Tolerance induction in the liver after
T and NKT cell activation
Toleranzinduktion in der Leber nach T- und NKT-Zellaktivierung
Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades
vorgelegt von
Annette Erhardt
aus Münchberg
Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 11.07.2008
Vorsitzender der Prüfungskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Gisa Tiegs
Zweitberichterstatter: PD Dr. Robert Slany
M einem D addyM einem D addyM einem D addyM einem D addy
CONTENTS
CONTENTS
Publication List
Abbreviations
1 Introduction ........................................................................ 7
1.1 The liver: anatomy, physiology, and diseases........................................... 7
1.2 Animal models of immune-mediated liver injury ..................................... 11
1.3 Immunological tolerance and the outstanding role of the liver.............. 14
1.4 General overview of regulatory T cell subsets......................................... 17
1.5 Gender-specific differences in autoimmunity.......................................... 25
1.6 Aims of this study....................................................................................... 28
2 Materials and Methods..................................................... 30
2.1 Mice.............................................................................................................. 30
2.2 Animal treatment ........................................................................................ 31
2.2.1 Treatment schedules and Con A administration ................................ 31
2.2.2 Depletion of cells (KCs and CD25+ Tregs) ........................................... 31
2.2.3 Blockade of the IL10-receptor............................................................ 32
2.3 Sampling of material .................................................................................. 32
2.4 Isolation of cells.......................................................................................... 33
2.4.1 Isolation of primary hepatocytes ........................................................ 33
2.4.2 Isolation of intrahepatic mononuclear cells and splenocytes ............. 34
2.4.3 Isolation of CD4+CD25+ Tregs and responder cells ............................. 34
2.5 In vitro experiments.................................................................................... 36
2.5.1 Co-culture of responder cells and Tregs .............................................. 36
2.5.2 Specific inhibition of cAMP by a PKA inhibitor ................................... 36
2.5.3 CFSE labelling................................................................................... 37
CONTENTS_______________________________________________________
2.5.4 Neutralization of IL-10........................................................................ 37
2.6 Analysis of plasma transaminases ........................................................... 38
2.7 Real time RT- PCR ...................................................................................... 38
2.8 Cytokine determination by enzyme-linked immunosorbent assay
(ELISA)....................................................................................................... 39
2.9 Flow cytometry ........................................................................................... 40
2.10 Immunofluorescent staining and confocal laser imaging..................... 41
2.11 Haematoxylin/eosin staining of liver sections ....................................... 41
2.12 Analysis of hCD2-∆∆∆∆kTββββRII mice by tail biopsies .................................... 42
2.13 Statistical analysis.................................................................................... 42
3 Results .............................................................................. 43
3.1 Characterization of Con A-induced tolerance.......................................... 43
3.1.1 Con A pretreatment results in reduction of transaminase levels after
Con A rechallenge ........................................................................... 43
3.1.2 Con A pretreatment ameliorates Con A-induced necrosis ................. 44
3.1.3 Induction of an anti-inflammatory cytokine profile .............................. 45
3.1.4 Determination of the frequency of cell subpopulations ...................... 48
3.1.5 Investigation of the time point of tolerance induction ......................... 51
3.1.6 Induction of Con A tolerance ex vivo ................................................. 54
3.2 Identification of IL-10 as central mediator of Con A tolerance ............... 55
3.2.1 Loss of Con A-mediated tolerance in male IL10 KO mice and after
anti-IL10R-treatment ........................................................................ 55
3.2.2 Detection of gender-related differences in IL10 KO mice .................. 60
3.3 Importance of Kupffer cells as IL-10-producing cells ............................. 61
3.4 Involvement of CD4+CD25+ regulatory T cells during Con A tolerance . 62
3.4.1 Identification of Tregs as source of IL-10 ............................................. 62
3.4.2 Special characteristics of tolerized Tregs............................................. 65
CONTENTS
3.4.3 Therapeutic potential mediated by tolerized Tregs .............................. 72
3.4.4 Dispensability of IL-10 on Treg activity in vitro .................................... 74
3.5 Oppositional regulation of IL-10 and IL-17 during Con A tolerance...... 77
3.6 Relevance of NKT cells in Con A hepatitis and during tolerance........... 79
4 Discussion ........................................................................ 81
4.1 The role of IL-10-producing CD4+CD25+FoxP3+ regulatory T cells......... 81
4.1.1 ...as cellular immunotherapy in vivo................................................... 81
4.1.2 ...as suppressor cells in vitro.............................................................. 86
4.2 The conversion of Kupffer cells from type I to type II MΦΦΦΦ....................... 91
4.3 Proposed mechanism of Con A-mediated tolerance............................... 93
4.4 Outlook ........................................................................................................ 95
5 Summary........................................................................... 98
References
Deutschsprachige Zusammenfassung
Danksagung
Lebenslauf
PUBLICATION LIST
PUBLICATION LIST
Abstracts:
Biburger M, Erhardt A, Tiegs G. Concanavalin A induced tolerance in a murine
model of immune mediated hepatitis is a multifactorial process involving CD4+
CD25+ regulatory T cells but not depending on Interleukin-10. Immunobiology
2005; 210(6-8):400 (Abstract E.7)
Biburger M, Erhardt A, Tiegs G. The central role of tumor necrosis factor in the
murine –galactosylceramide model of immune mediated hepatitis.
Immunobiology 2005; 210(6-8):493 (Abstract L.20)
Erhardt A, Biburger M, Tiegs G. Concanavalin A-induzierte Toleranz im Maus-
Immunhepatitis-Modell wird unter Beteiligung von CD4+CD25+ regulatorischen T-
Zellen, Kupffer-Zellen und IL-10 vermittelt. Z Gastroenterol 2006; 44:128 (Abstract
4.38).
Erhardt A, Biburger M, Tiegs G. Con A-induced tolerance involves Tregs, Kupffer
cells, IL-10 and non-responsiveness in IL-2 producing cells. J Hepatol 2006; 44
(Suppl 2): S9 (Abstract 16).
Erhardt AL, Biburger M, Tiegs G. Untersuchungen zum Zeitverlauf der
Toleranzinduktion im Concanavalin A-Immunhepatitis-Modell. Z Gastroenterol
2007; 45:123 (Abstract 4.09).
Erhardt A, Biburger M, Tiegs G. IL-10 und regulatorische T-Zellen sind die
Hauptmediatoren der Concanavalin A-induzierten Toleranz im Maus-
Immunhepatitis Modell. Z Gastroenterol 2008; 46: 142 (Abstract 4.51).
PUBLICATION LIST
Erhardt A, Biburger M, Tiegs G. Oppositional effects of IL-10 and IL-17 during
immunological tolerance against concanavalin A. J Hepatol 2008; 48 (Suppl 2):
S69 (Abstract 152).
Journal article:
Erhardt A, Biburger M, Papadopoulos, T, Tiegs G. IL-10, regulatory T cells, and
Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice.
Hepatology 2007; 45(2):475-485.
Further presentations:
Erhardt A, Biburger M, Tiegs G. Immunological tolerance against concanavalin A
involves Tregs, Kupffer cells, IL-10, and impaired IL-2 production. 16th European
Congress of Immunology, Paris 2006
Erhardt A, Biburger M, Tiegs G. Long-lasting tolerance against concanavalin A
involves regulatory T cells, Kupffer cells and IL-10. 1st World Immune Regulation
Meeting, Davos 2007
Erhardt A, Biburger M, Tiegs G. IL-10 and regulatory T cells – the main mediators
of immunological tolerance against concanavalin A. 37th Annual Meeting of the
German Society of Immunology, Heidelberg 2007
ABBREVIATIONS
ABBREVIATIONS
Ab antibody
Ag antigen
α-GalCer α-galactosylceramide
AIH autoimmune hepatitis
ALT alanine aminotransferase
ANOVA analysis of variance
APC antigen-presenting cell
AST aspartate aminotransferase
B6 C57BL/6
BSA bovine serum albumine
cAMP cyclic adenosine monophosphate
cDNA copy DNA
Cl2MBP dichloromethylene-bisphosphonate
Con A concanavalin A
Ct threshold cycle
CTL cytotoxic T lymphocytes
CTLA-4 cytolytic T lymphocyte-associated antigen 4
DC dendritic cell
DNA deoxyribonucleic acid
dNTP deoxynucleosidtriphosphate
EAE experimental autoimmune encephalomyelitis
EDTA ethylenediaminetetraacetic acid
ELISA enzyme-linked immunosorbent assay
FACS fluorescence-activated cell sorter
FITC fluorescein isothiocyanate
FCS fetal calf serum
FoxP3 forkhead box P3
GalN D-galactosamine
GFP green fluorescence protein
HBSS Hanks balanced salt solution
ABBREVIATIONS___________________________________________________
HE haematoxylin/eosin
HBV hepatitis B virus
HCC hepatocellular carcinoma
HCV hepatitis C virus
HSC hepatic stellate cell
ICER inducible cAMP early repressor
IFN interferon
Ig immunoglobulin
IL interleukin
i.p. intraperitoneal
iTregs induced regulatory T cells
i.v. intravenous
KC Kupffer cell
KO knock out
LPS lipopolysaccharide
LSEC liver sinusoidal endothelial cell
mAb monoclonal antibody
MACS magnetic activated cell sorter
MHC major histocompatibility complex
MNC mononuclear cells
MΦ macrophage
mRNA messenger ribonucleic acid
MS multiple sclerosis
NK natural killer
nTregs naturally occurring regulatory T cells
ORF open reading frame
OVA ovalbumin
PBMC peripheral blood mononuclear cells
PBS phosphate buffered saline
PCR polymerase chain reaction
PE R-phycoerythrin
PEA Pseudomonas aeruginosa exotoxin A
PKA protein kinase A
RA rheumatoid arthritis
ABBREVIATIONS
RNA ribonucleic acid
Rp-cAMPS adenosine 3’,5’-cyclic phosphorothioate-Rp (inhibitor of PKA)
RT reverse transcription
RT-PCR reverse transcriptase-polymerase chain reaction
SEB Staphylococcus aureus enterotoxin B
SLE systemic lupus erythematosus
TCR T cell receptor
tg transgenic
Th cell T helper cell
TNF tumor necrosis factor
TNFR tumor necrosis factor receptor
Tregs CD4+CD25+ regulatory T cells
wt wild type
INTRODUCTION
7
1 INTRODUCTION
1.1 The liver: anatomy, physiology, and diseases
In a healthy adult the liver normally weighs between 1.4 and 1.6 kilograms being
the second largest organ beside the skin and the largest gland within the human
body (1). It is located in the upper right quadrant of the abdomen. Interestingly, the
liver is capable of natural regeneration: as little as 25% of remaining liver can
regenerate in a whole liver again. It is divided in four lobes: the left lobe, the right
lobe, the caudate lobe, and the quadrate lobe. Each lobe is further divided into
lobules that are approximately 2mm high and 1mm in circumference. These
hepatic lobules are the functioning units of the liver. They consist of hexagonal
rows of hepatic cells called hepatocytes.
The liver has a special anatomical location, since it is supplied by two blood
vessels: on the one hand by the liver artery carrying oxygen-enriched blood, on
the other hand by the portal vein bearing venous blood which is rich in nutrients
absorbed from the small intestine. Hence, the liver is permanently exposed to
intestinal antigens including pathogens, toxins, or harmless dietary antigens (2).
To cope with these different challenges, the liver produces cytokines, chemokines,
complement components, or acute phase proteins and harbours large amounts of
immune cells. A human liver contains a population of approximately 1 x 1010
lymphocytes comprising conventional and unconventional subpopulations of the
innate (NK and NKT cells) and adaptive immune system (T and B cells; Fig. 1.1 A;
[3, 4]). Conventional T cells include CD4+ and CD8+ T cells. However, the common
ratio of CD4+ : CD8+ T cells in the blood is usually reversed in the liver, with more
CD8+ cells than CD4+ cells (1). Unconventional T cells comprise NK cell-marker-
positive T cells, namely classical and non-classical NKT cells, and NK cell-marker-
negative γδ T cells. NKT cells are more abundant in the liver than in other organs
(20-30% of the intrahepatic lymphocyte population; [4]). The migration and
expansion of NKT cells is controlled by NK cells which are also enriched up to
30% among liver-resident lymphocytes (Fig. 1.1 A). NK cells participate in innate
INTRODUCTION____________________________________________________
8
stellate cells
5%
endothelial cells
50%
Kupffer cells
20%
billary cells
<0.5%
lymphocytes
25%
T cells
35%
B cells
10%Others
5%
NKT cells
20%
NK cells
30%
immune responses against viruses, intracellular bacteria, parasites, and
transformed cells. The higher numbers of hepatic NK cells is reflected by
increased NK cytotoxic activity in the liver.
A
B
Fig. 1.1: Cell composition of a healthy liver. (A) percentages of hepatic lymphocyte subsets;
(B) percentages of hepatic non-parenchymal cells [modified from (2)]
Hence, the liver is a pool of an unusual and unique mixture of lymphocytes in
comparison to peripheral blood. Beside the liver-associated lymphocytes, the liver
contains parenchymal hepatocytes (≈ 60-80%) and nonparenchymal cells (≈ 20-
40%; [4]) containing sinusoidal endothelial cells (LSEC), intrahepatic
macrophages, namely Kupffer cells (KCs), and stellate cells (Fig. 1.1 B; [5]).
LSECs represent the population with the highest frequency among non-
parenchymal cells in the liver (≈ 50%) lining sinusoids and forming a fenestrated
endothelium, hence being in direct contact with blood cells of the immune system.
LSECs express many different pattern recognition receptors (PRRs) in order to
scavenge macromolecules and scan foreign and harmful agents resulting in
hepatic clearance.
Kupffer cells are derived from circulating monocytes and constitute the largest
population of resident macrophages in the body. They are well-positioned in the
INTRODUCTION
9
sinusoidal space and are in close contact with LSECs, thus fulfilling their functions
such as phagocytosis of apoptotic cells and microorganisms, antigen-presentation,
and involvement in tolerance (6). Beside antigen-trapping mediated by KCs and
LSECs, antigen-presentation to T cells is also maintained by ‘professional’ APCs
in the form of dendritic cells (DC; [6]). In the healthy liver, DCs are mostly
immature and reside around portal areas. Since IL-10 and TGFβ are constitutively
expressed by KCs and LSECs, the liver offers a cytokine milieu that might render
resident DCs tolerogenic (4). A small proportion of the non-parenchymal cells is
allocated to stellate cells/Ito cells (5%) found in the perisinusoidal space (Fig. 1.1
B and Fig. 1.2). The granular stellate cells (HSC) are described as being in a
quiescent state containing vitamin A-rich lipid droplets (7). After liver damage,
HSCs are activated characterized by adoption of a myofibroblast-like phenotype,
proliferation, contractility, expression of interstitial collagen I and III, and
chemotaxis. In chronic injury, activated HSCs are the major source of the
collagens that comprise fibrosis and cirrhosis (8).
Fig. 1.2: Immune cells in the healthy liver [from (4)]
Remarkably, the liver carries out many important physiological functions including
production and excretion of bile, metabolism of drugs, lipids, and carbohydrates,
enzyme activation, storage of glycogen, vitamins A, D, B12, iron, and copper,
synthesis and turnover of clotting factors and plasma proteins such as albumin
and globulin, and immunological interactions with intestinal antigens transported
via the portal vein.
INTRODUCTION____________________________________________________
10
In more detail, bile salts facilitate fat digestion and absorption. Bile is continuously
secreted by the liver (from 250 to 1000 mL/day) and stored in the gallbladder until
a meal. Furthermore, the liver removes potentially harmful substances by making
toxic substances more water-soluble. Hence, they can be more easily excreted
from the body to the urine. An important function of the liver is the synthesis of
plasma proteins including most of the clotting factors. Prothrombin and fibrinogen,
substances needed to help blood coagulate, are both produced by the liver.
Beside metabolizing fats and proteins, the liver takes part in the carbohydrate
metabolism in three ways: firstly, through the process of glycogenesis (convertion
of glucose, fructose, and galactose to glycogen); secondly through the process of
glycogenolysis (catabolism of stored glycogen to maintain blood glucose level);
and thirdly, through the process of gluconeogenesis (synthesis of glucose from
proteins or fats to maintain the blood glucose level). Hence, the liver supports the
body to store sugars and to transport and save energy. Last but not least, the liver
helps the body to fight infections by producing immune factors and removing
bacteria. The hepatic phagocytes produce acute-phase proteins in response to
microbes. These proteins are associated with inflammation processes, tissue
repair, and immune cell activities (9). Due to these diverse roles of the liver,
hepatic deficiency or damage would result in dramatic consequences.
Several causes might lead to hepatic damage comprising intensive alcohol abuse,
viral infections, bacterial invasion, drugs and toxins as well as foreign antigens
during transplantation and autoantigens. Symptoms of liver disease may be acute,
occurring suddenly, or chronic, developing slowly over a long period of time.
Symptoms depend on the type and severity of the liver disease. However, some
common signs are jaundice, nausea, darkened urine, unusual weight shifts,
abdominal pain, fatigue, or diarrhea. Finally, liver injury might result in fatty liver,
liver fibrosis, liver cirrhosis or in the worst case in hepatocellular carcinoma (HCC).
At last, the liver is not able to perform its normal synthetic and metabolic functions.
These hepatic failures are often associated with an inadequate immune response,
since T cells are activated and consequently, pro-inflammatory cytokines like
TNFα and IFNγ are released initiating inflammation and unspecific immune attack
against hepatocytes.
In particular, autoimmune hepatitis (AIH) with an incidence of 1 to 2:10.000 is
characterized by a misdirected immune reaction against autoantigens leading to
INTRODUCTION
11
the high titers of a wide range of circulating autoantibodies,
hypergammaglobulinemia, and endocrine abnormalities (10). Common
autoantibodies measured during AIH include antinuclear- (ANA), smooth muscle-
(SMA), type 1 liver-kidney microsomal- (LKM-1), soluble liver antigen- (SLA), and
perinuclear staining antineutrophil cytoplasmic- (pANCA) antibodies. Since AIH (a)
generally shows a marked female predominance (70-80% of affected individuals
are women) and (b) is especially induced in peri- and postmenopausal women, it
is possible that changes in hormonal regulation of the immune system might
contribute to AIH development beside environmental factors and genetic
predisposition regarding certain haplotypes of HLA-antigens (10). Moreover, in
patients with AIH (a) peripheral Treg numbers and functions are depressed
compared with controls, (b) the percentage of Tregs inversely correlates with
autoantibody titers, and (c) Treg numbers are lower in patients at the time of
diagnosis than during remission (11).
At present, the treatment of choice is the corticosteroid prednisone alone or a
combination with prednisone and azathioprine aiming at a downmodulation of an
overactive immune system. Both treatment protocols show high survival rates and
work best when AIH is diagnosed early. However, a rate of 13% of treatment
failures and the failure to induce permanent remission in most patients underlines
the urgent need to develop additional treatment regimens (12). Furthermore,
management of side effects such as weight gain, high blood pressure, anxiety,
osteoporosis, or diabetes is very important.
1.2 Animal models of immune-mediated liver injury
Nearly all of the above mentioned hepatic innate immune cells were intensively
investigated and were accounted for being involved in diverse liver injuries both in
humans and in experimental animal models. Until now, some meaningful models
of immune-mediated liver injury are already existing resembling and mimicking
human liver disorders such as steatohepatitis, autoimmune hepatitis, alcohol-
induced hepatitis, or ischemia/reperfusion liver injury. These dysfunctions are
often associated with a Th1 cytokine response characterized by IFNγ and TNFα
INTRODUCTION____________________________________________________
12
release. Hence, the development of models of T cell-dependent liver damage
might be necessary. In Figure 1.3, the most important models of immune-mediated
liver injury are summarized graphically.
Firstly, injection of an anti-CD3 Ab induces apoptosis in the liver followed by
necrosis as a consequence of T cell activation, macrophage activation, TNFα
production, and caspase-3 activation (13, 14). Secondly, T cell activation is
completely evaded and dispensable upon administration of bacterial
lipopolysaccharides (LPS) which primarily act on KCs followed by TNFα release
and hepatic damage (15, 16). Lastly, KC activation could also be circumvented by
administration of TNFα itself triggering a direct attack and apoptosis of
hepatocytes (17). Liver-specific inhibition of transcription und thus lack of
synthesis of anti-apoptotic signals is induced by administration of D-galactosamine
(GalN) in the three mouse models mentioned above (18). Further models
comprise the model of anti-CD95- (19) and PEA-induced (Pseudomonas
aeruginosa exotoxin A [20]) liver injury not requiring previous sensitization with
GalN. NK and T cells, but not NKT cells, are also involved in PEA-induced
hepatotoxicity. However, a moderate immune-mediated liver damage strictly
depending on NKT cells is induced by intravenous injection of α-
galactosylceramide (α-GalCer). Upon α-GalCer injection, rapid expression of
different cytokines including IL-2, IL-4, IL-6, TNFα, and IFNγ is detectable both in
liver tissue and plasma (21). The simultaneous production of Th1 and Th2
cytokines is an effect of NKT cell activation (see Chapter 1.4).
