CLEC7A-Dectin-1 attenuates the immune response against dying and dead cells [Elektronische Ressource] = (CLEC7A-Dectin-1 verringert die Immunantwort gegen sterbende und tote Zellen) / vorgelegt von Connie Hesse

De
CLEC7A/Dectin-1 attenuates the immuneresponse against dying and dead cells(CLEC7A/Dectin-1 verringert die Immunantwortgegen sterbende und tote Zellen)Der Naturwissenschaftlichen Fakultätder Friedrich-Alexander-Universität Erlangen-NürnbergzurErlangung des Doktorgrades Dr. rer. nat.vorgelegt vonConnie Hesseaus Eberswalde-FinowAls Dissertation genehmigt von derNaturwissenschaftlichen Fakultät derFriedrich-Alexander-Universität Erlangen-NürnbergTag der mündlichen Prüfung: 21.12.2010Vorsitzender der Promotionskommision: Prof. Dr. Rainer FinkErstberichterstatter: Prof. Dr. Lars NitschkeZweitberichterstatter: PD Dr. Reinhard VollTable of ContentsTable of ContentsTable of Contents ............................................................................................ 1Abstract ............................................................................................................ 3Zusammenfassung.......................................................................................... 41 Introduction ............................................................................................... 61.1 C-type lectins................................................................................................. 81.1.1 CLEC4L/DC-SIGN ................................................................................ 111.1.2 CLEC7A/Dectin-1.................................................................................. 121.1.3 CLEC9A/DNGR1 ...........
Publié le : vendredi 1 janvier 2010
Lecture(s) : 53
Tags :
Source : D-NB.INFO/1010108832/34
Nombre de pages : 120
Voir plus Voir moins

CLEC7A/Dectin-1 attenuates the immune
response against dying and dead cells
(CLEC7A/Dectin-1 verringert die Immunantwort
gegen sterbende und tote Zellen)
Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Connie Hesse
aus Eberswalde-FinowAls Dissertation genehmigt von der
Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 21.12.2010
Vorsitzender der Promotionskommision: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Lars Nitschke
Zweitberichterstatter: PD Dr. Reinhard VollTable of Contents
Table of Contents
Table of Contents ............................................................................................ 1
Abstract ............................................................................................................ 3
Zusammenfassung.......................................................................................... 4
1 Introduction ............................................................................................... 6
1.1 C-type lectins................................................................................................. 8
1.1.1 CLEC4L/DC-SIGN ................................................................................ 11
1.1.2 CLEC7A/Dectin-1.................................................................................. 12
1.1.3 CLEC9A/DNGR1 .................................................................................. 13
1.2 Cell death: apoptosis, primary necrosis, secondary necrosis...................... 14
1.3 Cell death and clearance............................................................................. 17
1.3.1 “Find-me” signals .................................................................................. 18
1.3.2 “Eat-me” signals (PS dependent).......................................................... 19
1.3.3 “Eat-me” signals (PS independent)....................................................... 19
1.3.4 “Tolerate-me” signals ............................................................................ 22
1.4 Clearance deficiency and autoimmunity...................................................... 22
2 Materials & Methods ...............................................................................26
2.1 Materials...................................................................................................... 26
2.2 Methods....................................................................................................... 30
2.2.1 Cells, culture conditions, isolations and generations............................. 30
2.2.2 Induction and detection of apoptosis and necrosis ............................... 33
2.2.3 Cell stainings......................................................................................... 33
2.2.4 Binding Experiments............................................................................. 34
2.2.5 Phagocytosis Assays ............................................................................ 37
2.2.6 In vitro cytokine release assays ............................................................ 38
2.2.7 Immunization experiments .................................................................... 39
2.2.8 Antibody determination ......................................................................... 41
2.2.9 Cytokine determination ......................................................................... 42
2.2.10 Flow cytometry analyses ................................................................... 43
2.2.11 Microscopy anaylses ......................................................................... 44
1Table of Contents
2.2.12 Statistical analyses ............................................................................ 44
