains 43
2.2 Methods
2.2.1 Isolation, in vitro cultivation and activation of T cells from peripheral blood (PB)
and synovial fluid (SF) of arthritis patients and healthy donors and their
subsequent analysis 44
2.2.1.1 Isolation of mononuclear cells from peripheral blood and synovial fluid 44
+ + + +
2.2.1.2 Separation of CD45RO CD4 T cells and CD45RO CD8 CTL 44H
+ +
(a) Isolation of CD4 T cells and CD8 CTL from purified PBMC & SFMC 44H
+ - + +
(b) and CD45RO CD4 T cell & CD8 CTL fractions 45H
2.2.2 In vitro cultivation of experimentally-used cells 45
(a) PBMC and SFMC 45
(b) 293T cells and Jurkat T cells 45
2.2.3 Analysis of isolated PB T cells and SF T cells. 45
2.2.3.1 Flow cytometric analysis (FACS) of cell surface markers 45
2.2.3.2 Activation of T cells with anti-human-CD3-mAb & anti-human-CD28-mAb 46
2.2.3.3 Assessment of changed marker expression in T cells after TCR stimulation 46
2.2.3.4 Analysis of cytokine expression upon TCR stimulation 46
2.2.3.5 Lymphocyte proliferation assay: VybrantCFDA SE Cell Tracer Kit 47
3Index
2.2.3.6 Immunoblotting of tyrosine-phosphorylated cytoplasmic proteins 47
(a) Sample preparation and in vitro stimulation of TCR 47
(b) Separation and detection of tyrosine-phosphorylated proteins 48
2.2.3.7 Analysis of in vitro stimulated PBL upon pre-incubation with synovial fluid 48
2.2.3.8 Analysis of CD95/CD95L-mediated apoptosis in lymphocyte 49
(a) In vitro induction of apoptosis 49
(b) Analysis of induced after in vitro cultivation with human CD95L 49
(c) Assessment of cell survival after in vitro 49
2.2.3.9 Amplification of plasmid DNA for experimental use 50
(a) Amplification of plasmid DNA 50
(b) Cultivation of E. coli 50
(c) Generation of transformation-competent E. coli 50
(d) Transformation of competent cells XL1-blue (E. coli) 50
2.2.3.10 Generation of human CD95L-enriched supernatant 51
(a) Transient transfection of 293 T cells 51
3 Results
+ + 3.1 SF CD4 T cells and SF CD8 CTL from many RA patients do notH
proliferate upon in vitro stimulation of T cell receptor and CD28 52
+ +3.2 Phenotypic characterization of SF CD4 T cells and SF CD8 CTL isolated fromH
RA patients and patients with other forms of chronic synovitis 54
+ + +3.2.1 SF CD4 T cells have a uniform effector/memory phenotype (CD95 CD45RO ) 54H
+ - + +3.2.2 There are more CD45RO than CD45RO CD95 CD8 CTL present in SF 56
+ +3.2.3 Activation status of SF CD4 T cells and SF CD8 CTL 56H
+ +3.2.4 αβ-TCR and CD3 are present on SF CD4 T cells and SF CD8 CTL cells 59H
+ +3.2.5 CD28 is found on most SF CD4 T cells, but subsets of SF CD8 CTL lack theH
co-stimulatory receptor 61
3.2.6 Conclusions: phenotypes of SF T cells.63
+ + 3.3 Analysis of SF CD4 T cells and SF CD8 CTL upon in vitro stimulation 64H
3.3.1 Anergic SF T cells from RA patients display a functional T cell receptor complex 64
+ +3.3.2 In vitro stimulated anergic SF CD4 T cells and SF CD8 CTL upregulate theH
expression of the activation marker CD69 and CTLA-4 67
+3.3.3 Subsets of non-proliferating SF CD8 CTL upregulate expression of CD45RO 71
+ + 3.3.4 Subsets of in vitro stimulated anergic SF CD4 T cells and SF CD8 CTLH
express IFN-γ and IL-13 72