However, in the present study the mouse model of concanavalin A (Con A)-
mediated hepatitis has been chosen, since it reflects the process of autoimmune
hepatitis very adequately (22), although Con A is not an autoantigen. Similarities
between the murine model and the human disease are (a) the good
responsiveness to immunosuppressive drugs (22), (b) the genetic prevalence of
certain mouse strains with respect to susceptibility (23), (c) the prevalence of CD4+
T cells, and (d) the immunosuppression in state of remission (24). Concanavalin A
is a mitogenic plant lectin isolated from the jack bean and often used in vitro to
activate T cells. It binds mannose residues of different glycoproteins and thus
activates lymphocytes in an antigen-unspecific manner. In vivo, a single
intravenous injection of a sublethal Con A dose induces an immune-mediated liver
INTRODUCTION
13
damage in mice (22) and rats (25) by local activation of liver-resident NKT cells
which mainly secrete IFNγ (26-28). Consecutively, CD4+ T cells and
polymorphonuclear cells are attracted and activated KCs produce large amounts
of TNFα resulting in necrotic cell death of hepatocytes and release of the
transaminases ALT and AST from the cytoplasm of hepatocytes into the blood (14,
29, 30). Additionally, IL-12 (31) and IL-18 (32) are important for disease
development. In contrast to the pro-inflammatory cytokines TNFα and IFNγ, the
immunosuppressive and anti-inflammatory IL-10 plays a protective role in this
model (33-35). Finally, CD4+ T cells are indispensable for the development of liver
injury in vivo confirmed by the usage of SCID (Severe Combined
Immunodeficiency Disorder) and RAG (Recombinase Activating Gene) KO mice,
both lacking T and B cell, and athymic nude mice, lacking only T cells and by
experiments with depletion of CD4+ T cells (22, 27). In contrast, depletion of CD8+
T cells did not prevent Con A-induced liver injury (22). Furthermore, transfer of
splenocytes (14) or intrahepatic mononuclear cells (36) from sensitive wt mice to
resistant nude mice restored the susceptibility of these mice towards Con A with
respect to establishment of liver damage and the capacity to produce TNFα and
IL-2 (14). NKT cell-deficient CD1dKO mice were also highly resistant to Con A-
induced liver damage indicating CD4+ T cells might largely refer to CD4+ NKT cells
(26, 27).
Twenty-four hours after Con A challenge transaminase levels start to decline and
the liver begins to regenerate (37). Interestingly, Con A-pretreated mice developed
tolerance against Con A rechallenge within eight days lasting over several weeks
(35). The mechanisms of this immunosuppressive and tolerogenic state and
potential involvement of regulatory cell types limiting the detrimental T cell
response has not been elucidated so far in this mouse model and is the main
matter in this study.
In conclusion, animal models of immune-mediated liver injury indeed reflect
several steps of human liver disorders; however, none of these models completely
comprises all aspects of the whole course of disease.
INTRODUCTION____________________________________________________
14
ConA
MΦΦΦΦ
TNFαααα
HC
HC
anti-CD3GalN
anti-CD95LPS
TNF ααααGalCer
PEA
Fig. 1.3: Summary of different models of immune-mediated liver injury and the relevant point of
attack [modified from (38)]
1.3 Immunological tolerance and the outstanding role of the
liver
It was a longstanding mystery of immunology how the immune system produces a
nearly universal repertoire, while at the same time avoiding reacting to self. Daily,
everybody is confronted with countless microbial challenges. To counteract these
challenges, the vertebrate adaptive immune system represented by T and B cells
has evolved a highly organized interaction and displays extensive diversity
generated by rearrangement of genes encoding antigen-specific receptors during
T and B cell differentiation in the thymus and bone marrow, respectively (39).
Sometimes, lymphocytes could recognize self antigens by such a receptor leading
to autoimmunity followed by autoimmune diseases. To prevent such fatal
consequences, mechanisms had been developed guaranteeing the maintenance
INTRODUCTION
15
of self-tolerance. Hence, immunological tolerance occurs when an
immunocompetent host fails to respond to the presence of a specific antigen. The
process of immunological tolerance is divided into two types: (a) central tolerance
occurring during lymphocyte development and (b) peripheral tolerance emerging
after leaving the primary lymphoid organs (40).
During T cell development in the thymus the process of negative selection leads to
deletion of self-reactive thymocytes whose T cell receptors have high affinity to the
MHC complex (Burnet’s theory of clonal deletion). As a consequence, the
thymocytes die by apoptosis (41). Furthermore, no binding to MHC also results in
apoptosis of the cells, since these cells did not undergo positive selection. Only
moderately binding T cells representing a population of only ~3-5% will survive
and leave the thymus due to positive selection (42).
During B cell development in the bone marrow immature B cells are screened for
autoreactivity. Early studies of B cell selection suggested that autoreactive B cells
are eliminated by clonal deletion in the bone marrow. However, subsequent
studies showed that autoreactive B cells specific for membrane-bound
autoantigens can also undergo the process of receptor editing, which abolishes
the autoreactive specificity without eliminating the cell (43). A new antigen-
receptor is generated with harmless specificity using the familiar machinery of
VDJ-recombination (44).
However, tolerance to self antigens has also to be ensured in the body’s periphery
preventing autoimmunity (39). In fact, several selective mechanisms are existing
outside of the primary lymphoid organs. Depending on co-stimulation, location,
antigen-dose, or timing the adequate tolerogenic process is chosen (41). In the
former case, lack of the second co-stimulatory signal between T cells and antigen-
presenting cells (APC) results in T cell inactivation and subsequently, in anergy. If
tissue cells present peptides from their endogenously synthesized proteins on self
MHC molecules in the absence of co-stimulation, interaction of such cells with
autoreactive T cells leads to non-responsiveness (41).
Furthermore, antigen concentration plays an important role in maintaining
tolerance: on the one hand, high doses of autoantigen lead to repeated stimulation
and hence, to deletion of autoreactive T cells by programmed cell death called
AICD (activation-induced cell death), on the other hand in case of low antigen
concentrations the threshold of receptor occupancy is not exceeded for triggering
INTRODUCTION____________________________________________________
16
an immune response. This process of ignorance is particularly pronounced in so-
called immunologically privileged sites like CNS (central nervous system), eyes or
testis. In these locations the antigens are sequestered from the immune system
(45).
Fig. 1.4: Mechanisms that prevent potentially autoreactive T cells from reacting inappropriately to
autoantigens [from (46)]
Ultimately, a specialized population of T cells called regulatory T cells is involved
in peripheral tolerance and posses the ability to produce anti-inflammatory
cytokines like IL-10 suppressing any tendency of self attack (47, 48). Hence, lack
of regulatory T cells results in the outbreak of autoimmune diseases like multiple
sclerosis (MS), type 1 diabetes or AIH (49, 50). In the thymus, regulatory T cells
interact agonisticly with self-antigens and then contribute in a dominant and active
fashion to self-tolerance in the periphery. Hence, positively selected T cells with
the highest avidity that escape deletion are activated and irreversibly committed
for regulatory effector functions (42, 51). The pool of regulatory T cells contains
both naturally arising T cells (nTregs) and adaptive or induced regulatory T cells
(iTregs). The main characteristics and differences between these suppressive cell
subpopulations are discussed in the next chapter. A summary of the noted
tolerance mechanisms is given in Figure 1.4.
INTRODUCTION
17
The liver appears to be a privileged organ regarding immune regulation and
tolerance, since it occupies a particular position within the human body linking the
gastrointestinal tract and the systemic venous circulation (52). As the liver takes
the position of a “scavenger” organ and is involved in clearance of foreign antigens
as well as bacterial and toxic products from the gut, it is compulsory to circumvent
any dispensable and inadequate immune activation to prevent liver damage.
However, gut-derived antigens are not ignored by the immune system rather the
liver has been considered to favour the induction of peripheral tolerance.
Furthermore, the overall predisposition of the intrahepatic immune response might
also account for long-term survival of allogeneic liver transplants despite MHC
discrepancies and even in the absence of immunosuppression (53). Additionally,
the presence of a liver allograft can suppress the rejection of other solid tissue
grafts from the same donor whereas further organ transplants from another donor
lead to graft rejection indicating antigen-specific induction of tolerance by the
transplanted liver (5, 54). This clearly indicates that active immune regulation
occurs in the liver, promoting the development of peripheral tolerance. In contrast,
pathogens settling the liver might exploit this benefit of local tolerance; therefore,
infections of the liver by pathogens (e. g. viruses) require induction of an effective
immune response to break down the infection and to prevent progression of
persistence and chronic infections (5).
In conclusion, the liver is an organ with paradoxical immunological properties: On
the one hand immune reactions against innocuous antigens have to be avoided
and on the other hand immune responses against harmful pathogens have to be
intact and effective. Therefore, the liver lymphocytes have to switch rapidly from a
tolerant to a responsive state (52).
1.4 General overview of regulatory T cell subsets
For many years, different working groups have identified lymphocytes that
suppress immune responses. The most potential and promising candidates are
the naturally arising CD4+CD25+FoxP3+ regulatory T cells (nTregs), NKT cells, Tr1
INTRODUCTION____________________________________________________
18
cells (type 1 regulatory T cells), and Th3 cells. In contrast to nTregs already
generated in the thymus, Tr1 and Th3 cells are induced in the periphery (iTregs
[55]). The different CD4+ T cell subsets including the above mentioned regulatory
T cell populations are depicted in Fig. 1.5.
Fig. 1.5: Development of different subsets of regulatory T cells [from: (55)]
In the last few years Tregs have become a popular subject of immunological
research. It has been shown that naturally occurring CD4+CD25+FoxP3+ T cells
identified by Sakaguchi and co-workers (56) in the mid 90s provide a further
mechanism in order to maintain self tolerance and thus to suppress autoreactive T
cells beside other protective mechanisms like negative selection in the thymus and
anergy in the periphery (see above [39]). Tregs are able to suppress the
proliferation of a wide variety of immune cells. They have been shown to prevent
the development of autoimmune diseases and they also play an important role in
transplantation tolerance by preventing graft rejection. Hence, a dysfunction of
these regulatory cells leads to severe immune-pathology including autoimmune
diseases like type 1 diabetes, multiple sclerosis, autoimmune gastritis, and
autoimmune hepatitis (57-59). This idea is supported by the following
observations: (a) in mice, depletion of the Treg population spontaneously results in
autoimmune diseases; (b) nude mice (which have no T cells of their own) develop
autoimmune disease if CD4+ T cells were administered that have been depleted of
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the CD25+ population (60); (c) both humans and mice with mutations in their
FoxP3 gene, the most specific marker of nTregs until now, suffer from autoimmune
diseases (61).
Tregs are generated in the thymus, since neonatal thymectomy of mice (d3Tx)
leads to the development of a wide spectrum of organ-specific autoimmune
manifestations including gastritis, oophoritis, or thyroiditis (62, 63). Tregs may arise
from relatively high-avidity interactions with self-peptide – MHC complexes, at an
avidity range between positive and negative selection, namely just below the
threshold for negative selection (63). The CD25+ subset constitutes about 5-10 %
of the peripheral CD4+ T cells in normal naive mice and healthy humans. CD25
(IL2-receptor α-chain) is a specific cell surface marker of Tregs (49). But it should be
noted that CD25 is not an absolute marker for Tregs, since it is also expressed on
activated conventional non-regulatory T cells. Other expressed cell surface
markers are CTLA-4 (cytolytic T lymphocyte-associated antigen 4) and GITR
(glucocorticoid-induced TNF-receptor family related gene [64]). But unfortunately,
none of these markers are uniquely expressed by Tregs. Further investigations
showed that the transcription factor Foxp3 is the most specific marker of nTregs
([65]; Fig. 1.6).
Fig. 1.6: Expression of surface and intracellular markers on CD4+CD25
+FoxP3
+ Tregs [from: (66)]
Together with IL2, FoxP3 is essential for development, maintenance and function
of CD4+CD25+ Tregs (66). Mutations in FoxP3 lead to depletion of CD4+CD25+ Tregs.
The scurfy-deficient mouse strain shows a frame-shift mutation in the FoxP3 gene
INTRODUCTION____________________________________________________
20
resulting in severe autoimmunity and fatal lymphoproliferative disorder. The
human equivalent to the scurfy pathogenesis is called IPEX (Immune
dysregulation, Polyendocrinopathy, Enteropathy, X-linked) syndrome
characterized by a set of autoimmune diseases. Hence, FoxP3 might be a master
gene controlling the development and function of Tregs which was also confirmed
by retroviral transduction of FoxP3 to non-regulatory CD4+ T cells (67).
CD4+CD25+FoxP3+ T cells require a first T-cell-receptor stimulation, but once
activated, they are suppressive in an antigen-non-specific manner (66). In vitro,
FoxP3+ Tregs appear to be anergic, when stimulated via the TCR (68). CTLA-4 is
responsible for mediating in vitro inhibition of T-cell proliferation and IL-2
expression via cell-cell contact (62). Nevertheless, there is still an ongoing debate
about the suppressive mechanism of Tregs in vivo and in vitro (66). Inhibition of T
cells in vitro seems to be CTLA-4-dependent (50), but independent of soluble
factors such as IL-10. Upon TCR stimulation, CTLA-4 is expressed on the surface
of Tregs followed by interaction with B7 on responder cells and overexpression of a
potent inhibitor of IL-2 transcription in responder cells, namely ‘inducible cAMP
early repressor’ (ICER; [69]). Subsequently, activated FoxP3- responder cells
themselves express CTLA-4 on their surface engaging neighbouring CD4+FoxP3-
T cells. In an ‘infectious manner’, ICER expression is induced in these cells
resulting in a successive attenuation of IL-2 expression (69). The expression of
ICER is stimulated by cyclic adenosine 5´-monophosphate (cAMP)-activated
transcription factors (70). Recently, it has been shown that cAMP takes part in
Treg-mediated suppression. In more detail, transfer of the second messenger
cAMP from regulatory T cells into responder cells is mediated via gap junctions,
since the suppressive activity of naturally occurring regulatory T cells was
abolished by a specific cAMP inhibitor, called Rp-cAMPS, as well as by a gap
junction inhibitor (71).
CD25- T cells are the cellular target of Tregs. Activated CD25+ cells strongly
suppress the proliferation and IL-2 production of co-activated conventional
CD4+CD25- responder cells in vitro. A cell-cycle arrest is induced often followed by
cell death.
The in vivo mechanisms of Tregs are far more complicated and immunosuppressive
cytokines like IL-10 or TGFβ seem to be implicated (63, 72, 73). The importance of
these cytokines could be easily demonstrated by using cells from IL10-/- mice or
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21
blocking TGFβ and IL10R with mAb. Hence, the mechanism of Treg-mediated
suppression still remains controversial, with a lot of conflicting findings regarding
the suppressive mechanism in vitro and in vivo (66).
Finally, nTregs might be interesting for potential clinical and therapeutic
applications. On the one hand, Treg function has to be enhanced during excessive
immune reactions, namely in case of organ transplantation, autoimmune diseases
and allergy, e.g. by ex vivo mechanisms such as ex vivo gene transduction of
Foxp3 or ex vivo expansion of regulatory cells using cytokines, pharmacological
agents, or modified DCs (66, 74). Thus, the aim of this therapeutic strategy might
be the suppression of immune responses by higher frequencies of Tregs. However,
on the other hand FoxP3+ Treg function has to be reduced during infectious
diseases and cancer allowing a proper attack of effector T cells and anti-tumoral T
cells, respectively. This can be achieved e.g. by transient removal of Tregs or by
blocking their function through monoclonal antibodies. In place, the aim is a
stronger immune response and thus less suppressive Tregs have to be present
(Fig. 1.7; [75]).
Fig. 1.7: Summary of immune responses influenced by CD4+CD25
+ Tregs [from: (66)]
Another T cell subpopulation with overt regulatory activities are the natural killer T
cells (NKT). NKT cells exhibit similarities to NK cells and T cells thus describing a
INTRODUCTION____________________________________________________
22
specialized subset of T cells. Namely, NKT cells express both NK surface markers
and the typical T cell marker CD3 (Fig. 1.8). The TCR does not interact with
peptide antigen presented by the classical MHC-encoded class I or II molecules;
rather it recognizes glycolipids presented by the non-classical, MHC class I-like
molecule CD1d (76). At least two classes of CD1d-dependent NKT cells have
been defined: a) type I NKT cells (invariant NKT cells, iNKT) expressing an
invariant TCR α-chain in combination with limited Vβ chains and recognizing the
glycosphingolipid antigen α-galactosylceramide (α-GalCer) isolated from a marine
sponge and b) type II NKT cells expressing more diverse TCR Vα chains. Hence,
α-GalCer mimics a natural ligand. Recently, the physiological ligand has been
identified called isoglobotrihexosylceramide (iGb3; [77]).
Fig. 1.8: Comparison of NKT cells, NK cells, and T cells [from: (76, 78)]
Most NKT cells are thymus-dependent (79), but some scientists argue for an
extrathymic origin, although NKT cells are absent in nude mice and do not develop
in thymectomized animals (80). However, it is obvious that NKT cells have to be
distinguished developmentally and functionally from CD4+ and CD8+ T cells.
Murine NKT cells are CD4+ or double negative whereas in humans CD4+, DN and
CD8+ NKT cells are present. In mice, NKT cells are found at the highest frequency
in liver (20-40% of liver lymphocytes; [3]), but they are present at lower
frequencies in thymus, bone marrow, spleen, lymph nodes and blood (< 1%).
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Interestingly, in humans they are clearly less frequent in the liver (4% of hepatic T
cells). The reasons for these species-specific differences are unknown (78).
Furthermore, human NKT cells can recognize mouse CD1d and vice versa,
indicating highly conserved specificity. Once activated, NKT cells respond with a
rapid and high cytokine production within 1-2 hour. On the one hand they release
Th1 cytokines like INFγ and TNFα, on the other hand they produce Th2 cytokines
like IL-4 (81). The range of actions and the role of NKT cells in the immune
response is extremely diverse and multifunctional: firstly, they play an important
role in the regulation and suppression of autoimmune diseases (82, 83), secondly,
they control viral, bacterial and parasite infections (e.g. Mycobacterium, Listeria,
Plasmodium) by enhancing microbial immunity, and thirdly, they play a central role
in tumor rejection. Yet NKT-cell activity can also been deleterious, e. g. in allergy
and atherosclerosis (76). Nevertheless, NKT cells might be attractive targets for
immunotherapy. However, there is much to be learned before these cells can be
effectively manipulated in the clinic. Probably, techniques have to be developed to
expand NKT cells in vivo or in vitro followed by reinjection to prevent diseases
(81).
The third identified suppressive T cell subpopulation is the subset of extra-
thymically generated T regulatory type 1 (Tr1) cells characterized by high IL-10
secretion. Interleukin-10 is expressed by a variety of immune cells, including CD4+
T cells, monocytes and macrophages (84), B cells, natural killer (NK) cells, and
dendritic cells (DC; [85]). IL-10 binds to the IL10-receptor expressed by most
haematopoietic cells. Initially identified as a factor produced by murine Th2 cells
IL-10 was primarily named cytokine-synthesis inhibitory factor (CSIF) due to its
capacity to inhibit IL-2, IFNγ and TNFα production by Th1 cells responding to
antigen and APC (86). IL-10 has anti-inflammatory and suppressive effects on
most haematopoietic cells.
Interestingly, allogeneic stimulation of CD4+ T cells after repetitive stimulation in
the presence of IL-10 induces a T cell population which secretes high amounts of
IL-10 and moderate amounts of TGFβ. These Tr1 cell clones suppress the
immune response of antigen-specific T cells both in vitro and in vivo (87) and are
induced by an IL-10 dependent process both in humans and mice. The unique
cytokine profile of Tr1 cells upon TCR-mediated activation is as followed: high
INTRODUCTION____________________________________________________
24
levels of IL-10 and TGFβ, normal levels of IL-5 and INFγ could be detected; low
levels of IL-2 and the absence of IL-4 release distinguish them from Th1 and Th2
cells (87-89). IL-10 secretion is detectable as soon as 4h after stimulation and is
the true hallmark of Tr1 cells. Stimulated Tr1 cell express activation markers such
as CD25, CD69, CD28, CD40L and CTLA-4 at higher levels. Unfortunately, a
specific cell marker could not be identified until now. However, Tr1 cells do not
constitutively express the transcription factor FoxP3, the most specific marker of
naturally occurring Tregs. Tr1 cells are anergic and proliferate poorly upon
activation. Their anti-inflammatory and suppressive mechanisms on naïve and
memory Th1 and Th2 cells are definitely ensured by the high levels of IL10, both in
vivo and in vitro. Hence, this cytokine is required for both the function and
differentiation of Tr1 cells (46, 86). Once activated, they suppress cytokine-
dependent, but in an antigen-non-specific manner by mediating bystander
suppressive activity against other antigens (87). The released IL-10 downregulates
expression of co-stimulatory molecules and pro-inflammatory cytokine production
by APCs and directly inhibits IL-2 and TNFα production by CD4+ T cells.