3 Results .....................................................................................................45
3.1 Cell death is reflected by characteristic morphological changes ................. 45
3.2 Plant lectin binding ...................................................................................... 47
3.3 C-type lectin CLEC9A/DNGR1 .................................................................... 50
3.4 C-type lectin CLEC4L/DC-SIGN.................................................................. 61
3.5 C-type lectin CLEC7A/Dectin-1 ................................................................... 66
4 Discussion...............................................................................................82
4.1 Lectin binding is a special feature of late apoptotic cells ............................. 82
4.2 C-type lectin CLEC9A/DNGR1 binds late apoptotic PMN endowed with intact
membranes........................................................................................................... 84
4.3 C-type lectin CLEC4L/DC-SIGN binds apoptotic cells endowed with intact
membranes........................................................................................................... 86
4.4 C-type lectin CLEC7A/Dectin-1 downregulates the response against late
apoptotic and primary necrotic cells...................................................................... 88
5 Concluding remarks ...............................................................................95
6 References...............................................................................................96
7 List of abbreviations.............................................................................109
8 List of figures ........................................................................................112
Acknowledgements.....................................................................................114
Curriculum Vitae..........................................................................................116
2Abstract
Abstract
Analysing apoptotic cell death it has been observed that late apoptotic cells expose
internal membranes with heavily altered glycocalyx. The latter is the target for a
plethora of sugar-epitope recognizing proteins such as pentraxins, collectins,
galectins, and, less physiological, plant lectins. These lectins bind to late apoptotic as
well as to primary and secondary necrotic cells. Strong binding to late apoptotic cells
with intact membranes was observed for the plant lectins Narcissus pseudonarcissus
(NPn) and Griffonia simplicifolia (GSL II). Within this thesis the C-type lectins
CLEC4L/DC-SIGN, CLEC9A/DNGR1, CLEC7A/Dectin-1 and their role in recognition,
uptake of apoptotic and necrotic cells and/or their influence on the immunogenicity of
dead and dying cells has been investigated. Enhanced binding for CLEC4L/DC-SIGN
and CLEC9A/DNGR1 to late apoptotic cells was observed. CLEC9A/DNGR1 binding,
which until now was only reported for necrotic cells, could also be demonstrated for
late apoptotic PMN endowed with intact membranes. CLEC9A/DNGR1 could,
therefore, act as recognition receptor of dying cells at the edge of late apoptosis and
secondary necrosis. Its physiological role is yet unkown, but might become clear,
when CLEC9A/DNGR1 ligands are revealed. For CLEC7A/Dectin-1 no direct binding
to apoptotic or necrotic cells was observed. However, a co-operation with other
receptors is proposed, since phagocytosis assays revealed differences in uptake
and/or degradation in the absence or presence of CLEC7A/Dectin-1 receptor. In vivo
studies revealed an attenuated immune response in the presence of CLEC7A/Dectin-
1 against dead and dying cells. This thesis points out that the role of lectin receptors
in the clearance process seems to be much more important than assumed. The anti-
inflammatory PS-dependent clearance of early apoptotic cells is well characterized.
Hence, if apoptotic cells escape clearance, the recognition of their altered surface
glycosylation pattern with exposed modified autoantigens by C-type lectin receptors
on professional phagocytes and antigen presenting cells is very likely. The role of C-
type lectins in immune stimulation in co-operation with other phagocytic receptors is
subject to deeper investigation. If involved in the clearance process of late apopotic
cells, as the data in this thesis indicate, C-type lectins might be targets for a
therapeutic concept for the chronic inflammatory autoimmune diseases SLE, which is
linked with clearance deficiencies.
3Zusammenfassung
Zusammenfassung
Untersuchungen zum Zelltod haben gezeigt, dass spät apoptotische Zellen innere
Membranen mit stark veränderter Glykokalyx exponieren. Letztere ist Zielstruktur für
zahlreiche Zucker-Epitop-erkennende Proteine, wie z.B. Pentraxine, Kollektine,
Galektine, und weniger physiologisch Pflanzenlektine. Diese Moleküle binden sowohl
spät apoptotische als auch primär und sekundär nekrotische Zellen. Starke Bindung
an spät apoptotische Zellen mit intakter Zellmembran wurde für die Pflanzenlektine
Narcissus pseudonarcissus (NPn) and Griffonia simplicifolia (GSL II) beobachtet. Im
Rahmen dieser Arbeit wurde die Rolle der C-typ Lektine CLEC4L/DC-SIGN,
CLEC9A/DNGR1, CLEC7A/Dectin-1 bei der Erkennung sowie der Aufnahme
apoptotischer und nekrotischer Zellen und/oder ihr Einfluss auf die Immunogenität
toter und sterbender Zellen untersucht. Es wurde verstärkte Bindung von
CLEC4L/DC-SIGN und CLEC9A/DNGR1 an spät apoptotische Zellen beobachtet.