3.3.5 Anergic RA SF T cells upregulate expression of CD25, but fail to express IL-2 74
3.3.6 Conclusions: TCR stimulation of SF T cells 77
4Index
3.4 Is the hyporesponsive status in SF T cells induced by a soluble factor? 78
3.4.1 PB T cells from healthy donors alter their TCR-mediated proliferation response
upon pre-incubation with SF from different arthritis patients 78
+ +
3.4.2 PB CD4 T cells and PB CD8 CTL attenuate their proliferative response afterH
in vitro cultivation with SF from an RA patient with anergic SF T cells 80
3.4.3 Conclusions: SF-induced suppression of PB T cells 83
3.5 CD95-mediated apoptosis in SF T cells 84
3.5.1 Jurkat T cells are highly susceptibility to CD95/CD95L-mediated apoptosis 84
3.5.2 There is no evidence for excessive induction of programmed cell death
+ + + +
in CD95L-treated CD95 SF CD4 T cells and CD95 CD8 SF CTL. 85H
3.5.3 PBL from RA patients were more susceptible to in vitro-induced CD95/CD95L-
mediated apoptosis than their counterparts isolated from the synovial fluid. 88
+
3.5.4 SF CD8 CTL from RA patients seem to be better protected against
+
CD95L-induced apoptosis than SF CD4 T cells 90H
3.5.5 Conclusions: CD95/CD95L-mediated apoptosis in SFL and PBL. 93
3.6 Analysis of chemokine receptors present on PB T cells and SF T cells 94
3.6.1 CCR7 is made by T cells with homing potential to secondary lymphoid tissue 94
3.6.1.1 CCR7 is expressed on different subsets of peripheral blood T cells 94
- +
3.6.1.2 Does the concentration of CCR7 CD45RO effector memory T cells in peripheral
blood reflect the presence of chronic inflammatory processes? 96
+ + - +
3.6.1.3 Both central memory (CCR7 CD45RO ) and effector memory (CCR7 CD45RO )
SF T cell subsets are present in synovial fluid 99
3.6.1.4 In vitro-stimulated proliferating PB T cells upregulate the expression CCR7 103
3.6.1.5 TCR-stimulated anergic RA SF T cells upregulate the expression of CCR7 103
3.6.1.6 Conclusions: CCR7 106
3.6.2 CXCR6 is made by subsets of effector T 1 cells and T 1 cells 107H C
3.6.2.1 Only a few PB T cells express CXCR6 107
+ +
3.6.2.2 CXCR6 is found on subsets of SF CD4 T cells and SF CD8 CTL 107H
+ + +
3.6.2.3 T cells & CXCR6 CD8 CTL accumulate in SF of arthritis patients 109H
3.6.2.4 Conclusions: CXCR6 111
3.7 KLRG1 is a potential marker for replicatory senescence of human T cells 112
+
3.7.1 PB CD4 T cells with an effector/memory phenotype express KLRG1 112H
+ - +
3.7.2 KLRG1 is found on CD45RO and CD45RO PB CD8 CTL 112
+ +
3.7.3 Subsets of SF CD4 T cells and CD8 CTL express KLRG1 114H
+
3.7.4 KLRG1 T cells do not accumulate in the synovial fluid of arthritis patients 115
3.7.5 Conclusions: KLRG1 117
5Index
4 Discussion
+ +
4.1.1 SF CD4 T cells and SF CD8 CTL from many RA patients are hyporesponsiveH
to recall antigen. 118
4.1.2 In vitro-stimulated hyporesponsive SF T cells from RA patients do not proliferate,
but they do upregulate the expression of activation marker and express different
cytokines 118
4.1.3 Anergy, bystander suppression or proliferative senescence: Does one of the
suppression models explain the hyporesponsive status in RA SF T cells? 119
4.1.4 Molecular mechanisms that might play a role in the state of proli