In healthy individuals, Tr1 cells contribute to immunological tolerance by
suppressing undesired immune responses toward self antigens, food antigens and
allergens. Therefore, the induction of oral tolerance to enteric antigens and
systemic tolerance to self antigens is the central function of Tr1 cells.
Finally, the last population to be mentioned is the Th3 subpopulation. They are
prevalent in the intestine like Tr1. Therefore, Th3 cells might be another cell
population responsible for oral tolerance beside Tr1 cells. The mainly produced
lymphokine is TGFβ (46). They mainly emerge after uptake of foreign antigen via
the oral route and require TGFβ, IL4 and IL10 and inhibition of IL12 for their
maturation (90). Once activated in an antigen-specific manner, the suppression is
antigen-non-specific, but depends on the cytokine TGFβ (91, 92). They inhibit the
development of immune-pathology in several animal models (90).
Hence, depending on the cytokine milieu, CD4+ T cells can differentiate into
regulatory IL-10-producing Tr1 or TGFβ-secreting Th3 cells, thereby representing
adaptive Treg-populations. The differentiation to Tr1 or Th3 cells probably depends
on natural CD4+CD25+ Tregs (93). A descriptive summary of thymically generated
and peripherally generated adaptive Tregs is given in Fig. 1.9.
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Fig. 1.9: Development of different subsets of regulatory T cells [from: (55)]
1.5 Gender-specific differences in autoimmunity
Interestingly, females have a higher incidence compared to males to develop
autoimmune diseases such as rheumatoid arthritis (RA), myasthenia gravis,
multiple sclerosis (MS), systemic lupus erythematosus (SLE), or autoimmune
hepatitis (AIH; [94]). This disparity also exists in autoimmune disease models (95).
Apart from inherent genetic susceptibility, several animal models suggest a role for
sex steroids. In more detail, females have been found to display heightened
immune reactions including a more pronounced B-cell-mediated immunity, higher
Ig levels, more vigorous T cell activation or a faster skin allograft rejection.
Consequently, gender differences in cytokine production have been observed with
increased Th1 cytokine release in females after challenge with an infectious agent
or antigen, except during pregnancy when a Th2 environment predominates (96,
97). Hence, it is postulated that gender and sex hormones have an effect on
INTRODUCTION____________________________________________________
26
various autoimmune responses, but the mechanisms of action are still unknown.
Most attention has been directed toward sex steroids. However, it has been shown
that the effect of estrogens on immune responses and in autoimmune diseases
was contradictory, since lower levels enhance whereas higher levels inhibit
immunological activities (97). Sex hormones, both androgens and estrogens,
influence the onset and severity of immune-mediated pathologic conditions by
modulating lymphocytes at all stages of life (98). For example, fluctuating
lymphocyte responses are observed during normal menses, pregnancy, and the
use of oral contraceptives (99). Indeed, differences of MS and SLE disease activity
and severity during pregnancy suggest a modulation of autoimmunity by sex
hormones (100). Interestingly, MS is triggered by a Th1 driven immune response
directed against autoantigens in the central nervous system and joints,
respectively. In contrast, pregnancy and SLE favour a Th2 environment. Sex
hormones (such as progesterone) that promote the development of a Th2
response antagonize the emergence of Th1 cells. This might be an explanation
why in MS symptoms improve during pregnancy, whereas in lupus, they do not
((97]; Fig. 1.10).
Fig. 1.10: Hormonal influences on cytokine secretion of Th1 and Th2 cells [from: (97)]
Noteworthy, pregnancy constitutes a major challenge to the maternal immune
system, since on the one hand the paternal alloantigens have to be tolerated and
on the other hand defence mechanisms against pathogens have to be maintained
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27
contemporaneously. Thus, pregnancy and the menstrual cyclus might affect the
severity of autoimmune disease (99). In fact, immunoregulatory T cells appear to
be most sensitive to sex hormone action and concentration among lymphoid cells
(101-103). Investigation of Treg numbers during the menstrual cycle revealed
significant changes in the different menstrual phases with an expansion of
CD4+CD25+FoxP3+ Tregs in the late follicular phase and a dramatic decrease in the
luteal phase (103).
In summary, it is hypothesized that androgens as well as estrogens give rise to an
anti-inflammatory cytokine profile thereby suppressing Th1-driven autoimmune
pathologies (104, 105), e. g. during pregnancy (100, 106), whereas reduced
hormone levels correlate with exacerbations of the disease. Indeed, androgens
promote oral tolerance induction (107) and estrogens have been shown to expand
the regulatory T cell compartment and to enhance their function (108, 109).
Hence, it seems that sex hormones from both genders have similar effects on
immunoregulation. However, studies with male and female mice revealed that
female mice are more prone to develop chronic relapsing-remitting disease in
response to immunization with myelin basic protein (110). This strongly suggests
that females may suffer from defects in immunoregulation though a direct
regulation by hormones seems to be excluded. Indeed, it has been shown recently
that CD4+CD25+ regulatory T cells contribute to gender differences in susceptibility
to experimental autoimmune disease (102). In conclusion, in comparison to males,
females are not only more sensitive to inflammation accompanied by increased
pro-inflammatory cytokine production, a phenomenon which is also present in the
model of Con A hepatitis used in this study (111), but also seem to possess
different mechanisms of adaptive tolerance, which can be broken more easily e. g.
during autoimmune processes.
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1.6 Aims of this study
Injection of the plant lectin concanavalin A induces pronounced T and NKT cell
activation followed by the onset of an acute liver injury in mice. Con A-induced
hepatitis has often been described as a murine model of immune-mediated
hepatitis in humans (22). Interestingly, it has been shown that Con A-pretreated
mice developed tolerance against Con A rechallenge within eight days manifested
by significantly decreased plasma ALT and AST levels.
In the first part of the study, the cytokine profile during Con A tolerance was
analysed in liver tissue and plasma measured by quantitative real-time RT-PCR
and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, the
study was intended to identify the mode of action and role of the
immunosuppressive IL-10, since it was upregulated in Con A tolerized mice.
Therefore, experiments with IL-10-/- mice and anti-IL-10-receptor mAb were
performed. Additionally, the tolerogenic, IL-10-producing cells had to be assessed
by depletion experiments.
Moreover, up to now studies regarding Con A-mediated immune-pathology and
tolerance were carried out predominantly in male mice. Since gender differences
with respect to regulation of autoimmune disease by CD4+CD25+ Tregs have been
described recently (102), induction of Con A hepatitis and tolerance was also
studied in female animals.
In the second part, development of Con A-mediated tolerance was established in
time course experiments, since the question arises, whether tolerance can also be
induced at other points of time than day eight. In this context, the intrahepatic
composition was analysed: the modification of the frequency of liver-resident
lymphocyte subsets after Con A challenge was evaluated by FACS analysis in
time kinetics. Furthermore, secondary lymphoid organs such as spleen and lymph
nodes (portal) were also included into the study design.
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In the third part, special characteristics and possible therapeutic applications of
Tregs isolated from tolerized and non-tolerized mice were compared in vivo and in
vitro. Firstly, expression of surface markers on Tregs had to be identified by FACS
analysis; secondly, cytokine response and a potential suppressive capacity of
CD4+CD25+FoxP3+ T cells had to be determined by co-culturing with responder
cells; finally, a possible therapeutic approach triggered by adoptively transferred
regulatory T cells prior to Con A administration had to be explored in vivo. Positive
effects of regulatory T cells had to be disclosed with respect to reduced liver
damage represented by decreased plasma ALT levels. Hence, Con A tolerance
appears to be an appropriate model for evaluation of therapeutic intervention
strategies in complex immunoregulatory system.
Lastly, the role of the recently identified pro-inflammatory cytokine IL-17 produced
by Th17 cells was investigated. Until now, CD4+ effector T cells have been
categorized into two subsets: T helper type 1 (Th1) with IFNγ and TNFα secretion
and T helper type 2 (Th2) with IL-4, IL-5, and IL-13 release (112). However,
another subset of T cells which produces IL-17 has been identified: Th17 cells.
Induced Th17 cells with specificity for self-antigens are highly pathogenic and lead
to the development of autoimmune diseases such as multiple sclerosis (MS) or
rheumatoid arthritis (RA; [113]). Hence, it might be interesting to investigate the
role of the pro-inflammatory IL-17 in contrast to the anti-inflammatory IL-10 in the
murine immune-mediated model of Con A hepatitis and during Con A tolerance.
The mechanisms of a potential immunosuppressive role and possible involvement
of regulatory cell types have not been elucidated so far in this mouse model of
immune-mediated liver injury and is the main matter in this study.
MATERIALS AND METHODS_________________________________________
30
2 MATERIALS AND METHODS
2.1 Mice
For this study female and male C57BL/6 wild-type, IL10-/- (114), Rag-/- (115, 116),
CD1d-/- (117), or hCD2-∆kTβRII mice (6-10 wk old) weighing 20 to 25 g were used.
Transgenic mice expressing a dominant-negative TGFβ type II receptor in T cells
under the control of the human CD2 promoter/locus control region (hCD2-∆kTβRII)
were a gift from Christoph Schramm, Hamburg, and Manfred Blessing, Leipzig,
Germany (118). C57BL/6 wt mice were obtained from the internal animal facilities
of the Institute of Experimental and Clinical Pharmacology and Toxicology,
University of Erlangen-Nuremberg or were purchased from Charles River
Laboratories (Sulzfeld, Germany). IL10-/- and Rag-/- were obtained from Janvier, Le
Genest-St-Isle, France, or Jackson Laboratory, Bar Habor, ME, USA; CD1d-/- mice
(C57BL/6 background) were a gift from Luc van Kaer, Department of Microbiology
and Immunology, Vanderbilt University School of Medicine (Nashville, TN, USA).
Animals received humane care according to the criteria outlined in the “Guide for
the Care and Use of Laboratory Animals" prepared by the US Academy of
Sciences and published by the National Institutes of Health. All mice also received
humane care according to the guidelines of the National Institute of Health and
legal requirements in Germany. Animals were maintained under controlled
conditions (22°C, 55% humidity, 12-hour day/night rhythm) and fed standard
laboratory chow.
MATERIALS AND METHODS
31
2.2 Animal treatment
2.2.1 Treatment schedules and Con A administration
The murine model of Con A-induced liver injury was used in the present study. T
cell-dependent liver damage was induced by concanavalin A (Sigma-Aldrich,
Taufkirchen, Germany) which was administered intravenously in pyrogen-free
saline. Mice received a sublethal dose of 20 mg/kg (wt and TGFβRII transgenic
mice), 25 mg/kg (CD1d-/- mice) or 12 mg/kg (IL10-/- mice) in a total volume of 100
µL/10 g mouse, respectively. Control mice were injected with saline. Animals were
restimulated with Con A on day 3, 8, 14 or 42, respectively.
2.2.2 Depletion of cells (KCs and CD25+ Tregs)
For depletion of Kupffer cells (KCs), 100 µL of liposome-encapsulated
dichloromethylene-biphosphonate (Cl2MPB, clodronate liposomes; kindly provided
by Dr. van Rooijen, Vrije Universiteit, Amsterdam, The Netherlands) were injected
intravenously 48 hours before Con A rechallenge. Dichloromethylene-
biphosphonate for their preparation itself was a gift of Roche Diagnostics
(Mannheim, Germany). As a control, mice were injected with liposome-
encapsulated phosphate-buffered saline.
In vivo depletion of CD4+CD25+ Tregs was achieved by intraperitoneal injection of
250 µg anti-CD25 mAb (clone PC61.5) or isotype-control rat IgG 24 hours before
Con A restimulation. The anti-CD25 mAb was prepared by our own working group
using the hybridoma cell line PC61.5, which was kindly provided by the
Department of Dermatology, University Hospital of Erlangen, Germany. The
efficiency of depletion was verified by FACS analysis of splenocytes using PE-
labelled anti-CD25 mAb 7D4 (Miltenyi Biotec, Bergisch Gladbach, Germany;
dilution 1:200) recognizing a different epitope than PC61.5. The detailed
procedure of flow cytometric analysis is explained in chapter 2.9.
MATERIALS AND METHODS_________________________________________
32
2.2.3 Blockade of the IL10-receptor
To block IL-10 responses, 500 µg anti-IL10-receptor mAb (DNAX/Schering-Plough
Biopharma, Palo Alto, CA, USA) were injected intravenously per mouse one hour
prior to Con A pretreatment resembling IL10-/- mice and one hour prior to Con A
restimulation, respectively.
2.3 Sampling of material
Mice were anesthetized lethally (150 mg/kg i.v. methohexital + 15 mg/kg heparin)
8 hours after Con A injection. In time course experiments, further time points were
chosen such as 3 hours and 6 hours after Con A challenge. After opening the
abdomen, cardiac blood was withdrawn for plasma cytokine determination and
analysis of plasma transaminases. The liver was removed and small liver samples
were frozen in liquid nitrogen for RNA isolation and subsequent RT-PCR, a
second part was embedded in GSV-1 tissue-embedding medium (Slee Technik
GmbH, Mainz, Germany) and frozen at –75°C for preparation of liver sections,
immunofluorescent stainings and confocal laser imaging. For preparation of
leukocytes and subsequent T and NKT cell enrichment complete fresh livers were
used. Complete fresh spleens and portal lymph nodes were also removed and
stored in Hanks balanced salt solution (HBSS: 5.4 mM KCl; 0.3 mM Na2HPO4 x 7
H2O; 0.4 mM KH2PO4; 4.2 mM NaHCO3; 1.3 mM CaCl2; 0.5 mM MgCl2 x 6 H2O;
0.6 mM MgSO4 x 7 H2O; 137 mM NaCl; 5.6 mM D-glucose; pH 7.4; all chemicals
were purchased from Carl Roth GmbH, Karlsruhe, Germany) for subsequent
isolation of splenocytes or lymphnodal cells.
MATERIALS AND METHODS
33
2.4 Isolation of cells
2.4.1 Isolation of primary hepatocytes
For isolation of hepatocytes, the two-step collagenase perfusion method of Selgen
(119) modified by our own working group was used. Mice were anesthetized by
i.p. injection of methohexital and in situ hepatectomy was performed as follows:
The abdomen was opened. The hepatic portal vein was cannulated and the liver
was perfused for 5 min with pre-perfusion medium modified by our working group
(5.36 mM KCl; 0.44 mM KH2PO4; 4.17 mM NaHCO3; 138 mM NaCl; 0.38 mM
Na2HPO4; 5 mM glucose; 0.5 mM EGTA; 50 mM Hepes; pH 7.35; all chemicals
were purchased from Carl Roth GmbH). Thereafter, perfusion was carried out for
20min with 150 mL perfusion medium (5.36 mM KCl; 0.77 mM MgSO4; 0.34 mM
Na2HPO4; 0.94 mM MgCl; 138 mM NaCl; 0.44 mM KH2PO4; 10 mM glucose; 2 mM
CaCl; 10 mM Hepes; 100 U/L penicillin; 100 U/L streptomycin [Carl Roth GmbH,
Karlsruhe, Germany]; 20% BSA [purchased from Serva, Heidelberg, Germany])
containing 0.04 mg/mL Liberase (Roche, Mannheim, Germany) digesting the liver.
The liver was removed und minced carefully in a dish with 25 mL Liberase-
perfusion medium. Cells were further individualized by gentle pipetting. The cell
suspension was filtered through a 100 µm-nylon mesh (Becton Dickinson GmbH,
Heidelberg, Germany), and filled up to 50 mL. After a 20 min precipitating period at
room temperature, 25 mL of the supernatant were removed; the remaining 25 mL
were gently agitated and layered on a 90% Percoll density solution (GE
Healthcare, Munich, Germany). After a centrifugation step at 50 x g for 10 min at
4°C, supernatant was discarded and the pellet was washed two times with
Williams E medium (Invitrogen, Gibco Cell Culture Products, Karlsruhe, Germany),
for 3 min at 50 x g at 4°C. Finally, the hepatocytes were suspended in FACS buffer
containing 1% BSA (Serva) and 0.05% NaN3 (Carl Roth GmbH) in phosphate
buffered saline (PBS) for subsequent flow cytometric analysis.
MATERIALS AND METHODS_________________________________________
34
2.4.2 Isolation of intrahepatic mononuclear cells and splenocytes
Hepatic leukocytes were isolated as described previously by Liu and co-workers
(120). Briefly, livers were pressed through 100 µm nylon meshes (Becton
Dickinson GmbH) in HBSS and centrifuged for 5 min at 500 x g. The cell pellet
was resuspended in isotonic 36% Percoll/HBSS (Percoll; GE Healthcare) solution
containing 100 U/L heparin, vortexed vigorously and centrifuged at 800 x g for 20
min. Thereafter, the cell pellet was resuspended in red blood cell lysis solution
containing 139 mM NH4Cl and 19 mM Tris (Carl Roth GmbH), incubated for 10
min at room temperature and centrifuged for 5 min at 500 x g. After a final washing
step with HBSS containing fetal calf serum (FCS; Invitrogen, Gibco Cell Culture
Products), the cell pellet was resuspended in cold FACS buffer for flow cytometric
analysis or medium for subsequent cultivation, respectively.
Single cell suspensions were prepared from spleens and lymph nodes by pressing
the organs through 100 µm nylon meshes in HBSS. After centrifugation for 5 min
at 500 x g, the pellet was subjected to red blood cell lysis, washed twice in HBSS
and resuspended in FACS buffer, HBSS, or RPMI medium (Invitrogen, Gibco Cell
Culture Products), accordingly to the following procedure.
2.4.3 Isolation of CD4+CD25+ Tregs and responder cells
To isolate CD4+CD25+ Tregs, a combined sorting procedure was carried out using
magnetic-bead separation (MACS, CD4+CD25+ Regulatory T-Cell-Isolation Kit,
mouse; Miltenyi Biotec) and FACS sorting. Briefly, untouched CD4+ T cells were
enriched using a biotinylated antibody cocktail depleting all other blood-cell types
and anti-biotin microbeads. CD4+CD25+ T cells were isolated by positive selection
using PE-labelled anti-CD25 mAb (clone 7D4) and anti-PE microbeads (Miltenyi
Biotec). Purity was controlled by flow-cytometry and reached ~85%. Subsequently,
splenic CD4+CD25+ Tregs and CD4+CD25- responder cells or liver-derived
CD3+CD25-NK1.1- T cells were purified to ~98% by FACS-sorting using a MoFlo™
Cellsorter (Dako Cytomation; Freiburg, Germany). For this purpose, untouched
splenic CD4+ responder cells and CD4+CD25+ Tregs which were pre-isolated by
MACS columns and still labelled with anti-CD25-PE mAb (clone 7D4; Miltenyi
MATERIALS AND METHODS
35
Biotec), were additionally stained with anti-CD4-Tricolor mAb (clone RM4-5;
dilution 1:200; Caltag-Laboratories, Hamburg, Germany). Afterwards,
contaminating cells (~10-15%) were eliminated in the respective sample by FACS-
sorting resulting in high responder- and Treg-purity of ~98%. Furthermore, hepatic
responder cells characterized as CD3+CD25-/NK1.1- were also purified to high
grade, since CD25+ and NK1.1+ cells were depleted to guarantee no
contamination of the responder cell pool with any known regulatory or suppressor-
cell type. Briefly, intrahepatic mononuclear cells isolated by Percoll density
gradient (GE Healthcare; see chapter 2.4.2) were labelled with anti--CD3ε-
Cychrome (clone 145-2C11; dilution 1:200; BD Pharmingen), anti-NK1.1-FITC
mAb (clone PK136; diluted 1:100; BD Pharmingen, Heidelberg, Germany), and
anti-CD25-PE mAb (clone 7D4; dilution 1:200; Miltenyi Biotec). Subsequently,
liver-derived responder cells were sorted using a MoFlo™ Cellsorter (Dako
Cytomation) and by positioning the gate on CD3+CD25-/NK1.1- cells.
Interestingly, it could be demonstrated that a further stimulus given to MACS-
isolated, but untouched splenic CD4+ T cells by an anti-CD4 mAb (clone RM4-5;
dilution 1:200; Caltag-Laboratories) increased the suppressive capacity of Tregs in
the same manner as the above mentioned combined sorting procedure with
MACS and subsequent FACS sorting.
To further characterize the isolated Treg population, FoxP3 expression was
checked by intracellular FoxP3 staining (clone FJK-16s; dilution 1:100;
ebiosience/Natutec, Frankfurt, Germany) and reached ~93% for Tregs from both
tolerized and non-tolerized mice, respectively.