CLEC9A/DNGR1 Bindung, die bisher nur für nekrotische Zellen berichtet wurde,
konnte in dieser Arbeit auch für spät apoptotische PMN mit intakter Membran
nachgewiesen werden. CLEC9A/DNGR1 könnte demnach als Erkennungsrezeptor
für sterbende Zellen an der Grenze von später Apoptose und sekundärer Nekrose
agieren. Die physiologische Bedeutung von CLEC9A/DNGR1, die bis jetzt noch
unbekannt ist, könnte aufgeklärt werden, wenn dessen Liganden analysiert sind. Für
CLEC7A/Dectin-1 wurde keine direkte Bindung an apoptotische oder nekrotische
Zellen beobachtet. Eine Kooperation mit anderen Rezeptoren ist aber denkbar, da
Phagozytoseversuche Unterschiede in der Aufnahme und/oder der Degradation
sterbender und toter Zellen in Abwesenheit oder Gegenwart vom CLEC7A/Dectin-1
Rezeptor aufwiesen. In vivo Studien zeigten eine verringerte Immunantwort in
Gegenwart von CLEC7A/Dectin-1 gegen tote und sterbende Zellen. Diese Arbeit
zeigt, dass die Rolle von Lektinrezeptoren im Clearance-Prozess viel bedeutender ist
als bisher angenommen. Die anti-inflammatorische PS-abhängige Clearance früh
apoptotischer Zellen ist gut charakterisiert. Wenn jedoch apoptotische Zellen ihrer
Beseitigung entkommen, ist die Erkennung ihrer veränderten Glykokalyx mit
exponierten, modifizierten Autoantigenen durch C-typ Lektinrezeptoren auf
professionellen Phagozyten und antigenpräsentierenden Zellen sehr wahrscheinlich.
Die Rolle von C-typ Lektinen bei der Immunstimulation in Kooperation mit anderen
phagozytischen Rezeptoren ist Gegenstand weiterer Untersuchungen. Wenn C-typ
4Zusammenfassung
Lektine am Clearance-Prozess spät apoptotischer Zellen beteiligt sind, worauf die
Ergebnisse dieser Arbeit hinweisen, könnten C-typ Lektine therapeutische Targets
für die chronische Autoimmunerkrankung SLE, welche mit Clearance-Defizienz
verknüpft ist, darstellen.
51 Introduction
1 Introduction
Lectins are proteins that bind sugar epitopes of glycoconjugates like glycoproteins or
glycolipids. This group of proteins do not act enzymatically on their ligand nor do they
belong to the class of antibodies [1], but due to their protein-carbohydrate-interaction
they are involved in a variety of cellular processes. Soluble and cell bound lectins
serve different functions including intracellular trafficking, cell adhesion, cell-cell
signalling, glycoprotein clearance, and modulation of innate immunity [2]. They are
ubiquitous in nature and they are to be found in plants, bacteria, viruses, and animals
[3]. Their history started over 100 years ago with Silas Weir Mitchell’s studies on
snake venoms. He observed agglutination of erythrocytes by rattlesnake venom. A
few years later H. Stillmark isolated ricin within his doctoral thesis, an extremely toxic
hemagglutinin, from seeds of the castor plant (Ricinus communis). The word “lectin”
(Latin: legere, meaning, among other things, "to select") was coined in 1954 by
William C. Boyd who discovered that plant agglutinins were able to distinguish
between erythrocytes of different blood types [4]. The term was generalized by
Sharon et al. to embrace all sugar-specific agglutinins of non-immune origin,
irrespective of source and blood type specificity [5]. Purification of various lectins led
to the modern era of “lectinology” providing new tools for cancer research and for
studying polysaccharides, glycoproteins, and cell surfaces. Table 1 depicts an
overview of the history of the "lectinology".
Analysing apoptotic cell death it has been observed that late apoptotic cells expose
internal membranes with heavily altered glycocalyx [6]. The latter is the target for a
plethora of sugar-epitope recognizing proteins that bind to late apoptotic as well as to
primary and secondary necrotic cells, such as C1q, PTX-3, galectins, SP-A, and, less
physiological, plant lectins. The biological function of animal lectins with respect to
apoptosis, clearance, clearance failure, and inflammation is a wide field that remains
to be elucidated.
61 Introduction
Table 1: Milestones in lectinology (adapted from [7] and [8])
1860 Description of blood coagulation as indication for lectin activity in rattlesnake
venom (S.W. Mitchell)
1888 Detection of erythrocyte agglutination by a toxic protein fraction from castor
beans (termed ricin) and seeds of related plants (H. Stillmark)
1890 Detection of a toxic lectin in the bark of black locust (Robinia pseudoacacia)
(O. Power, O. Cambier)
1891 Application of toxic plant agglutinins as model antigens (P. Ehrlich)
1898 Introduction of the term ‘haemagglutinin’ or ‘phytohaemagglutinin’ for plant
proteins that agglutinate red blood cells (M. Elfstrand)
1902 Detection of bacterial agglutinins (R. Kraus, S. Ludwig) and confirmation of
lectin presence (seven to nine decades later shown to depend on the
presence of a C-type lectin) in snake venom (S. Flexner, H. Noguchi)
1906 Detection of an agglutinin in bovine serum (later characterized as the C-type
lectin conglutinin) by use of activated complement-coated erythrocytes (H.-J.