Additional and helpful information regarding the procedure of flow cytometry is
noted down in chapter 2.9.
MATERIALS AND METHODS_________________________________________
36
2.5 In vitro experiments
2.5.1 Co-culture of responder cells and Tregs
1 x 105 splenic responder cells (CD4+CD25-) or CD25/NKT-cell-depleted (protocol
of FACS-sorted depletion see chapter 2.4.3) hepatic lymphocytes were cultured
alone or with 1 x 105 CD4+CD25+ Tregs from tolerized or control animals for 72
hours in 96-well round-bottom plates (Nunc GmbH & Co. KG, Thermo Fisher
Scientific, Wiesbaden, Germany), in presence of either Con A (5 µg/mL; Sigma-
Aldrich) or immobilized anti-CD3 mAb (5 µg/mL; clone 145-2C11, Immunotools,
Friesoythe, Germany). Cytokine concentrations in supernatant were measured by
ELISA.
To check the general and well-known ability of Treg-mediated suppression of T cell-
proliferation, Tregs and CFSE-labelled responder cells were mixed at different ratios
ranging from 1:1 to 1:10. Finally, co-cultures were stimulated with the strong agent
TPA (25 ng/mL)/Ionomycin (1 µM; Sigma-Aldrich; see chapter 2.5.3 for further
information).
2.5.2 Specific inhibition of cAMP by a selective PKA inhibitor
To estimate the role of cAMP and ‘infectious tolerance’ in Treg-mediated
suppression, sorted CD4+ wt responder cells were preincubated with 1 mM Rp-
cAMPS (Calbiochem, Darmstadt, Germany), a specific inhibitor of protein kinase A
(PKA), for 30 min. Control responder cells were preincubated with the solvent of
Rp-cAMPS. After washing, responder cells were cultured solely or in co-culture
with wt Tregs and stimulated with 5 µg/mL anti-CD3 mAb (clone 145-2C11,
Immunotools) for 3 days. Total RNA was prepared from the sorted CD4+ T cells
and quantitative real-time RT-PCR for quantification of IL-2, FoxP3 and ICER
mRNA expression was performed.
MATERIALS AND METHODS
37
2.5.3 CFSE labelling
To investigate the proliferation status of CD4+CD25- responder cells, they were
labelled with carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) using
“Molecular Probes Vybrant CFDA-SE Cell-Tracer Kit” (Invitrogen) and cultured
alone or together with Tregs in 96-well round-bottom plates (Nunc GmbH & Co. KG,
Thermo Fisher Scientific) for 3 days under different stimulation conditions such as
Con A (5 µg/mL; Sigma-Aldrich), anti-CD3 mAb (5 µg/mL; clone 145-2C11,
Immunotools) or TPA (25 ng/mL)/Ionomycin (1 µM; Sigma-Aldrich). CD4+CD25-
responder cells were diluted to 2 x 107 cells/mL in PBS and labelled with a CFSE
working solution of 2.5 µM for 15 min at room temperature. To quench unbound
CFSE, FCS (Invitrogen) was added to the assay. The cells were washed with ice-
cold PBS two times. Proliferation (reflected by successive diminution of
fluorescence-intensities by dye-distribution to daughter cells) was measured by
flow-cytometry.
2.5.4 Neutralization of IL-10
Co-culture experiments were performed with responder cells and Tregs as
mentioned above. The effect of IL-10 was investigated by neutralization of IL-10
with an anti-IL-10 mAb. Immediately, the antibody was added to the culture in
soluble form in a concentration of 20 µg/mL (clone JES5-2A5, Serotec, Dusseldorf,
Germany). To further check the participation of IL-10 regarding the suppressive
capacity of Tregs in vitro, experiments with wt responder cells co-cultured with Tregs
from IL10-/- mice were performed.
MATERIALS AND METHODS_________________________________________
38
2.6 Analysis of plasma transaminases
Liver injury was quantified by automated measurement of plasma-activities of
alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) 8 hours
after Con A administration according to Bergmeyer (121) using reagents from
Roche diagnostics and a COBAS Mira System (Roche).
2.7 Real time RT- PCR
Total RNA was isolated from liver tissue using the NucleoSpin RNA II Isolation Kit
(Macherey-Nagel, Düren, Germany) or from sorted CD4+ lymphocytes with
TRIZOL (Invitrogen) according to the manufacturer’s protocol. One µg of total RNA
was transcribed using SuperScript™ II RnaseH– reverse transcriptase,
oligonucleotides and oligo(dT) primers from Invitrogen. Real-time RT-PCR was
performed using a LightCycler™ system and LightCycler™-FastStart DNA-Master
SYBR-Green-1 mix (Roche) or Absolute™QPCR SYBR Green mix (Abgene,
Thermo Fisher Scientific, Hamburg, Germany). Primer-pairs were ordered from
Eurogentec (Cologne, Germany) and used as listed in table I.
Reactions were performed in a 10 µL volume. To confirm amplification specificity,
melting curves of PCR products were analyzed. Relative mRNA levels were
calculated by means of 2∆CP (∆CP=difference of crossing points of test samples
and respective control samples as extracted from amplification curves by the
LightCycler™ software) after normalization to reference β-actin levels.
Quantification is reported as the x-fold differences relative to a calibrator cDNA
from the respective control mice.
MATERIALS AND METHODS
39
Table I: List of used primer pairs
2.8 Cytokine determination by enzyme-linked immunosorbent
assay (ELISA)
Sandwich ELISAs for murine plasma TNFα, IFNγ, IL-2, IL-6, IL-10, and IL-17 were
performed using Nunc-Immuno 96-well flat-bottom high-binding Maxisorb™-
polystyrene microtiter plates (Nunc GmbH & Co. KG, Thermo Fisher Scientific).
Abs were purchased from BD Pharmingen (Heidelberg, Germany) for IL-2, IL-6,
and IL-10. IL-17, IFNγ and TNFα were quantified using DuoSet ELISA-
Development Systems (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany)
primer sequence
ββββ-actin 5’ TGG AAT CCT GTG GCA TCC ATG AAA
ββββ-actin 3’ TAA AAC GCA GCT CAG TAA CAG TCC G
TNFα α α α 5’ GAA TGG GTG TTC ATC CAT TCT
TNFα α α α 3’ ACA TTC GAG GCT CCA GTG AAT TCG
IFNγ γ γ γ 5’ GAA CGC TAC ACACTG CAT C
IFNγγγγ 3’ GAG CTC ATT GAA TGC TTG G
IL-2 5’ ATG TAC AGC ATG CAG CTC GCA TCC TGT GTC A
IL-2 3’ AGT CAA ATC CAG AAC ATG CCG CAG AGG TCC A
IL-6 5’ GCC TAT TGA AAA TTT CCT CTG
IL-6 3’ GTT TGC CGA GTA GAT CTC
IL-10 5’ GTT ACT TGG GTT GCC AAG
IL-10 3’ TTG ATC ATC ATG TAT GCT TC
IL-17 5’ TCC AGA AGG CCC TCA GAC TA
IL-17 3’ AGC ATC TTC TCG ACC CTG AA
ICER 5’ ATG GCT GTA ACT GGA GAT GAA ACT
ICER 3’ CTA ATC TGT TTT GGG AGA GCA AAT GTC
FoxP3 5’ GCA ATA GTT CCT TCC CAG AG
FoxP3 3’ TTC ATC TAC GGT CCA CAC TG
MATERIALS AND METHODS_________________________________________
40
and the TMB-Substrate Reagent Set (BD Pharmingen) according to manufacturers’
instructions. Briefly, microtiter plates were coated with diluted capture/primary Abs
and incubated over night at 4°C. After washing with a buffer containing 0.05%
Tween20 in PBS (pH 7.2-7.4), plates were blocked for at least two hours with
blocking solution (1% BSA [Serva], 0.05% NaN3 [Carl Roth GmbH] in PBS) to
avoid unspecific binding on the surface. After further washing steps, standard and
samples were applied for additional two hours. The immobilized antigens formed a
complex with the capture Ab and the added detection/secondary antibody. Finally,
addition of streptavidin-peroxidase (R&D Systems GmbH) and TMB substrate (BD
Pharmingen) resulted in a visible signal which indicates the quantity of antigen in
the sample.
2.9 Flow cytometry
Typically 4 x 105 leukocytes were stained using a standard protocol including pre-
blocking of Fc-receptors. The following mAbs were used: anti-CD16/32 (“Fc-
block”; clone FCR4G8; Serotec), FITC-labelled anti-mouse-NK1.1 (clone PK136;
dilution 1:100), anti-mouse-CD103-FITC (clone M290; dilution 1:100), anti-mouse-
CD45RB-biotin (clone 16A, dilution 1:500), CyChrome-labelled anti-mouse-CD3ε
(clone 145-2C11; dilution 1:200; all BD Pharmingen), anti-mouse CD4-Tricolor
(clone RM4-5; dilution 1:200; Caltag-Laboratories), anti-mouse-CD62L-FITC
(clone MEL-14; dilution 1:100), anti-mouse-CD4-FITC (clone YTS.1.2; dilution
1:200; both purchased from Immunotools) and anti-mouse-CD25-PE (clone 7D4;
dilution 1:200; Miltenyi Biotec).
For intracellular FoxP3 staining biotinylated anti-FoxP3 (clone FJK-16s; diluted
1:100; eBioscience/NatuTec) and streptavidin-CyChrome (diluted 1:300; BD
Pharmingen) were used together with “FoxP3-Staining Buffer Set” (FixPerm-
solution and permeabilization wash-buffer; eBioscience/Natutec) according to
manufacturers instructions. Intracellular IL-10 staining was carried out using anti-
mouse-IL10-FITC (clone JES5-2A5; diluted 1:10; Caltag-Laboratories)
MATERIALS AND METHODS
41
concomitant with FoxP3 detection. Data were recorded and analyzed using a
FACScanTM Flow Cytometer (BD Biosciences) and CellquestTM software.
2.10 Immunofluorescent staining and confocal laser
imaging
For immunohistochemistry with cryostat sections, liver samples were embedded
with GSV 1 tissue-embedding medium (Slee Technik GmbH, Mainz, Germany),
frozen in 2-methyl-butane (Carl Roth GmbH), and stored at -20°C until use.
Cryostat sections of 10 µm were thawed on glass slides, air dried, fixed for 10
minutes at 4°C in acetone/methanol (1+1; Carl Roth GmbH), and used
immediately or stored at -20°C. After washing with PBS the sections were blocked
with PBS containing 3% BSA (Serva) at room temperature for 1 hour.
Subsequently, slides were incubated with a primary Ab in PBS/3% BSA at 4°C
overnight. Macrophages were detected with a rat monoclonal antibody directed
against a murine pan-macrophage marker (clone BM8; dilution 1:100; Dianova,
Hamburg, Germany). After rinsing with PBS, binding sites were detected with a
secondary Ab (rabbit anti-rat immunoglobulin G tagged with fluorescein
isothiocyanate; dilution 1:100; Dako, Hamburg, Germany) for one hour at room
temperature. After prolonged rinsing with PBS, slides were coverslipped using
PBS/glycerol (pH 8.6; Carl Roth GmbH) and examined by confocal laser scanning
microscopy (Axiovert 100M, Carl Zeiss, Oberkochen, Germany).
2.11 Haematoxylin/eosin staining of liver sections
For histological analysis of tissue structure livers were fixed over night in 4%
formalin (Carl Roth GmbH) and subsequently embedded in paraffin. Sections were
stained with Haematoxylin/Eosin using a standard procedure and analyzed by light
microscopy with the aid of Prof. Dr. T. Papadopoulos (at that time Institute of
MATERIALS AND METHODS_________________________________________
42
Pathology, University of Erlangen-Nuremberg; now Vivantes Klinikum Spandau,
Berlin).
2.12 Analysis of hCD2-∆∆∆∆kTββββRII mice by tail biopsies
Three weeks after birth, offspring were biopsied at tail. Each biopsy was incubated
in 150 µL tail buffer containing 1 x SSC, 1 mM Tris-HCl (pH 8.0), 20 mM EDTA
(pH 8.0), 1% SDS, and proteinase K (1mg/mL; all chemicals were purchased from
Carl Roth GmbH) over night at 56°C. After centrifugation at 15 000 x g for 10 min),
supernatant was mixed gently with isopropanol (Carl Roth GmbH) to precipitate
DNA. After a further centrifugation step (15 000 x g, 15 min), supernatant was
discarded whereas the pellet was washed with 300 µL ethanol (70%, Carl Roth
GmbH). The pellet was air-dried after a final centrifugation step (15 000 x g, 5
min), dissolved in 200 µL H2O and stored at 4°C for subsequent genotyping. The
analysis for genotype was performed by PCR using an hCD2-specific primer (5’-
TTT GTA GCC AGC TTC CTT CTG -3’) and a human TGFβ type II receptor-
specific primer (5’- TGC ACT CAT CAG AGC TAC AGG- 3’). The expected gene
product consisted of 650 bp (118) and was analysed with Bio-Rad Gel Doc 2000
(Bio-Rad Laboratories GmbH, Munich, Germany).
2.13 Statistical analysis
The results were analyzed using Student’s t test, if two groups were compared or
by ANOVA followed by the Dunnett’s test if more groups were tested against a
control group. If variances were inhomogeneous in the Student’s t test, the results
were analyzed using the Welsh test. All data in this study are expressed as a
mean ± SEM. A p value of 0.05 or less was considered significant.
RESULTS
43
3 RESULTS
3.1 Characterization of Con A-induced tolerance
3.1.1 Con A pretreatment results in a reduction of serum transaminase
levels upon Con A rechallenge
A single injection of Con A induces an acute immune-mediated liver injury in mice
resembling human liver disorders like autoimmune hepatitis (AIH), alcohol-induced
hepatitis or ischemia/reperfusion injury (22). Hence, the murine model of
experimental liver injury might be appropriate to study pathophysiology of
immunologically mediated hepatic disorders. As early as 6 hours after Con A
challenge the levels of the liver-specific transaminase ALT were significantly
elevated. Peak levels of transaminase release were reached at about 8 hours
lasting for 24 hours (15). To analyze the potential of Con A-mediated immune
activation to induce a tolerogenic and immunosuppressive milieu, C57BL/6 mice
were pretreated with a sublethal Con A dose or saline as negative control. Eight
days later mice were restimulated with Con A.
In fact, Con A-pretreated mice were partially protected from liver injury in
comparison to saline-treated control mice reflected by significantly decreased
plasma ALT and AST levels measured 8 hours after Con A rechallenge (Fig. 3.1).
RESULTS_________________________________________________________
44
Fig. 3.1: Protection from Con A-induced hepatitis by a single Con A pretreatment: Con A or saline
were administered intravenously 8 days prior to Con A rechallenge. Plasma transaminase activities
were measured 8 hours after Con A restimulation. Data are expressed as the mean ± SEM (n ≥ 4; *
p ≤ 0.05 vs. saline-pretreated control).
3.1.2 Con A pretreatment ameliorates Con A-induced liver necrosis
To further confirm the amelioration of liver damage in Con A-pretreated mice, HE
staining was performed. Intravenous injection of saline into the lateral tail vein of
mice represented the negative control exhibiting an undamaged liver architecture
with accurate fenestrated sinusoids and intact binucleate hepatocytes well-
organized in plates (Fig. 3.2 A). In contrast, necrotic areas could be detected after
a single Con A challenge depicting the positive control with manifested liver
damage (Fig. 3.2 B). Additionally, inflammatory cell infiltrations could be detected
due to injection of the common T cell mitogen Con A. However, histological
staining of Con A-pretreated mice failed to display severe necrosis resembling the
negative control and correlating very well with significantly decreased ALT and
AST levels in tolerized mice. Interestingly, Con A rechallenge at day 8 led to
obvious mononuclear cell infiltration, not yet acting as inflammatory effector cells
any more (Fig. 3.2 C).
RESULTS
45
Fig. 3.2: Development of Con A tolerance within 8 days after a single Con A pretreatment: Saline-
treated (A) and Con A-treated (B) mice were used as negative and positive control, respectively. A
third group of mice was pretreated with Con A and restimulated after one week (C). Liver samples
taken 8 hours after Con A rechallenge were analyzed by HE staining. Necrotic liver damage is
indicated by black arrows, whereas monounuclear cell infiltration into liver tissue is marked by a
white arrow.
3.1.3 Induction of an anti-inflammatory cytokine profile
After injection, Con A locally activates T and NKT cells in the liver. These cells
interact with intrahepatic macrophages, i.e. Kupffer cells (KCs), which is followed
by strong production of a broad range of pro-inflammatory cytokines, including
TNFα and IFNγ mainly produced by KCs and NKT cells, respectively. This
cooperative cytokine signalling is indispensable for the onset of Con A hepatitis
(27-29). It is noteworthy, that TNFα expression is elevated especially in liver tissue
over a long period (24 hours) in comparison to plasma (1 to 4 hours; [15]).
In contrast to induction of hepatitis, protection from liver injury in Con A-pretreated
mice was associated with an anti-inflammatory cytokine profile as measured by
ELISA in plasma (Fig. 3.3 A) and RT-PCR in liver tissue (Fig. 3.3 B) 8 hours after
rechallenge, i.e. at the time point of ALT quantification.
CBA
RESULTS_________________________________________________________
46
Fig. 3.3: Induction of an anti-inflammatory cytokine profile upon Con A restimulation: Mice were
pretreated with Con A or saline and restimulated after 8 days. Both plasma and liver samples were
taken 8 hours after Con A rechallenge. Cytokine expression was determined both (A) in plasma by
ELISA, and (B) in liver tissue by quantitative real-time RT-PCR. For RT-PCR β-actin was used as
reference gene. X-fold induction was calculated referring to mRNA levels of cytokines in saline-
pretreated animals. Data are expressed as the mean ± SEM (n ≥ 5; * p ≤ 0.05 vs. saline pretreated
control).
The plasma cytokine concentrations of IFNγ, IL-2 and IL-6 (122, 123) were
significantly decreased upon Con A rechallenge. Using quantitative real-time RT-
PCR analysis the significantly diminished IL-2 and IFNγ plasma levels were
partially reflected in the liver with their intrahepatic mRNA levels being lower in
Con A-pretreated than saline control mice 8 hours after rechallenge. Interestingly,
IFNγ expression was even significantly elevated shortly (1.5 hours) after
rechallenge in Con A-pretreated mice compared to saline controls (Fig. 3.4)
indicating that lymphocytes in pretreated mice were still able to respond to Con A
rechallenge and to release cytokines. Thus, passive mechanisms such as broad-
range anergy and non-responsiveness due to Con A pretreatment could not
explain Con A-induced tolerogenic effects, rather supposing an active process of
cytokine suppression, indicated by notably reduced plasma IFNγ production in Con
RESULTS
47
A-tolerized mice 8 hours after restimulation in contrast to the early time point 1.5
hours.
Fig. 3.4: Efficient cytokine response of lymphocytes
upon Con A restimulation: Mice were pretreated with
Con A or saline and restimulated after 8 days.
Plasma samples were taken 1.5 and 8 hours after
Con A rechallenge. IFNγ production was determined
by ELISA. Data are expressed as the mean ± SEM
(n = 5; * p ≤ 0.05 vs. saline pretreated control).
Tolerization did not significantly affect plasma TNFα concentrations at the
indicated time point (Fig. 3.3 A). However, intrahepatic TNFα expression - and
also IL-6 expression - was significantly lower in Con A-tolerized than saline-
pretreated mice 8 hours after Con A rechallenge (Fig. 3.3 B). Therefore, the
expression of pro-inflammatory cytokines responsible for Con A hepatitis was
downregulated in tolerized mice. In contrast, the anti-inflammatory and
immunosuppressive cytokine IL-10 revealed significantly higher expression in Con
A-pretreated than in control mice, both systemically in the plasma (Fig. 3.3 A) and
locally in the liver (Fig. 3.3 B). These observations point to a potential role of the
immunosuppressive cytokine IL-10 in the development of Con A tolerance.
RESULTS_________________________________________________________
48
3.1.4 Determination of the frequency of cell subpopulations
To clarify the mechanism of tolerance induction, different intrahepatic cell
populations were investigated, since tolerance might be attributed to Con A-
provoked depletion of certain cell types which are absolutely essential for induction
of liver injury. Con A-induced hepatitis is an immune-mediated process that
depends on T cells, NKT cells, and KCs as important sources of IFNγ, IL-6, and
TNFα. Depletion of these cells types as a result of the first Con A stimulus would
explain the repressed cytokine response in tolerized animals. Hence, the
composition of intrahepatic cell subpopulations was investigated by FACS analysis
and immunofluorescent stainings in time course experiments and especially on
day 8, the day of tolerance induction. In fact, NKT cells which are essential for Con
A-induced hepatitis (26, 27) transiently disappeared short-termly after Con A
pretreatment both in the spleen and the liver; subsequently, the frequency of NKT
cells characterized by surface expression of CD3+ and NK1.1+ returned to normal
and even reduplicated to ~40% in the liver on day 8, shown by FACS analysis
suggesting an important role of NKT cells in the onset of Con A tolerance (Fig.
3.5).
Fig. 3.5: Short-term disappearance of NKT cells in liver and spleen is followed by an increase of
these cells: Mice were treated with Con A; subsequently, NKT cell frequency was measured by
FACS analysis in the liver and spleen on day 1, 3, 8, and 14 after Con A challenge. The blue line
represents the absolute NKT cell number relating to countable cells, whereas the bars indicate the
NKT cell fraction among lymphocytes. Data are expressed as the mean ± SEM (n ≥ 4).
RESULTS
49
There might be two reasons for the transient and early-stage disappearance of
NK1.1 expression: firstly, NKT cells might undergo apoptosis due to activation-
induced cell death supported by the fact that Fas (CD95) is upregulated on NKT
cells after Con A challenge (27), or secondly, NKT cells downregulate the NK1.1
marker due to Con A activation, hence becoming undetectable in FACS analysis
with anti-NK1.1 mAbs.
In contrast, the number of T cells also relevant for Con A-induced hepatitis was
mostly unchanged 8 days after Con A treatment demonstrated by flow cytometry
(data not shown).
The existence of liver-resident macrophages, the KCs, was also checked on day
8, since KCs are the principal TNFα-producing cells in Con A-mediated hepatitis.
Nevertheless, KCs were still apparent in Con A-pretreated mice demonstrated by
immunofluorescence staining with a murine pan-macrophage marker (clone BM8,
Fig. 3.6).
Fig. 3.6: Presence of Kupffer cells in both Con A- and saline-pretreated mice on day 8: Mice were
pretreated with either Con A or saline. For Kupffer cell detection, immunofluorescent staining was
performed 8 days later. 10 µm cryostat sections of liver tissue were stained with rat anti-mouse
macrophage mAb (clone BM8) and anti-ratIgG-FITC on glass-slides and subsequently examined
by confocal laser-scanning microscopy. The left picture depicts the negative or isotype control.
In conclusion, all three cell types essential for Con A-mediated liver damage,
namely KCs and the effector lymphocyte populations of T cells and NKT cells,
were still detectable on day 8 in saline- as well as in Con A-treated mice, thereby
RESULTS_________________________________________________________
50
excluding Con A-induced cell depletion. Together with the finding that IFNγ
production was inducible at early time points during Con A tolerance these data
again exclude passive tolerance mechanisms such as cell depletion and non-
responsiveness /anergy, rather arguing for active tolerogenic processes, which
might be mediated by suppressive and regulatory cell types and molecules.
It is well known that naturally arising CD4+CD25+FoxP3+ regulatory T cells play an
important role during tolerance induction in the periphery (see Introduction chapter
1.3 and 1.4). To address the question, whether Con A tolerance might be
associated with an expansion of local Treg populations, the frequency of Tregs was
investigated in the liver, spleen and liver-draining portal lymph-nodes. A transient
increase of CD4+CD25+FoxP3+ frequencies was detected in all three organs
(mainly 24 hours after Con A treatment), especially at the site of inflammation,
namely in the liver; however, tolerance establishment on day 8 was not associated
with increased intrahepatic Treg frequencies suggesting qualitative rather than
quantitative changes regarding the Treg population (Fig. 3.7). A slight, but not
significant upregulation of FoxP3 could be detected in the portal lymph nodes on
day 14.
Fig. 3.7: Occurrence of FoxP3+ Tregs in liver, spleen and liver-draining portal lymph-nodes after Con
A treatment: Organs were excised on day 1, 3, 8, or 14 after Con A treatment. Saline injection was
regarded as negative control. Lymphocytes were isolated and stained for CD4 and FoxP3.
Frequencies of FoxP3+ T cells among CD4
+ T cells were calculated. To enable direct comparison
of frequencies at the different time points, the relative rate of FoxP3+ Tregs from Con A-treated mice
was normalized to the saline controls at the same time point, with the latter defined as “1”. Data
are calculated as the mean ± SEM of the measured frequencies in saline controls from all time
points (n ≥ 4).
RESULTS
51
3.1.5 Investigation of the time point of tolerance induction
Tolerance with respect to inhibition of liver damage and production of inflammatory
cytokines was induced 8 days after the first Con A stimulation. The time course of
FoxP3+ Tregs infiltration into liver and liver draining portal lymph nodes raised the
question, whether tolerance could be also induced on other time points. Hence,
time course experiments were performed aiming at the identification of the
progress of tolerance.
Interestingly, liver damage upon Con A rechallenge as early as 3 days after Con A
pretreatment was even more pronounced than in control mice demonstrated on
the one hand by HE staining (Fig. 3.8 A-E) and on the other hand by increased
production of pro-inflammatory cytokines measured by ELISA (Fig. 3.9 A) and RT-
PCR (Fig. 3.9 B). HE staining clearly illustrated an aggravated liver damage in
mice restimulated on day 3, since massive necrotic areas, even extended to
bridging necrosis (Fig. 3.8 C), were detectable in comparison to a moderate liver
injury after a single Con A injection (positive control, Fig. 3.8 B) and in comparison
to mice restimulated at day 8 (Fig. 3.8 D) and 14 (Fig. 3.8 E).
Fig. 3.8: Development of long-lasting Con A tolerance within one week after a single Con A
pretreatment: Saline-treated (A) and Con A-treated (B) mice were used as negative and positive
control, respectively. Moreover, mice were pretreated with Con A and restimulated after 3 (C), 8
(D), or 14 days (E). Liver samples taken 8 hours after Con A rechallenge were analyzed by HE
staining. Necrotic liver damage is indicated by black arrows, whereas cell infiltration into liver tissue
is marked by white arrows.
A B C D E
RESULTS_________________________________________________________
52
Fig. 3.9: Cytokine responses to Con A restimulation in Con A –pretreated mice: Mice were
pretreated with Con A and restimulated after 3, 8, or 14 days. To enable direct comparisons of
cytokine responses at the different time points due to normal experimental day-to-day variations,
the relative plasma cytokine levels from Con A-treated mice were normalized to saline controls of
the same time point, with the latter being defined as “1”. Intrahepatic cytokine mRNA levels were
normalized to β-actin and calculated as x-fold induction to cDNA from control mice. All data
presented as mean ± SEM.
A striking increase of IL-6, TNFα and IFNγ - the main parameters of Con A-
induced liver inflammation - was detectable in liver tissue upon restimulation 3
days after Con A pretreatment (Fig. 3.9 A). This cytokine response was also
reflected by the plasma cytokine concentrations of the pro-inflammatory mediators
IFNγ and TNFα. (Fig. 3.9 A) thereby matching the severity of liver damage as
demonstrated by HE staining (Fig. 3.8 C). In contrast, a reduced Th1 responses
and an increased IL-10 expression were detected at later time points (8 and 14
days).
RESULTS
53
It is worth mentioning that Con A-pretreated mice still developed tolerance upon
Con A restimulation after two weeks verified by an intact liver architecture as
demonstrated by HE staining (Fig. 3.8 E) and measurement of an anti-
inflammatory cytokine expression in plasma (Fig. 3.9 A) and liver tissue (Fig. 3.9
B). In a further long-time experiment, mice were restimulated even six weeks after
the first Con A challenge. Intriguingly, tolerance was fully inducible in Con A-
pretreated mice characterized by significantly reduced ALT levels (Fig. 3.10 A) and
an anti-inflammatory cytokine profile in the plasma (Fig. 3.10 B) and liver tissue
(Fig. 3.10 C): IFNγ and IL-6 production were strongly downregulated in plasma
which was partially reflected in the liver. Intrahepatic TNFα expression was
significantly repressed, whereas IL-10 was strongly induced.
Fig. 3.10: Establishment of long-lasting
tolerance: Mice were pretreated with saline
or Con A and restimulated after 6 weeks.
Plasma and liver samples were taken 8
hours after rechallenge. Transaminase
activities were measured in plasma (A).
Cytokine expression was determined (B) in
plasma by ELISA, and (C) in liver tissue by
real-time RT-PCR; β-actin was used as
reference gene (mean ± SEM, n ≥ 4; * p ≤
0.05 vs. saline pretreated control).
C
A B
RESULTS_________________________________________________________
54
Due to this time course experiment the time point ‘8 days’ was chosen for further
experimental settings analyzing characteristics of Con A tolerance, when a
tolerogenic state was already reproducibly reached.
Earlier time points than day 3 were not analyzed, since within few hours after Con
A injection NKT cells, which are essential for Con A-induced hepatitis, are well
known to transiently disappear or at least become undetectable and incapable of
being stimulated for a few days (see Fig. 3.5).
3.1.6 Induction of Con A tolerance ex vivo
To test whether the in vivo effect of reduced pro-inflammatory cytokine response in
Con A tolerized mice was reproducible in vitro, ex vivo were performed:
splenocytes from Con A- or saline -pretreated mice were isolated 8 days after pre-
treatment and restimulated ex vivo with Con A or anti-CD3 mAb. Consequently,
splenocytes from saline-pretreated mice received the first stimulus, whereas
splenocytes from Con A-treated mice received the second stimulus in vitro.
Indeed, splenocytes from Con A-treated mice responded with significantly reduced
IL-2, IFNγ and TNFα expression and conversely increased IL-10 production
reflecting definitely the cytokine profile measured in tolerized mice in vivo (Fig. 3.3
and 3.11).
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55
Fig. 3.11: Modified ex vivo cytokine responses of splenocytes from Con A-tolerized mice in contrast
to control splenocytes after in vitro restimulation: Mice were pretreated with Con A or saline.
Splenocytes from tolerized and non-tolerized animals were isolated on day 8 and restimulated ex
vivo with either Con A (5 µg/mL) or anti-CD3 mAb (5 µg/mL) for 72 hours. Cytokine concentration
was measured in supernatant by ELISA. Data are expressed as the mean ± SEM (n = 4; * p ≤ 0.05
vs. cells from saline-treated control animals).
3.2 Identification of IL-10 as central mediator of Con A tolerance
3.2.1 Loss of Con A-mediated tolerance in male IL10-/- mice and after anti-
IL10R treatment
IL-10 plays a protective role in Con A-induced immune-mediated liver injury due to
its immunosuppressive capacity (33, 34). It is well known that IL-10 inhibits the
production of pro-inflammatory cytokines like TNFα, IFNγ, and IL-6 and exerts
inhibitory action on a variety of cell types (86). Due to the increased IL-10 release
in Con A-pretreated wt mice experiments with IL-10-/- mice were performed
examining the development of tolerance in complete absence of IL-10. Indeed,
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56
Con A tolerance was totally reversed in male IL10-/- mice with respect to liver
damage indicating a critical role of IL-10 in the onset of tolerance (Fig. 3.12).
Fig. 3.12: Loss of Con A tolerance in male IL10 knock out
mice regarding pathophysiology, but not IL-2 suppression:
Con A (�) or saline (����) were injected intravenously in male
C57BL/6 wt mice and (weight and age-matched) IL10-/-
mice. Animals were restimulated with Con A after 8 days.
ALT transaminase activities and plasma cytokine
concentration were determined 8 hours later. All data are
represented as the mean ± SEM (n ≥ 4; * p ≤ 0.05 vs. saline-
pretreated control animals).
In contrast to male wt animals, Con A-pretreated IL10-/- mice developed fulminant
liver injury comparable to saline-pretreated animals as manifested by increased
ALT-, IFNγ, and IL-6-levels, i.e. by parameters of hepatocyte damage. However,
IL-2 production upon Con A challenge was largely suppressed also in Con A-
tolerized IL-10-/- mice suggesting an IL-10 independent suppression of IL-2
production.
To further elucidate the role and mode of action of IL-10 during development of
Con A tolerance, experiments with monoclonal antibodies directed against the IL-
10 receptor, specifically blocking the binding site of IL-10, were performed.
Injection of anti-IL10R mAb one hour prior to Con A pretreatment (→ 1. stimulus)
imitating IL-10-/- mice confirmed the importance and participation of the anti-
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57
inflammatory IL-10 during differentiation processes and establishment of Con A
tolerance. In contrast to significant attenuation of liver damage in tolerized control
animals, ALT levels were comparable in Con A-pretreated and saline-pretreated
mice upon administration of anti-IL-10R mAb (Fig. 3.13).
Fig. 3.13: Reversal of Con A tolerance with respect to liver damage after treatment with anti-IL10R
mAb. C57BL/6 wt mice were pretreated with Con A or saline 8 days before Con A rechallenge.
Anti-IL10R mAb was injected (500 µg/mouse; i.v.) either 1 hour prior to Con A/saline pretreatment
(1. stimulus) or 1 hour prior to Con A restimulation (2. stimulus). Measurement of ALT was
performed 8 hours after Con A rechallenge. Data are expressed as mean values ± SEM (n = 4; *, p
≤ 0.05 vs. saline-pretreated control).
With respect to cytokine release, IFNγ and IL-6 expression – representing
parameters of aggravated liver damage – were not diminished in antibody-/Con A-
pretreated mice in comparison to saline-pretreated mice measured both in plasma
by ELISA (Fig. 3.14 A) and in liver tissue by RT-PCR (Fig. 3.14 B).
RESULTS_________________________________________________________
58
Fig. 3.14: Detection of a pro-inflammatory Th1 cytokine response in anti-IL10R-treated animals.
Con A or saline were injected intravenously to C57BL/6 wt mice. Half of the animals were injected
with anti-IL10R antibody, either prior to Con A/saline pretreatment (1. stimulus) or prior to Con A
restimulation (2. stimulus). Cytokine expression in anti-IL10R-treated vs. control-treated and Con
A- vs. saline-pretreated mice was measured A) in plasma by ELISA, or B) from total liver RNA by
quantitative real-time RT-PCR 8 hours after Con A rechallenge. For RT-PCR analysis β-actin
mRNA was used as an internal standard to normalize for equal levels of total RNA. x-fold induction
was calculated referring to mRNA levels of the respective cytokines in saline-pretreated control
animals (mean ± SEM; n = 4; *, p ≤ 0.05 vs. saline-pretreated control).
Moreover, administration of the anti-IL-10R mAb one hour prior to the first Con A
challenge resulted in a strong upregulation of intrahepatic TNFα expression both
in saline- and Con A-pretreated animals clearly pointing to loss of tolerance
RESULTS
59
induction after blocking the IL-10 binding site and confirming the results in IL10-/-
mice (Fig. 3.12).
In a parallel experiment, the temporary effect of IL-10 was inhibited on day 8,
namely by injection of anti-IL10R mAb one hour prior to the second Con A
challenge. Again, the results show significant reduction of Con A-mediated
tolerance as antibody/ Con A-pretreated mice displayed elevated ALT values,
even dramatically pronounced in comparison to saline-pretreated animals (Fig.
3.13). In addition, IFNγ, TNFα, and IL-6 levels in both plasma (Fig. 3.14 A) and
liver tissue (Fig. 3.14 B) were enhanced in Con A-pretreated mice as in saline-
pretreated animals suggesting that IL-10 might not only participate in long-term
differentiation processes, but also acts as short-term protective and
immunosuppressive mediator in vivo. Hence, a critical role of IL-10 in the onset of
Con A tolerance was demonstrated by reversal of Con A tolerance regarding
suppression of hepatocyte damage, IFNγ- and IL-6-production in IL-10-/- mice as
well as by blocking the binding site of IL-10.
In contrast, IL-2 production upon Con A challenge was largely suppressed also in
Con A-tolerized IL-10-/- (Fig. 3.11) and anti-IL-10R-treated mice (Fig. 3.14 A)
indicating that IL-2 impairment strictly works in an IL-10-independent manner.
Tolerization-induced IL-2-downmodulation might be caused by induction of non-
responsiveness in IL-2-secreting cells or increased consumption. This clearly
shows that IL-2 diminution, which is often used as main indicator for
immunoregulation, is obviously not related to the pathophysiology in this model.
The tolerization-induced raise of IL-10 production was still detectable in plasma
(Fig. 3.14 A) and liver (Fig. 3.14 B) of the antibody/Con A-pretreated groups,
however, to a lesser extent than in the tolerant controls.
RESULTS_________________________________________________________
60
3.2.2 Detection of gender-related differences in IL10-/- mice
Gender often plays an important factor regarding the onset of autoimmune
diseases, since females show a higher incidence to develop Th1-related
autoimmune diseases. Hence, female mice were included in the experimental
design. Female and male wt and IL-10-/- mice were pretreated with saline and Con
A, respectively, and restimulated with Con A 8 days later. Both female and male wt
mice developed Con A-mediated tolerance showing attenuated liver damage with
decreased plasma ALT levels and an anti-inflammatory cytokine profile
characterized by downregulated IL-6 and IFNγ expression (Fig. 3.15) and
increased IL-10 production.
Fig. 3.15: Gender-related differences of Con A
tolerance in IL10 KO mice. To further confirm the
importance of IL-10 during Con A tolerance, Con A or
saline were injected intravenously into both female and
male wt or (weight and age-matched) IL10-/-
mice 8
days prior to Con A restimulation. Liver injury was
quantified by measuring plasma transaminase activity
and cytokine expression 8 hours after Con A
rechallenge. Data are represented as the mean ± SEM
(n ≥ 4; *, p ≤ 0,05 vs. saline-pretreated control).
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61
Interestingly, female wt mice developed tolerance despite a notably higher basic
liver damage which has already been observed in other studies (111).
Surprisingly, tolerance, as denoted by reduced ALT and pro-inflammatory cytokine
levels, was still inducible in female IL-10-/- animals, although they developed more
severe liver damage due to the lack of IL-10 which plays a protective role in this
model of immune-mediated hepatitis. In contrast, male IL10-/- mice, showing even
enhanced IFNγ levels and high IL-6 release upon Con A restimulation, failed to
establish tolerance (Fig. 3.15). These results suggest gender-related differences
regarding mechanisms and onset of peripheral tolerance.
Again, IL-2 production was still suppressed in Con A-restimulated female and male
IL10-/- mice, confirming an IL-10-independent suppression of IL-2 release which
might have been mediated by induction of partial non-responsiveness in IL-2-
secreting cells or higher IL-2 consumption.
3.3 Importance of Kupffer cells as IL-10-producing cells
Since IL-10 was identified as an important mediator of Con A tolerance, the
question regarding the IL-10-producing cell population arose.
Kupffer cells, the liver-resident macrophages, represent a cell population able to
produce significant amounts of IL-10 and IL-6 (124). Interestingly, IL-6 production
by KCs and LSECs being suppressed by high IL-10-concentrations (84). This
profile markedly resembles the anti-inflammatory cytokine response found during
Con A tolerance. To investigate a possible participation of KCs in producing IL-10,
KCs were depleted by clodronate-liposomes 48 hours prior to Con A rechallenge.
Successful KC depletion was verified by staining of cryostat liver sections with the
BM8 mAb as described before. In KC-depleted, Con A-pretreated mice the relative
tolerization-induced IL-10 augmentation was reduced in plasma (Fig. 3.16 A) and
particularly impaired regarding intrahepatic IL-10 mRNA levels measured by
quantitative real-time RT-PCR (Fig. 3.16 B). This clearly indicates that KCs
contribute to IL-10 production in Con A tolerance, although a additional cell
population must be involved in tolerization-induced IL-10 upregulation.
RESULTS_________________________________________________________
62
Fig. 3.16: Important role of Kupffer cells for IL-10
production upon Con A tolerization. Prior to Con A
rechallenge both saline-pretreated and Con A-
pretreated mice were either mock-treated or
subjected to KC depletion by injection of clodronate
liposomes. The tolerization-induced IL-10 boost was
measured both in the plasma by ELISA (A) and in
liver tissue via real-time RT-PCR (B). All data are
presented as the mean ± SEM (n ≥ 3; *, p ≤ 0.05).
3.4 Involvement of CD4+CD25+ regulatory T cells during Con A
tolerance
3.4.1 Identification of Tregs as source of IL-10
Since KC-depletion still led to an increased IL-10 release in Con A-pretreated
animals, albeit with significantly diminished expression, another source of IL-10
beside KCs had to be identified. CD4+CD25+Foxp3+ naturally arising Tregs and
especially induced Tr1 cells are well known to produce the anti-inflammatory
cytokine IL-10. To investigate the potential role of CD4+CD25+ Tregs in Con A
tolerance in vivo, these cells were depleted by injection of anti-CD25 mAb (clone
PC61.5) 24 hours prior to Con A rechallenge. The efficiency of this depletion (>
A
B
RESULTS
63
95%) was verified by FACS analysis of the splenic Treg population using anti-CD25
mAb (clone 7D4) recognizing a different epitope than PC61.5 (Fig. 3.17).