Bordet, F.P. Gay); detection of a haemagglutinin in mushrooms (Amanita
spp.) (W.W. Ford)
1907/09 Detection of non-toxic plant agglutinins (K. Landsteiner, H. Raubitschek)
1913 Use of intact cells for the purification of lectins, like ricin (R. Kobert)
1919 Crystallization of a globulin from jack bean, concanavalin A, which was later
defined as lectin and used in pioneering studies (J.B. Sumner)
1935/36 Discovery of a carbohydrate as a ligand for concanavalin A (J.B. Sumner,
S.F. Howell)
1941 Detection of viral agglutinins (G.K. Hirst, L. McClelland, R. Hare)
1947/48 Detection of blood group-specific lectins (W.C. Boyd, K.O. Renkonen)
1952 Carbohydrate nature of blood group determinants detected by lectin-
mediated agglutination (W.M. Watkins, W.T.J. Morgan)
1954 Introduction of the term “lectin” for plant (antibody-like), carbohydrate-binding
proteins/agglutinins (W.C. Boyd)
1960 Detection of the mitogenic potency of lectins toward lymphocytes (P. C.
Nowell)
1963-65 Introduction of affinity chromatography for the isolation of lectins (I.J.
Goldstein, B.B. L. Agrawal)
1972 Determination of the amino acid sequence and the three-dimensional
structure of a lectin – concanavalin A or ConA (G.M. Edelman, K.O.
Hardman, C.F. Ainsworth)
1974 Isolation of the first mammalian lectin (asialoglycoprotein receptor) from liver
(G. Ashwell)
1978 First International Lectin Meeting (T.C. Bøg-Hansen)
1979 Detection of endogenous ligands for plant lectins (H. Rüdiger)
1983 Detection of the insecticidal action of a plant lectin (L.L. Murdock)
71 Introduction
1984 Isolation of lectins from tumors (H.-J. Gabius; R. Lotan, A. Raz)
1985 Immobilized glycoproteins as pan-affinity adsorbents for lectins (H. Rüdiger)
1989 Detection of the fungicidal action of a plant lectin (W. J. Peumans)
1992/93 Detection of impaired synthesis of lectin (selectin) ligands by defective
fucosylation as cause for leukocyted adhesion deficiency type II, a
congenital disorder of glycosylation (CDG IIc) (A. Etzioni and colleagues)
1995 Structural analysis of a lectin-ligand complex in solution by NMR
spectroscopy (J. Jiménez-Barbero and colleagues)
1996-2003 Detection of differential conformer selection by plant, bacterial, and animal
lectins (H.-J. Gabius and colleagues; L. Poppe and colleagues)
2001-2005 Development of glycan/lectin microarrays for specifity analysis of
lectins/structural analysis of glycans and glycoproteomics (various
laboratories worldwide)
2001- today Advances in lectinology and glycosciences honoured by devoting special
issues in internationally renowned journals
1.1 C-type lectins
Several families of glycan-binding proteins or lectins have been implicated in a wide
variety of immunological functions including first-line defense against pathogens, cell
trafficking, cell differentiation, and immune regulation. These include, among others,
the C-type lectins (collectins, selectins, mannose receptor, and others), S-type lectins
(galectins), I-type lectins (siglecs and others), P-type lectins (phosphomannosyl
receptors), pentraxins, and tachylectins (Table 2). Many biological effects of complex
carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition
domains. At least seven structurally distinct families of carbohydrate-recognition
domains are found in lectins that are involved in intracellular trafficking, cell adhesion,
cell-cell signalling, glycoprotein turnover, and innate immunity.
Table 2: Most important structural families of animal lectins and their classification (from [9]
and [8], with modifications)
Family Structural motif Carbohydrate Examples
ligand(s)
C-type conserved CRD variable (mannose, collectins, selectins,
glucose, galactose, endocytic lectins
fucose)
S-type conserved CRD ß-galactosides galectins
I-type or Ig- immunoglobulin-like sialic acids or other siglecs and others
type CRD sialylated structures
8

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.