Fig. 3.17: Efficient depletion of CD25+ T cells after injection of anti-CD25 mAb: Con A or saline
were injected intravenously into mice 8 days prior to Con A restimulation. Twenty-four hours before
Con A rechallenge half of the Con A- or saline-pretreated mice were injected with anti-CD25 mAb
PC61.5 to deplete CD25-positive Tregs. Efficient depletion of CD25+ Tregs was verified by FACS
analysis of splenocytes. Cells were gated on viably lymphocytes by their light-scatter
characteristics and on CD4-positive cells. Mean ± SEM of percentages of CD4+CD25
+ Tregs within
the CD4+ T-cell population is depicted.
It is often criticized that depletion by anti-CD25 mAbs affects activated effector T
cells; however, anti-CD25-treatment did not influence effector T cells that had
been activated by the first Con A stimulus, since the transient activation-induced
CD25 upregulation expires within ~3-5 days (own observations and demonstrated
for rats [25]).
Firstly, CD25-positive Treg depletion caused lower IL-2 suppression factors,
suggesting that CD25+ Treg cells were involved in Con A-induced, IL-10-
independent IL-2 suppression in vivo (Fig. 3.18) beside an induction of partial non-
responsiveness in IL-2 producing T cells. Secondly, plasma IL-2 concentrations
were generally higher in both saline- and Con A-pretreated mice upon CD25-
depletion, since CD25 represents the IL-2 receptor alpha chain (IL2Rα). Hence,
more unbound IL-2 was detectable in the plasma of CD25-depleted mice.
RESULTS_________________________________________________________
64
Fig. 3.18: Involvement of Tregs in IL-2 suppression in Con A-tolerized mice. Con A or saline were
injected intravenously into mice 8 days prior to Con A restimulation. 24 hours before Con A
rechallenge half of the Con A- or saline-pretreated mice were injected with anti-CD25 mAb PC61.5
to deplete CD25-positive Tregs. IL-2 concentrations were measured in plasma of Con A tolerized
mice (�) and saline-pretreated mice (����) 8 hours after Con A rechallenge (mean ± SEM; n ≥ 4; *, p
≤ 0.05). The suppression factor describes the IL-2 cytokine values of saline-pretreated mice divided
by those of Con A-pretreated mice.
Finally, depletion of CD4+CD25+ Tregs caused reduced plasma IL-10 levels in
saline-pretreated mice and a partial but significant reduction in Con A-pretreated
mice in comparison to non-depleted animals, suggesting that Tregs were involved in
the IL-10 response (Fig. 3.19 A). Consequently, the following experiment had to
include double depletion of both Tregs and KCs verifying the participation of these
cell types in tolerization-induced IL-10 release and onset of Con A tolerance.
Indeed, double depletion of Tregs and KCs prior to Con A restimulation caused a
largely diminished IL-10 response in both saline- and Con A-pretreated mice,
suggesting that CD4+CD25+ Tregs and KCs together are crucial for primary IL-10
production and notably for IL-10 augmentation in tolerized mice (Fig. 3.19 B).
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65
A
B
Fig. 3.18: Critical role of Tregs and Kupffer cells for IL-10 production upon Con A tolerization. Prior to
Con A rechallenge both saline-pretreated (����) and Con A-pretreated (�) mice were either mock-
treated or subjected to A) Treg depletion by injection of anti-CD25 mAb or B) double depletion of
Tregs and KCs (mean ± SEM; n ≥ 3; *, p ≤ 0.05; n.s., not significant).
3.4.2 Special characteristics of tolerized Tregs
As already mentioned, the frequency of CD4+CD25+FoxP3+ Tregs was not
significantly increased in liver, spleen and liver-draining portal lymph-nodes during
tolerance (from day 8 on; see Fig. 3.7). Hence, Con A pretreatment might induce
predominantly qualitative rather then quantitative modulations in the Treg
population. Therefore, qualitative changes were investigated regarding modified
distributions of naïve and effector phenotypes among Tregs by measuring the
expression of the Treg markers CD62L (L-Selectin) (125) and CD103, respectively.
CD62L characterizes the naïve lymph-node-homing phenotype, whereas CD103 is
responsible for effector function and homing to inflamed tissues (126, 127). Again,
pronounced alterations were found shortly after Con A treatment, e.g with an
upregulation of CD103 at the site of inflammation, namely in the liver, and a
RESULTS_________________________________________________________
66
simultaneous downregulation of CD103 in secondary lymphoid organs, whereas
the frequency of CD62L expressing Tregs in portal lymph-nodes 24 hours after Con
A challenge was increased; however, in the tolerogenic state (day 8) frequencies
of both the CD62L+ and CD103+ Treg populations had reached their base levels
(Fig. 3.20).
Fig. 3.20: Appearance of CD103 and CD62L expression on FoxP3-positive Treg populations in liver,
spleen and liver-draining portal lymph nodes after Con A treatment: The corresponding organs
were excised at the indicated time points after Con A injection or saline injection as a negative
control, lymphocytes were isolated, stained for CD4, FoxP3, CD103, and CD62L and the
frequencies of CD62L+ and CD103
+ Tregs among CD4
+FoxP3
+ Tregs were calculated. Saline controls
were normalized and defined as “1” due to day-to-day variations, and the relative frequencies of the
respective population in Con A-pretreated samples were normalized and compared to the saline
controls. The percentages depicted in each panel represent the absolute frequencies of the
respective population in saline controls (calculated as mean ± SEM of the measured frequencies in
saline controls from all time points).
Nevertheless, Tregs from Con A-tolerized mice disclosed a higher immune-
modulatory potential than those from saline-pretreated mice as found in the
following in vitro experiments.
The features of CD4+CD25+ Tregs from Con A-tolerized and non-tolerized mice
were compared in co-cultures with equal numbers of responder cells of splenic or
hepatic origin. CD3+NK1.1-CD25- T cells were used as hepatic responder cells
from which Tregs and NKT cells had been removed by FACS-sorting excluding
RESULTS
67
contamination with any kind of regulatory and suppressive cell population.
CD4+CD25- T cells were used as splenic responder cells.
In fact, purified CD4+CD25+ T cells from non-tolerized mice were able to suppress
IL-2 production of CD4+CD25- splenic responder cells and also to significantly
suppress their IFNγ production. However, Tregs from Con A-tolerized mice revealed
significantly higher suppression than those of non-tolerized mice arguing for an
improved immunosuppressive capacity (Fig. 3.21).
Fig. 3.21: Tregs from Con A-pretreated mice reveal
increased suppressive capacity in vitro. CD4+CD25
+ Tregs
were isolated from either Con A-pretreated or saline-
pretreated mice and 1 × 105 Tregs/well were co-cultivated
with splenic responder T cells (CD4+CD25
-; 1 × 10
5/well).
Co-cultures were stimulated with anti-CD3 mAb (plate-
bound, 5 µg/mL) and cytokine concentrations in
supernatant was measured after 72 hours of cultivation by
ELISA (mean ± SEM; *, p ≤ 0.05).
The effect of Tregs on hepatic CD3+ responder cells depleted from CD25+ T cells
and NKT cells was also analyzed. Co-cultivation of hepatic responder cells from
control mice with Tregs from either Con A-tolerized or non-tolerized mice almost
completely abrogated the measurable IL-2 response. Interestingly, IL-2
concentration in supernatants of single-cultured responder cells from Con A-
pretreated mice was largely diminished compared to those from mock-treated
mice, even in the absence of Tregs, indicating that IL-2 impairment is largely
independent from Tregs after ex vivo restimulation and supporting the hypothesis of
induction of partial anergy. In contrast to IL-2, significant reduction of IFNγ
RESULTS_________________________________________________________
68
production by hepatic T cells was achieved only upon co-cultivation with Tregs.
Again, Tregs from Con A-tolerized mice revealed a significantly stronger
suppression than those from saline-pretreated animals already recognized in co-
cultures with splenic responder cells (Fig. 3.22).
Fig. 3.22: Con A-induced suppression of IL-2 and
tolerization-induced IL-10 boost in vitro: CD4+CD25
+
Tregs were isolated from either Con A-pretreated or
saline-pretreated mice and 1 × 105 Tregs/well were co-
cultivated with hepatic T cells, depleted from both
CD25+ regulatory T cells and NKT cells by FACS
sorting (CD3+NK1.1
-CD25
-; 1 × 10
5/well). Co-cultures
were stimulated with anti-CD3 mAb (plate-bound,
5µg/mL) and cytokine concentrations were measured
in supernatant after 72 hours of cultivation by ELISA
(mean ± SEM; *, p ≤ 0.05).
A more detailed investigation of Tregs from tolerized versus non-tolerized mice
supported the in vivo findings regarding IL-10 production. In Treg-single cultures IL-
10- concentration was significantly higher in the supernatant of tolerized than non-
tolerized Tregs. Furthermore, co-cultures of CD25- responder T cells with Tregs from
Con A-tolerized mice but not with Tregs from control mice resulted in a pronounced
IL-10 release, even higher than the sum of those of corresponding single cultures
(Fig. 3.22). To verify Tregs as IL-10-producing cells, intracellular cytokine-staining
was performed. The pronounced IL-10 production was not conferred by FoxP3-
responder cells cultured alone or with Tregs, but rather by CD4+CD25+FoxP3+ Tregs
RESULTS
69
from saline-pretreated or – to a higher degree – from Con A-pretreated animals.
The percentage of Tregs showing bright IL-10 expression increased upon ex vivo
Con A restimulation and for Tregs from Con A-pretreated mice significantly upon co-
culture with responder cells (Fig. 3.23). The enhanced IL-10 production might have
been due to increased expression by original Tregs, or – in a manner of infectious
tolerance – Treg-mediated engagement of originally FoxP3-CD25- negative cells.
Fig. 3.23: Detection of increased IL-10 production by Tregs from Con A-pretreated mice in vitro: To
detect IL-10-producing cell populations, sorted CD4+CD25
- responder cells from non-tolerized mice
and/or CD4+CD25
+ Tregs from saline- or Con A-pretreated mice were cultivated and stimulated for
14 hours in the presence of Con A (5 µg/mL) and BD GolgiStop™ containing Monesin, to achieve
intracellular cytokine accumulation. Finally, IL-10-production of FoxP3- responders or FoxP3
+ Tregs
was assessed by combined intracellular staining for FoxP3 and IL-10 and subsequent FACS-
analysis gating on either responder cells or Tregs (mean ± SEM; *, p ≤ 0.05).
In order to identify whether the suppressive activity of Tregs from tolerized mice was
due to infectious tolerance, CD4+ responder cells were preincubated with Rp-
cAMPS, a specific PKA/cAMP antagonist, and co-cultured with CD4+CD25+ Tregs
from tolerized and non-tolerized wt mice, respectively. After TCR-stimulation for 3
days, total RNA of sorted CD4+ T cells was isolated and quantitative RT-PCR was
performed for measurement of IL-2, FoxP3, and ICER mRNA induction in
responder cells. After blockade of PKA, both IL-2 mRNA expression (2.1 ± 0.75 vs.
1.0 ± 0.18 fold) and IL-2 protein concentration (315.6 ± 16.9 vs. 170.3 ± 47.7
pg/mL) of CD4+ responder cells were two-fold enhanced compared to untreated
responder cells supporting the involvement of cAMP in IL-2 synthesis (128).
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70
However, upon co-culture with Tregs and without addition of the cAMP antagonist
IL-2 mRNA and protein expression was downregulated as expected. Again, Tregs
from tolerized animals showed slightly higher suppressive capacity in contrast to
Tregs from control animals (Fig. 3.24 A), confirming data obtained by IL-2 ELISA
(Fig. 3.21, 3.22). Surprisingly, addition of the cAMP inhibitor resulted in an
upregulation of IL-2 mRNA expression only in co-cultures of CD4+ responder cells
and Tregs from control animals, whereas Tregs from tolerized mice were still
suppressive despite blockade of cAMP (Fig. 3.24 A). Additionally, a strong
upregulation of ‘inducible cAMP early repressor’ (ICER) and FoxP3 in Rp-cAMPS
pretreated CD4+ lymphocytes co-cultured with Tregs from tolerized animals could
be detected (Fig. 3.24 B and C) suggesting a cAMP-independent suppression
mediated by Tregs from Con A tolerized mice. Conversely, FoxP3 and ICER were
down-modulated in presence of cAMP antagoniszation in co-cultures with Tregs
from non-tolerized animals, correlating very well with the increased IL-2
expression and confirming a conventional cAMP-dependent suppression mediated
by naive control Tregs. These findings indicate that Tregs from tolerant mice
suppress IL-2 production by a novel cAMP-independent, yet unknown mechanism
in comparison to naive control Tregs.
Fig. 3.24: Unconfined suppressive activity of Tregs
from tolerized mice despite inhibition of cAMP
activity. CD4+CD25
+ Tregs from Con A- or saline-
pretreated wt mice were co-cultured with CD4+CD25
-
responder cells at a ratio of 1:1. CD4+CD25
-
responder cells were preincubated for 30min with the
cAMP inhibitor Rp-cAMPS or its solvent as control.
After stimulation quantitative real-time RT-PCR for
the expression of IL-2 (A), FoxP3 (B), and ICER
mRNA (C) was performed (mean ± SEM; n = 3; *, p ≤
0.05).
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71
Eventually, there might be the objection that the reduced cytokine release of
responder cells was correlated with less proliferation of these cells. However, it
has to be emphasized that Treg-mediated suppressive effects on cytokine release
of responder cells in vitro were not caused by inhibition of proliferation of cytokine-
producing responders, since under these culture conditions (i. e. anti-CD3 mAb [5
µg/mL]; w/o APCs and w/o anti-CD28 mAb) responder-cell proliferation even
without Tregs was only marginal as measured by using CFSE-labelled responder
cells all the more suggesting a specific priming and existence of unique markers
on tolerized Tregs which are responsible for their increased immunosuppressive
activity. Finally, the principle capability of isolated Tregs to suppress proliferation of
responder cells was tested due to the uncommonly combined Treg sorting
procedure of MACS separation with subsequent FACS sorting: CD4+CD25- cells
were labelled with CFSE and cultivated alone or with Tregs under the strong
TPA/Ionomycin stimulation. Even at the low Treg:responder ratio of 1:10 analyzed
here, Tregs significantly suppressed responder-cell proliferation proving the
suppressive feature of Tregs (Fig. 3.25).
Fig. 3.25: Suppression of proliferation of responder cells by Tregs: To test, whether isolated Tregs
were in principle capable of suppressing proliferation of responder cells, CD4+CD25
- responders
were labelled with CFSE and cultivated alone or – at a responder/Treg ratio of 10:1 - together with
CD4+CD25
+ Tregs derived from Con A-pretreated mice for 3 days in the presence of 25ng/mL TPA
and 1µM Ionomycin. Proliferation is shown by means of the proliferative index (PI) which
represents a mathematical approximation to the median number of cell divisions the entirety of
responder-cells has passed through since the time point of labelling. PI was calculated using the
algorithm PI=Log[FInd/MFIall]/Log[2], with MFIall=median fluorescence-intensity of all responder-cells
and FInd=peak-fluorescence of non-proliferating cells (mean ± SEM; *, p ≤ 0.05 vs. unstimulated
control; #, p ≤ 0.05 vs. stimulated responder cells w/o Tregs)
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72
3.4.3 Therapeutic potential mediated by tolerized Tregs
Since CD4+CD25+FoxP3+ IL-10-producing T cells were identified as one of the
Con A tolerance-mediating cell populations (see Fig. 3.19, 3.22, 3.23), the
immune-therapeutic potential of Tregs was assed in the model of immune-mediated
Con A liver injury. A therapeutic capacity of regulatory T cells has already been
published in mouse models of colitis, EAE, and glomerulonephritis (50, 129, 130).
Hence, 1 x 106 sorted CD4+CD25+ Tregs from tolerized and non-tolerized wt, IL10-/-,
or TGFβR2 tg (hCD2-∆kTβRII) mice or CD4+CD25- control lymphocytes were
injected into C57BL/6 mice 24 hours prior to Con A treatment. To further
characterize the features of injected Tregs, an aliquot was picked and tested for
FoxP3 expression. Interestingly, the adoptively transferred CD4+CD25+Tregs of all
mouse strains were > than 93% Foxp3-positive.
Fig. 3.26: Measurement of the expression of FoxP3 on sorted CD4+CD25
+ T cells used for the
therapeutic approach: An aliquot of MACS-/FACS-sorted CD4+CD25
+ T cells was analysed for
FoxP3 expression by intracellular FACS staining. The isolated CD4+CD25
+ cell population showed
a purity of about 99% (left panel). The right panel demonstrates the expression of FoxP3 gated on
these cells. The percentage of CD4+CD25
+FoxP3
+ Tregs from non-tolerized IL10
-/- mice is depicted
representing the high FoxP3 expression of Tregs from all tested mouse strains exemplarily.
In principle, mice injected with wt Tregs exhibited considerably lower liver injury than
control mice. However, Tregs from Con A -pretreated wt mice appeared to be more
efficient, showing statistically significant suppression of liver injury thereby
RESULTS
73
supporting the above mentioned in vitro results regarding an increased
immunosuppressive potential and specialized features of tolerized Tregs.
In contrast, Tregs from IL10-/- mice failed to show any therapeutic effect against Con
A hepatitis, since the plasma transaminases of Con A-treated wt mice were not
reduced after injection of either tolerized or non-tolerized Tregs from IL10-/- mice
suggesting that CTLA-4 engagement is not sufficient to mediate the therapeutic
effect of Tregs (Fig. 3.27).
Fig. 3.27: Significant reduction of Con A-mediated liver damage by adoptively transferred Tregs from
tolerized wt mice in contrast to Tregs from IL10-/-
mice. CD4+CD25
+ Tregs and CD4
+CD25
- cells were
isolated from Con A- or saline-pretreated animals (wt and IL10-/-
mice) on day 8. Wt mice were
injected with 1 × 106 FACS-sorted Tregs from either Con A-tolerized or those from saline-pretreated
wt (white bars) or IL10-/-
mice (black bars), or with 1 × 106 CD4
+CD25
- control cells 24 hours prior to
Con A treatment. An additional control group did not receive any cell type (shaded bar). Plasma
transaminase activity denoting the degree of liver damage was measured 8 hours after Con A
challenge (mean ± SEM; n = 4; *, p ≤ 0.05 vs. control).
TGFβ has also been shown to be released by regulatory T cells and to possess
suppressive and regulatory activity (90, 118, 131). Hence, the therapeutic effect of
Tregs from saline- and Con A-pretreated hCD2-∆kTβRII mice expressing a
dominant-negative TGFβ type II receptor in T cells (118) were tested. Despite the
impaired TGFβ signalling in T cells Tregs from transgenic mice were still able to
suppress Con A-induced hepatitis in a similar manner like wt Tregs (data not
RESULTS_________________________________________________________
74
shown) denoting no relevance of T cell-produced TGFβ during Con A-mediated
immunosuppression. These findings correlate very well with an intact tolerance
induction in both female and male transgenic mice (Table II).
In conclusion, these results clearly indicate that IL-10- but not TGFβ-producing
Tregs have a therapeutic potential in this model of immune-mediated hepatitis.
Table II: Tolerance induction in hCD2-∆kTβRII mice: Female and male TGFβR2 transgenic mice
were pretreated with Con A or saline 8 days before Con A rechallenge. Measurement of the liver-
specific plasma transaminase ALT and the cytokine concentrations was performed 8 hours after
Con A rechallenge (mean ± SEM; *, p ≤ 0.05 vs. saline-pretreated control)
female TGFββββR2 tg mice male TGFββββR2 tg mice
parameter sal/Con A Con A/Con A sal/Con A Con A/Con A
ALT [U/L] 2293±933 965±411 1279±731 313±181
IFNγγγγ [pg/mL] 1675±418 865±173 497±76 198±22 *
IL-6 [pg/mL] 14018±2373 5710±901 * 5617±866 927±413 *
IL-2 [pg/mL] 340±78 76±20 * 84±22 6±2 *
IL-10 [pg/mL] 54±5 288±47 * 131±39 301±49 *
3.3.4 Dispensability of IL-10 on Treg activity in vitro
It has often been described that regulatory T cells possess different suppression
patterns in vivo and in vitro, namely cytokine-dependent versus cytokine-
independent suppression. During establishment of Con A-mediated tolerance and
in the therapeutic assay IL-10 was indispensable in vivo validated by the use of
IL10-/- mice and blocking anti-IL10R mAb. To investigate the potential role of IL-10
in vitro, splenocytes were isolated from saline- and Con A-pretreated mice and
sorted for CD4+CD25- responder cells and CD4+CD25+ Tregs. After in vitro co-
cultivation of Tregs and responder cells at a ratio of 1:1 and TCR-stimulation,
cytokine production was measured in the supernatant. Purified CD4+CD25+ T cells
from non-tolerized as well as from tolerized mice were able to significantly
suppress IL-2 and IFNγ production of CD4+CD25- responder cells as mentioned
RESULTS
75
above (see Fig. 3.21; Fig. 3.22). Neutralization of IL-10 by addition of anti-IL-10
mAb failed to reverse the immunosuppressive effect of Tregs from both control and
tolerized animals indicating an IL-10-independent suppression pattern of
regulatory T cells with respect to IL-2 and IFNγ secretion in vitro in contrast to the
in vivo experiments where IL-10 is absolutely essential for suppression of IFNγ
production and liver damage. The discrepancy of a cytokine-independent, but cell-
cell contact-dependent suppression of Tregs in vitro and cytokine-dependent
suppression in vivo has already been approved by several groups (68).
Interestingly, IL-2 concentrations in culture supernatants of responder cells from
Con A-pretreated mice were largely diminished compared to those from saline-
treated mice, even in the absence of Tregs (see Fig. 3.22) and after neutralization of
IL-10 (Fig. 3.28), suggesting that IL-2 impairment is independent from Tregs and IL-
10 following ex vivo restimulation. Hence, it seems that responder cells have
already been suppressed to produce IL-2 by the in vivo pretreatment regimen.
Fig. 3.28: IL-10 does not mediate the
suppressive capacity of regulatory T cells in
vitro. Splenic CD4+CD25
+ Tregs and
CD4+CD25
- responder cells were isolated from
either Con A-pretreated or saline-pretreated wt
mice and 1 × 105 Tregs/well were co-cultivated
with CD4+CD25
- wt responder cells at a ratio of
1:1. Neutralizing anti-IL-10 mAb (20µg/mL)
was added. Co-cultures were stimulated with
anti-CD3 mAb (5µg/mL, plate-bound) and
cytokine concentrations were measured in
supernatants after 72 h by ELISA (mean ±
SEM; n = 3; *, p ≤ 0.05).
RESULTS_________________________________________________________
76
This outcome is comparable to the in vivo findings, since IL-2 was still suppressed
both in Con A-pretreated IL-10-/- and in anti-IL-10R/Con A-pretreated wt mice. In
contrast, regulation of IFNγ release by responder cells required an intact IL-10
signalling, since neutralization of IL-10 and subsequent lack of the protective and
immunosuppressive IL-10 caused an increase of IFNγ production by responder
cells from tolerized as well as non-tolerized animals resembling the in vivo results
(Fig. 3.28).
To further confirm the dispensability of IL-10 in vitro, Tregs from tolerized and non-
tolerized IL-10-/- mice were co-cultured wit wt responder cells and stimulated under
the same conditions as mentioned above. Indeed, the suppressive capacity of Tregs
from IL-10-/- mice was not reversed in vitro, since these cells were still able to
inhibit the cytokine release of wt responder cells (Fig. 3.29) confirming the
experiments with neutralizing anti-IL10 mAb.
Fig. 3.29: Intact suppression pattern of
Tregs from IL10-/-
mice in vitro: Splenic
CD4+CD25
- responder cells were isolated
from control wt mice and CD4+CD25
+
Tregs were isolated from tolerized and
non-tolerized IL10-/-
mice. 1 × 105
Tregs/well were co-cultivated with
CD4+CD25
- wt responder cells at a ratio
of 1:1. Co-cultures were stimulated with
anti-CD3 mAb (5µg/mL, plate-bound) and
cytokine concentrations were measured
in supernatants after 72 h by ELISA
(mean ± SEM; n = 3; *, p ≤ 0.05).
RESULTS
77
The only difference was the increased IL-2 production of IL-10-/- responder cells
from saline and Con A-pretreated mice in the absence of co-cultivated Tregs,
indicating enhanced basal sensitivity towards T cell stimulation as a result of
sustained lack of IL-10.
3.5 Oppositional regulation of IL-10 and IL-17 during Con A
hepatitis and tolerance
In contrast to the anti-inflammatory IL-10 the recently identified IL-17 produced by
Th17 cells has pro-inflammatory activity. Until now, CD4+ effector T cells have
been categorized into two subsets: T helper type 1 (Th1) cells secreting IFNγ and
TNFα and T helper type 2 (Th2) cells producing IL-4, IL-5, and IL-13 release (112).
However, another subset of T cells that produce IL-17 has been identified, i. e.
Th17 cells. The Th17 response is initiated by IL-6, a differentiation factor of Th17
cells beside TGFβ and IL-21 (113). Induced Th17 cells with specificity for self-
antigens are highly pathogenic and lead to the development of inflammation and
autoimmune diseases such as multiple sclerosis or rheumatoid arthritis (113).
Hence, it might be interesting to investigate the role of the pro-inflammatory IL-17
in comparison to the anti-inflammatory IL-10 in the murine immune-mediated
model of Con A hepatitis and Con A tolerance, since the Con A model reflects
processes of autoimmune hepatitis very well.
The main mediators of Con A hepatitis are the pro-inflammatory cytokines IFNγ
and TNFα; however, IL-17 is also strongly upregulated after a single Con A
challenge, especially between 3 and 6 hours after Con A administration measured
both in the plasma by ELISA (saline-pretreated animals in Fig. 3.30 A) and in liver
tissue by quantitative RT-PCR (saline-pretreated animals in Fig. 3.30 B)
suggesting a harmful role of IL-17 during Con A hepatitis in contrast to the
protective IL-10. However, IL-17 is strongly downregulated during Con A tolerance
comparable to the Th1 cytokines IFNγ and TNFα. Again, an oppositional
development was detected regarding IL-17 and IL-10 in Con A tolerized animals,
RESULTS_________________________________________________________
78
since the IL-17 downregulation is definitely accompanied with an upregulation of
IL-10 expression (Con A-pretreated animals in Fig. 3.30 A and B). Interestingly,
expression of IL-17 correlates very well with the expression of IL-6: simultaneous
overexpression could be detected during Con A hepatitis, whereas both cytokines
were repressed in Con A tolerance. This phenomenon is explainable by the fact
that IL-6 is a differentiation factor of Th17 cells.
In conclusion, IL-10 seems to act in an immunoregulatory manner suppressing the
release of IFNγ, TNFα, IL-6 and IL-17.
Fig. 3.30: Opposite expression and release of IL-10 and IL-17 during Con A tolerance: Con A or
saline were injected intravenously into wt mice. On day 8 animals were restimulated with Con A.
Cytokine expression of anti-inflammatory IL-10 and pro-inflammatory IL-17 was measured A) in
plasma by ELISA, or B) in total liver RNA by quantitative real-time RT-PCR 3,6, and 8 hours after
Con A injection. For RT-PCR analysis ß-actin mRNA was used as an internal standard. x-fold
induction was calculated referring to mRNA levels of the respective cytokines in saline-treated
control animals (mean ± SEM; n = 4; *, p ≤ 0,05 vs. saline-pretreated control).
RESULTS
79
3.6 Relevance of NKT cells in Con A hepatitis and during
tolerance
Besides their cytotoxic and pro-inflammatory characteristics, NKT cells are also
well known for their immunoregulatory potential. Since both NKT-cell number and
frequency among hepatic and splenic lymphocytes was significantly increased in
Con A-tolerized mice after a short-term disappearance (see Fig. 3.5), a potential
role of NKT cells in the onset of Con A tolerance was hypothesized. To answer
this question, NKT-cell deficient CD1d-/- mice were used. The severity of liver
injury and the cytokine response (i.e. IFNγ, IL-2, IL-6 and IL-10 in plasma) was
measured in Con A- or saline-pretreated CD1d-/- mice after Con A restimulation.
Obviously, NKT cells are indispensable for Con A-induced liver injury, since ALT
and AST values of CD1d-/- mice were much lower than those of wild-type mice (26,
27). Thus, the investigation of these mice regarding Con A liver injury might be
informative only to a limited extend. However, a higher injected dose of Con A (25
mg/kg) resulted in a moderate liver damage in CD1d-/- mice.
Interestingly, Con A-pretreated CD1d-/- mice revealed reduced plasma ALT levels
(680 ± 338 U/L vs. 124 ± 17 U/L; mean ± SEM) and the typical cytokine-profile of
Con A-induced tolerance, i.e. reduced IL-2-, IFNγ-, IL-6-levels and higher IL-10-
production than saline-pretreated littermates upon rechallenge (Fig. 3.31). In
summary, this clearly indicates that NKT cells are dispensable for development of
Con A tolerance despite their necessity for Con A-induced hepatitis.
RESULTS_________________________________________________________
80
Fig. 3.31: Maintenance of the characteristic cytokine profile of Con A tolerance in CD1d knockout
mice: NKT-cell deficient CD1d-/-
mice were injected intravenously with Con A or saline 8 days prior
to Con A-restimulation. Eight hours after Con A rechallenge, plasma cytokine levels were
measured by ELISA. (mean ± SEM; n = 4; *, p ≤ 0.05 vs. saline controls)
DISCUSSION
81
4 DISCUSSION
4.1 The role of IL-10-producing CD4+CD25+FoxP3+ regulatory T
cells
4.1.1 ...as cellular immunotherapy in vivo
This study clearly demonstrated a crucial involvement of IL-10-producing
CD4+CD25+FoxP3+ regulatory T cells during complex immune-modulatory
processes in the liver. Interestingly, this cell population takes part in both triggering
therapeutic effects in experimental liver injury induced by the mitogenic plant lectin
Con A and in mediating tolerance against Con A restimulation. The immune-
regulatory role of the liver is of particular interest, since it fulfils scavenger function
by eliminating foreign antigen material from the intestinal tract. Hence, it is
compulsory to circumvent any dispensable and inadequate immune activation to
prevent chronic liver damage. However, gut-derived antigens are not ignored by
the immune system and infections of the liver by pathogens (e. g. viruses) require
induction of an effective immune response to break down the infection and to
prevent harmful progression of persistence and chronic infections (5, 52).
Therefore, liver lymphocytes have to switch rapidly from a tolerant to a responsive
state. The liver has been considered to favour the induction of peripheral tolerance
to self antigen probably by activation and differentiation of regulatory T cells which
are well-known to prevent autoimmune disease by production of anti-inflammatory
cytokines like IL-10 and to maintain peripheral tolerance by active suppression
beside the passive mechanisms of deletion, ignorance, or anergy (47, 48; see
chapter 1.3). Hence, lack or dysfunction of regulatory T cells results in severe
immune-pathology and outbreak of autoimmune diseases including type 1
diabetes, multiple sclerosis, autoimmune gastritis, and – most interestingly in this
context – autoimmune hepatitis (57-59). This idea is supported by the following
observations: (a) in mice, depletion of the Treg population spontaneously results in
autoimmune diseases; (b) T cell-deficient nude mice develop autoimmune
DISCUSSION______________________________________________________
82
disease, if CD4+ T cells were administered that have been depleted of the CD25+
population (60); (c) both humans and mice with mutations in their FoxP3 gene, the
most specific marker of nTregs until now, suffer from autoimmune diseases (61).
The murine model of Con A hepatitis is a well-known model of human autoimmune
hepatitis, although Con A activates a wide variety of T cells and does not
represent an autoantigen. Nevertheless, the mouse model and the human disease
have many features in common such as good responsiveness to
immunosuppressive drugs (22), genetic prevalence of certain mouse strains with
respect to susceptibility (23), prevalence of CD4+ T cells, and immunosuppression
in state of remission (24).
Remarkably, it has already been shown that in patients with AIH peripheral Treg
numbers and functions are depressed compared with controls. Moreover, the
percentage of Tregs inversely correlates with autoantibody titers, and – most
interestingly in this context – Treg numbers are higher in AIH patients during
remission than at the time of diagnosis (11, 59). Indeed, this kind of
immunosuppression in state of remission is also detectable during Con A-induced
liver damage: primarily, injection of a sublethal Con A dose induces acute liver
injury in mice depending on T cells, NKT cells, and KCs accompanied by a pro-
inflammatory Th1 response; however, this period of immune-activation goes along
with long-term differentiation processes characterized by an immunosuppressive
milieu and a shift to Th2 bias, which is even more pronounced after Con A
restimulation. In more detail, this stadium is characterized by an anti-inflammatory
cytokine profile, i.e. down-modulation of IFNγ, TNFα, IL-17, IL-6, and IL-2 and a
concomitant increase of IL-10 release. Interleukin-10 was first described as
cytokine-synthesis inhibitory factor (CSIF) due to its capacity to inhibit activation of
and cytokine production by Th1 cells (86). IL-10 is expressed by a variety of
immune cells, including CD4+ T cells, monocytes and macrophages (84), B cells,
natural killer (NK) cells, and dendritic cells (DC) (85). IL-10 binds to the IL10-
receptor expressed by most haematopoietic cells. The routine function of IL-10
appears to be to limit and terminate inflammatory responses of most
haematopoietic cells. In addition, IL-10 regulates growth and differentiation of B
cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, DCs,
keratinocytes, and endothelial cells (132). It is especially noteworthy, that IL-10
also plays a key role in differentiation and function of regulatory T cell populations.
DISCUSSION
83
Hence, it seems that the detrimental T cell response in Con A hepatitis is restricted
by immune-regulation during tolerance establishment which involves IL-10-
producing regulatory T cells.
Due to the above mentioned correlation between human AIH and Con A-induced
hepatitis regarding immunosuppression, Treg populations were analyzed in more
detail during Con A hepatitis and tolerance.
Treg populations described so far include the naturally occurring CD4+CD25+ Tregs
(nTregs) as well as antigen-driven IL-10-producing Tregs, and TGFβ-secreting Tregs
(87, 133, 134). Naturally occurring Tregs - generated in the thymus and
characterized by expression of the transcription factor FoxP3 - represent 5-10% of
CD4+ T lymphocytes and suppress T cell responses via cytolytic T-lymphocyte
associated protein 4 (CTLA-4)/B7 engagement (65). They are also able to secrete
TGFβ and IL-10. However, it has to be emphasized that this cytokine pattern is not
a unique feature of nTregs. Interestingly, IL-10 controls in vivo expansion of naïve
CD4+ T cells in lymphopenic hosts as well as wasting disease, autoimmune colitis
and allograft rejection (72, 73, 135) whereas inhibition of gastritis (135) and in vitro
suppression of responder cells is IL-10-independent (49). These controversial
findings might suggest different levels of immune regulation, with the most
‘primitive’ level in in vitro systems followed by low levels of (local) inflammation (e.
g. gastritis) and higher levels of (systemic) inflammation. The latter is always
accompanied by activation of cells of the innate immune response including DCs,
granulocytes, macrophages/monocytes, and NK cells, which ultimately require
stronger suppression comprising immunosuppressive cytokines such as IL-10 or
TGFβ (136, 137). Indeed, it has been recently shown that immunosuppressive
properties of CD4+CD25+ Tregs were not limited to influence effector T cells, but
also includes inhibition of immune-pathology mediated by cells of the innate
immune system (137).
In contrast to nTregs, antigen-driven ‘IL-10 Tregs’ can be induced both in vitro and in
vivo by different antigenic stimulation (129, 138-141). Again, their development
and function in vivo are IL-10-dependent, whereas inhibition of in vitro T cell
proliferation is a cell contact-mediated mechanism (136). ‘IL-10 Tregs’ have been
described very well in the model of experimental autoimmune encephalomyelitis
where a beneficial involvement of IL-10 was clearly demonstrated (138, 141). It is
DISCUSSION______________________________________________________
84
worth mentioning, that induced ‘IL-10 Tregs’ do not express FoxP3 (142), which has
often been implicated with upregulation of IL-10 mRNA expression (65). As they
inhibit naïve T cell proliferation with an efficiency similar to FoxP3+ nTregs (142),
FoxP3 expression seems not to be necessary for suppressor function. However, a
common and essential condition regarding the suppressive ability of both types of
Tregs is the lack of IL-2 production, since inhibition of T cell responses was
overcome by exogenous IL-2 (49, 142). The ability to generate ‘IL-10 Tregs’ through
antigenic stimulation and immunosuppressive drugs in the absence of FoxP3
(142) might be a useful approach for new therapeutic applications.
A second and probably distinct population of antigen-driven IL-10-dependent,
FoxP3-negative Tregs has been named T regulatory type 1 (Tr1) cells which have
been defined by their ability to secrete high amounts of IL-10 and TGFβ and to
suppress T cell responses in a cytokine-dependent, but cell contact-independent
manner in vivo and in vitro (87).
In conclusion, ‘IL-10 Tregs’, Tr1 cells, and also CD4+CD25+ nTregs have been shown
to control immune pathologies such as gastritis, autoimmune colitis or EAE.
However, until now, the nature and mechanism of IL-10-secreting Tregs in
experimental models of hepatic immune pathologies has not yet been
investigated.
To address the question whether Con A tolerance is associated with expansion of
local Treg populations, the frequency of CD4+CD25+FoxP3+ nTregs was analyzed in
liver, spleen and liver-draining portal lymph nodes. Strikingly, a pronounced
increase was detectable especially in the liver 24 hours after Con A injection
supposing that immune cell activation took place mainly in the liver and to lesser
extent in secondary lymphoid organs. During tolerance induction, namely from day
8 after the first Con A challenge, both Treg frequencies and the surface expression
of CD62L (lymph-node homing marker) and CD103 (inflammation homing marker)
on FoxP3+ Tregs turned to normal suggesting rather qualitative than quantitative
changes in the Treg population. Thus, the starting-point of investigation was the
causal research of the elevated IL-10 production in tolerized mice. To demonstrate
a general participation and role of CD4+CD25+ Tregs regarding establishment of
Con A tolerance by release of the immunosuppressive IL-10, CD25+ T cells were
depleted with anti-CD25 mAb prior to Con A rechallenge. In fact, reduced IL-10
expression in CD25-depleted, Con A-pretreated mice provided evidence for an
DISCUSSION
85
important role of Tregs in tolerization-induced IL-10 production. Additionally, several
mouse models of other human autoimmune diseases, e. g. rheumatoid arthritis,
colitis, or EAE, also displayed an immunoregulatory involvement of IL-10-
producing Tregs in therapeutic approaches (66, 134, 143, 144). To check a
therapeutic capacity of Tregs in Con A hepatitis, 1 x 106 CD4+CD25+ Tregs from
either tolerized or non-tolerized wt mice were injected one day prior to Con A
challenge. Indeed, adoptively transferred CD4+CD25+ Tregs from wt mice prevented
hepatitis in principle; however, Tregs from Con A-tolerant mice showed a higher
suppressive capacity resulting in a significant reduced liver damage upon Con A
administration. This clearly indicates that Tregs, which have once been exposed to
Con A, are primed specifically to recognize the same antigens faster and hence to
ameliorate the state of health more effectively. In contrast, Tregs from both tolerized
an non-tolerized IL10-/- mice did not attenuate Con A-induced hepatitis in wt mice,
correlating very well with findings in the EAE model, where transfer of CD4+CD25+
T cells from naive SJL mice, but not from IL10-/- mice, decreased the severity of
active EAE (143) indicating that CD4+CD25+ T cells may play an important role in
the down-regulation of the pathogenic T cell responses in EAE and Con A hepatitis
via a mechanism that involves IL-10. It seems noteworthy that the transferred IL-
10-producing CD4+CD25+ regulatory T cells were also positive for FoxP3, i.e. they
expressed the most specific marker of nTregs. Hence, these cells differ from the in
vivo functional FoxP3-negative ‘IL-10 Tregs’ (136, 142) and the induced Tr1 cells
(145) which also suppress immune-pathologies via IL-10. However, these Treg
subsets do not express FoxP3 and Tr1 cells suppress immune responses not only
by IL-10 but also by TGFβ. However, in the present model TGFβ produced by T
cells was not responsible for mediation of Con A tolerance (see experiments with
hCD2-∆kTβRII mice expressing a dominant-negative TGFβ type II receptor in T
cells) and again, Tregs from hCD2-∆kTβRII mice were suppressive in the same
manner like wt Tregs. Thus, TGFβ released by T cells might not have contributed to
the tolerogenic and therapeutic effects.
In summary, it is hypothesized that Con A induces peripheral conversion of CD4+
lymphocytes into CD4+CD25+FoxP3+ IL-10-producing regulatory T cells in vivo
which suppress Con A-induced immune-pathology efficiently. Indeed, the use of in
vivo induced Tregs would represent an advance in the treatment of immune-
DISCUSSION______________________________________________________
86
pathologies such as colitis, autoimmune gastritis, MS or AIH compared to in vitro
expanded and re-injected Tregs, since the in situ development of antigen-specific
Tregs in lymphopenic organisms would prevent generalized immunosuppression.
Nevertheless, possible side effects and induction of pro-inflammatory cytokine
release have to be dampened by immunosuppressive drugs in the early
development in immunotherapy. Later, immunosuppressive drugs have to be
withdrawn to guarantee an intact response to pathogens.
4.1.2 ...as suppressor cells in vitro
The discussion regarding the involvement of immunomodulatory cytokines such as
IL-10 or TGFβ in Treg-mediated suppression in vitro is still ongoing and was
therefore also investigated in the present study: The indispensable role of IL-10 in
vivo was clearly demonstrated by experiments with blocking anti-IL-10R mAbs and
IL10-/- mice, since firstly, tolerance was totally reversed in IL10-/- mice and
secondly, Tregs from IL10-/- mice were not protective in the immunotherapeutic
approach compared to wt Tregs. In contrast, the inhibitory potential of Tregs from
both tolerized and non-tolerized mice was not reversed in vitro by neutralization or
lack of IL-10 indicating a different suppression pattern of Tregs in vitro and in vivo.
These controversial findings have already been approved by several other groups
with the result that different in vivo models of disease suppression show different
patterns of dependency on various immunosuppressive cytokines and that in vitro
suppression of Tregs is most likely cytokine-independent, but cell contact-
dependent (49, 66, 68, 134).
To clarify the necessity of IL-10 in Treg-mediated suppression in vitro, splenocytes
were isolated from saline- and Con A-pretreated mice. CD4+CD25- responder cells
and CD4+CD25+ Tregs were co-cultured and stimulated in the presence or absence
of anti-IL10 mAb. Indeed, CD4+CD25+ T cells from non-tolerized as well as from
tolerized wt mice significantly suppressed IL-2 and IFNγ production of CD4+CD25-
responder cells despite neutralization of IL-10 indicating an IL-10-independent
suppressive activity of Tregs with respect to IL-2 and IFNγ secretion in vitro in
contrast to the in vivo experiments where suppression of IFNγ production strictly
DISCUSSION
87
depended on IL-10. Interestingly, IL-2 concentrations in supernatants of single-
cultured responder cells from Con A-tolerant wt mice were largely diminished
compared to those from saline-pretreated wt mice, even in the absence of Tregs
and after neutralization of IL-10, suggesting that in vitro inhibition of IL-2
production by responder cells was independent from Tregs and IL-10 following ex
vivo restimulation. Thus, these responder cells are characterized by a kind of non-
responsiveness, which was induced by in vivo pretreatment. These results are
comparable to the in vivo findings, where IL-2 was still suppressed in Con A-
pretreated IL10-/- and in anti-IL-10R mAb/Con A-pretreated mice.
These consistent results of in vivo and in vitro experiments provide clear evidence
of a downmodulation of IL-2 in response to restimulation of tolerized mice or of T
cells from such animals in comparison to non-tolerized controls. Tolerization-
induced, but Treg-/IL-10-independent IL-2-downmodulation may be caused by
presence of another non-CD25+ regulatory T-cell type in Con A-pretreated mice,
increased consumption, or induction of non-responsiveness. Recently, it has been
demonstrated that activation of T cells in the presence of IL-10 induces non-
responsiveness/anergy, which cannot be reversed by IL-2 or stimulation by anti-
CD3 mAb and anti-CD28 mAb (146). This observation corroborates the present
findings, since the first in vivo Con A challenge induces immune-activation and IL-
10 release. Afterwards, ex vivo restimulation of isolated splenic T cells by anti-CD3
mAb results in additional IL-10 release (see Fig. 3.11 Induction of Con A tolerance
ex vivo) with concomitant induction of anergy and loss of IL-2 production in Con A-
polarized T cells. Hence, IL-10 induces T cell anergy and therefore may play an
important role in induction and maintenance of antigen-specific T cell tolerance.
One factor which might also contribute to the reduced IL-2 response in tolerized
mice was identified upon analysis of changes in the phenotype of CD4+ liver T-
cells 8 days after a single Con A injection. A pronounced reduction of the
population of naïve-like CD4+CD45RBhigh cells could be detected, with a
concomitant increase of cells with the phenotype of antigen-experienced effector
CD4+CD45RBlow cells, which have been shown to reveal diminished IL-2
responses in comparison to naïve cells (73, 147). This alteration found in vivo was
also transferred to the in vitro assays. Thus, the tolerization-induced block of IL-2
response found in vitro may account on the same mechanisms as the in vivo
effect.
DISCUSSION______________________________________________________
88
In contrast, IFNγ production by responder cells is strictly regulated by IL-10, since
neutralization of IL-10 caused an increase of IFNγ release in single cultures of
responder cells from tolerized as well as non-tolerized animals resembling the in
vivo results. Moreover, Tregs from IL10-/- mice also failed to reverse the
immunosuppressive effect confirming the dispensability of IL-10 in vitro. Wt
responder cells were co-cultured with Tregs from both tolerized and non-tolerized
IL10-/- mice. Nonetheless, cytokine release of wt CD4+CD25- T cells was
significantly suppressed. In conclusion, an opposite role and necessity of IL-10
regarding in vitro vs. in vivo Treg-mediated suppression of IFNγ production was
identified. Hence, this raises the question for the exact mechanism and pathway of
the different suppression patterns, since IL-10 controls in vivo expansion of naïve
CD4+ T cells as well as wasting disease, EAE, and inflammatory bowel disease
(IBD; [72, 73, 135]) whereas inhibition of gastritis (135) and in vitro suppression of
responder cells is IL-10-independent (49). Possibly, the dispensability of IL-10
regarding the in vitro suppressive capacity of both nTregs and iTregs might be due to
different layers of immune regulation, since our in vitro assays do not reflect the
impact of APC activation and the complex in vivo environment and rather
represent a ‘simple’ microenvironment. The type and number of cells from the
innate immune system, especially APCs, might explain the dispensability of IL-10
in vitro in contrast to the in vivo situation, where a stronger immune response is
required due to increased inflammatory processes. In vivo cell-cell interactions are
typically complex, varying in time and location, whereas in vitro assays are often
short-lived and regional. Schematically, figure 4.1 represents a possible
explanation for the differential requirement of IL-10 due to the corresponding
inflammation status.
DISCUSSION
89
Fig. 4.1: Layers of regulation of the immune response and relevance of immunosuppressive
cytokines [from (136)]
In vitro systems represent the most primitive level followed by low levels of local
inflammation and higher levels of systemic inflammation. The latter is always
accompanied by potent activation of cells of the innate immune system including
DCs, granulocytes, macrophages/monocytes, and NK cells, which ultimately
require stronger suppression comprising immunosuppressive cytokines such as
IL-10 or TGFβ (136, 137).
Nevertheless, it might be interesting to identify tolerogenic markers of Tregs of Con
A-pretreated mice, since induced Tregs demonstrated special features both in vivo
and in vitro in contrast to naive Tregs: Beside significant attenuation of liver damage
mediated by tolerized Tregs, a higher suppressive potential with increased IL-10
release was noticed in vitro. This clearly indicates that Tregs, which have already
been exposed to Con A, are specifically primed: Tregs from tolerized mice
recognize the same antigens faster and hence suppress Con A-induced hepatitis
and in vitro cytokine release of responder cells more effectively.
To test the possibility of CTLA-dependent ‘infectious tolerance’ as an outstanding
suppressive mechanism of Tregs from Con A-tolerant mice, experiments with a
specific cAMP inhibitor were performed. More precisely, CD4+CD25+ nTregs
generated in the thymus are characterized by the expression of the transcription
factor FoxP3 (65), CTLA-4, and the glucocorticoid-induced TNF-receptor-related
DISCUSSION______________________________________________________
90
protein (GITR; [64]). CTLA-4 is responsible for mediating in vitro inhibition of
proliferation and IL-2 expression via cell-cell contact. Upon TCR stimulation,
CTLA-4 is expressed on the surface of Tregs followed by interaction with B7 on
responder cells and overexpression of a potent inhibitor of IL-2 transcription in
responder cells, namely ICER. Subsequently, activated FoxP3- responder cells
themselves express CTLA-4 on their surface engaging neighboring CD4+FoxP3- T
cells. In an ‘infectious manner’, ICER expression is induced in these cells leading
to successive attenuation of IL-2 expression. Recently, it has been demonstrated
that forced FoxP3 expression in CD25- responder cells induced constitutive
expression of ICER resulting in a regulatory phenotype. ICER is also upregulated
by cAMP-activated transcription factors. Recently, it has been shown that cAMP
takes part in Treg-mediated suppression in vitro by transfer of the second
messenger cAMP from regulatory T cells into responder cells via gap junctions;
the suppressive activity of nTregs was abolished by a specific cAMP inhibitor (Rp-
cAMPS) as well as by a gap junction inhibitor. However, in the present study co-
cultures of responder cells and Tregs displayed an interesting phenomenon: Tregs
isolated from Con A tolerized mice were still able to suppress IL-2 mRNA
expression of responder cells despite blockade of cAMP in contrast to naive Tregs,
suggesting differentiation of Tregs from tolerized mice to tolerogenic cells that
suppress IL-2 production by a mechanism different from infectious tolerance.
Notably, ICER, the cAMP- and FoxP3-inducible repressor of IL-2 transcription, as
well as FoxP3 were strongly upregulated in Rp-cAMPS-pretreated CD4+
responder cells co-cultured with Tregs from tolerized animals. These results
suggest a cAMP-independent, probably FoxP3 and ICER-dependent suppression
by Con A-polarized Tregs. Further experiments will be necessary to identify markers
responsible for the increased immunosuppressive efficiency of tolerized Tregs both
in vitro and in vivo.
In summary, the differential role of IL-10 in Treg-mediated suppression could be
confirmed in the present study, since IL-10 is essential in vivo, but not in vitro.
Moreover, IL-10-producing CD4+CD25+FoxP3+ Tregs from tolerized mice represent
a new population with increased suppressive activity and might be kept in mind for
the establishment of novel therapeutic approaches in complex immunoregulatory
systems.
DISCUSSION
91
4.2 The conversion of Kupffer cells from type I to type II
macrophages
In the last few years, more and more interest was directed to alternative activation
of macrophages. Classically activated macrophages (MΦ) require two signals to
become activated: The first signal is IFNγ, which primes MΦ for activation; the
second signal is TNFα itself or an inducer of TNFα (148), e. g. exposure to
microbes or microbial products such as LPS. After activation, these MΦ migrate to
sites of inflammation where they encounter pathogens and degrade them. Due to
their type 1 response, many have referred to these cells as MΦ1 or type I
macrophages, mirroring the Th1 nomenclature.
Recently, another type of MΦ was identified called “alternatively activated
macrophages” (MΦ2a; [149]. They are induced by treatment with IL-4 and IL-13
(150, 151). They are not efficient at antigen presentation and mainly produce IL-10
and IL-1 receptor antagonist (148).
Beside these two types, so-called type II-activated MΦ (MΦ2b; [149]) were initially
identified during an examination of ligation of FcγRs on activated MΦ.
Subsequently, a turn off of IL-12 synthesis and secretion of large amounts of IL-10
could be detected (152, 153). Similar to classically activated MΦ, type II activation
requires two signals: ligation to FcγRs and a stimulatory signal to influence
cytokine production (TLRs, CD40, CD44; [148]). The dramatic induction of IL-10
release by these MΦ suggested that they possess anti-inflammatory activities. And
indeed, in vitro-generated type II MΦ were able to rescue mice from lethal
endotoxemia in contrast to either control MΦ or type II MΦ from IL10-/- mice (154)
confirming that this effect was a result of IL-10 secretion.
Accordingly, in tolerized mice large amounts of the immunosuppressive IL-10 were
secreted by KCs supposing a Con A-induced conversion of KCs from TNFα/IL-6-
producing type I macrophages to IL-10-producing type II macrophages.
Normally, KCs fulfil diverse functions in the liver including clearance of endotoxin
from the portal circulation, antigen-presentation or the release of soluble mediators
such as cytokines (6). In response to LPS, KCs synthesize IL-1, IL-6, and TNFα
(84). On the one hand, this might be beneficial for host defense, since IL-6 induces
DISCUSSION______________________________________________________
92
the hepatic acute-phase response preventing propagation of unnecessary and
harmful inflammation in the liver sinusoid; however, production of the pleiotropic
cytokine IL-6 by LSECs and KCs was also found during acute and chronic human
liver disease (155). Comparably, IL-6 is also released by KCs in the murine model
of Con A immune-mediated liver injury inducing an acute hepatitis along with KC-
secreted TNFα (14, 29, 30) and NKT-cell-produced IFNγ (26-28) after a single Con
A challenge.
Beside IL-6 and TNFα, KCs produce significant amounts of the
immunosuppressive cytokine IL-10, with IL-6 and TNFα production by KCs and
LSECs being suppressed by high IL-10 concentrations (84). Hence, KCs with a
type II phenotype are able to control the release of pro-inflammatory cytokines by
an autoregulatory mechanism. These observations suggest an important role of
KCs in regulation of local immune response and inflammation in the liver.
Since LSECs and KCs in liver sinusoid are repeatedly exposed to endotoxin
present in the venous blood of the portal vein, this phenomenon was mimicked in
vitro by Knolle and colleagues (124): Interestingly, a second stimulation of LSECs
with LPS induced a state of tolerance, which resulted in a decreased IL-6
secretion. These findings are in accordance to Con A tolerance and resemble the
cytokine profile found here, since a second Con A challenge after one week
caused a reduction of IL-6 and TNFα expression both in plasma and liver tissue
accompanied by an increased IL-10 release. This might presume a Con A-induced
differentiation of Kupffer cells from type I to type II macrophages that instigates
them to increased IL-10-production upon restimulation in a similar manner to Con
A-tolerized Tregs, with IL-10 playing an important role in Con A tolerance. To
confirm the importance of KCs in tolerization-induced IL-10 production, KCs were
depleted by clodronate-liposomes prior to Con A rechallenge. Indeed, in KC-
depleted, Con A-pretreated mice IL-10 augmentation was reduced in plasma and
especially in the liver as detected by measurement of intrahepatic IL-10 mRNA-
levels. This indicates that KCs contribute to IL-10 production in Con A tolerance.
Double-depletion of both Tregs and KCs prior to Con A restimulation caused a
largely diminished IL-10 response in both saline- and Con A-pretreated mice,
verifying that CD4+CD25+ Tregs and KCs together are crucial for primary IL-10
production and especially for IL-10 augmentation in tolerized mice provoked by a
DISCUSSION
93
Kupffer cells Tregs T cells
IL-6 TNFα IFNγ IL-2IL-10
Tr1?
IL-17
Con A-induced differentiation to IL-10-producing CD4+CD25+FoxP3+ Tregs and to
type II macrophages, respectively.
Thus, cytokine-induced suppression by Con A-primed, IL-10-secreting Tregs and
KCs and development of tolerance might help to downregulate inflammatory
reactions in the liver. Hence, IL-10, autologous patients’ Tregs or still unidentified
differentiation factors may be promising tools for therapeutic intervention against
immune-mediated liver injury.
4.3 Proposed mechanism of Con A-mediated tolerance
Summarizing all results, figure 4.2 shows the proposed mechanism of Con A-
mediated tolerance.
Fig. 4.2: Mechanism of Con A-mediated tolerance
In the present study, the murine model of Con A-induced hepatitis was used. A
single intravenous injection of a sublethal dose of Con A induces acute immune-
mediated liver injury in mice. The first Con A challenge results in a pronounced Th1
response: the pro-inflammatory cytokine TNFα is mainly released by KCs, whereas
DISCUSSION______________________________________________________
94
IFNγ is largely secreted by NKT cells. Additionally, IL-2, IL-6, and IL-17 contribute to
induction of acute hepatitis manifested by liver necrosis and elevated transaminase
activities. In contrast, the immunosuppressive cytokine IL-10 is protective in this
model, since administration of recombinant IL-10 ameliorates Con A-induced liver
damage (33) and IL10-/- mice develop a more severe Con A hepatitis compared to
wt mice (see chapter 3.2.1). Interestingly, Con A restimulation induces a tolerogenic
state within one week characterized by an anti-inflammatory cytokine profile with
downregulation of IFNγ, TNFα, IL-2, IL-6, and IL-17 and a significant increase of IL-
10 production. Con A-induced conversion of CD4+CD25+FoxP3+ Tregs and KCs is
responsible for the tolerization-induced IL-10 augmentation. Finally, IL-10 exerts its
immunosuppressive function on effector T cells by inhibiting the release of pro-
inflammatory cytokines. In contrast, suppression of IL-2 production appears to be
IL-10 independent rather involves Tregs (see Fig. 3.18: reduced suppression factor
after CD25-depletion) and a kind of Con A-induced anergy (see chapter 4.2).
DISCUSSION
95
4.4 Outlook
It has been highlighted repeatedly in the present work that Con A-induced hepatitis
includes many factors and components of the immune system and hence
represents an adequate experimental model of immune-mediated liver injury
resembling autoimmune hepatitis in humans. In conjunction with the present work,
the most interesting consensus between the animal model and the human disease
is the detection of immunosuppression in state of remission. This fact might allow
identifying potential targets for therapy of complex human immune-mediated
diseases by means of Con A tolerance. Nevertheless, it is always difficult to
transfer observations and results from mouse to man.
At present, AIH is normally medicated with the corticosteroid prednisone alone or
in combination with azathioprine primarily aiming at downregulation of boosting
immune response. Both treatment protocols show high survival rates and work
best when AIH is diagnosed early. However, a rate of 13% of treatment failures
and the failure to induce permanent remission in most patients underlines the
urgent need to develop additional treatment regimens (12). Furthermore,
management of side effects such as weight gain, high blood pressure, anxiety,
osteoporosis, or diabetes is very important. Therefore, the identification and
characterization of IL-10-producing CD4+CD25+FoxP3+ Tregs from Con A-tolerized
mice might represent a novel therapeutic option. It is hypothesized that Con A
induces peripheral conversion of CD4+ lymphocytes into CD4+CD25+FoxP3+ IL-10-
producing regulatory T cells in vivo which suppress Con A-induced immune-
pathology more efficiently. Indeed, the use of in vivo differentiated Tregs would
represent an advance in the treatment of immune-pathologies such as
autoimmune gastritis, MS, or AIH compared to in vitro expanded and re-injected
patients’ Tregs, since the in situ development of antigen-specific Tregs in
lymphopenic organisms might prevent generalized and long-term
immunosuppression with prednisone and azathioprine. Nevertheless, possible
side effects and induction of pro-inflammatory cytokine release have to be
dampened by immunosuppressive drugs in the early phase of immunotherapy.
Later, immunosuppressive drugs have to be withdrawn to guarantee an intact
response to pathogens. The aim might be the induction of a complete response
DISCUSSION______________________________________________________
96
and permanent remission without adverse and harmful side effects in AIH patients.
Thus, the tolerance-mediating markers of Con A-polarized Tregs have to be
identified and compared to markers on naïve Tregs, e. g. by microarray analysis. A
promising candidate might be programmed cell death 1 (PD-1) and the interaction
with its ligand, PDL-1. It has been observed, that PD-1-/- mice spontaneously
develop autoimmune diseases (156). Therefore, PD-1 has been postulated to
have essential roles in the regulation of autoimmunity. Indeed, recent studies
clearly demonstrated that PD-1 plays an important role in induction and
maintenance of peripheral tolerance. PD-1 ligands on antigen-presenting cells
have been shown to switch off autoreactive T cells and induce peripheral
tolerance, whereas those on parenchymal cells prevent tissue destruction by
suppressing effector T cells to maintain tolerance. A possible involvement of PD-1
and its ligand in Con A tolerance can be assumed, since PDL-1 is also expressed
on hepatocytes (157). Hence, the use of PD-1-/- and PDL-1-/- mice and the study of
differences of further co-stimulatory/ inhibitory molecule expression (e. g. CTLA-4,
CCR5, TGFβ) will be helpful to gain further insights into mechanisms of liver
tolerance. Beside Tregs, potential candidates might be hepatocytes, LSECs or
hepatic stellate cells.
Moreover, the influence of gender and sex hormones has to be checked in more
detail, since (a) severity of Con A hepatitis differed in female and male mice (own
observations and [111]), (b) development of Con A tolerance varied in female and
male IL10-/- mice, and (c) AIH generally shows a marked female predominance
supporting the idea that changes in hormonal regulation of the immune system
might contribute to AIH development beside environmental factors and genetic
predisposition (10). Due to these aspects, systematic investigations of different
expression patterns on various cell populations have to be performed in female
and male humans with AIH as well as in mouse models of immune-mediated liver
injury to identify major gender-related differences of functional markers.
Genetic prevalence as cause of AIH disease is evident by restriction to certain
haplotypes of HLA-antigens. Interestingly, strain differences regarding disease
severity are also detectable in the murine model of Con A-induced liver damage
(23). Namely, the peak of ALT (a) was dramatically higher in the C57BL/6 strain in
contrast to the BALB/c strain and (b) was inducible as early as 8 hours after Con A
injection in the C57BL/6 strain and as late as 24 hours after Con A injection in the
DISCUSSION
97
BALB/c strain. Interestingly, Con A-induced protection from Con A liver damage
was not restricted to C57BL/6 mice but was also found in male Balb/c mice in a
single pilot experiment. Also in female Balb/c mice, IL-10 had been identified as
essential mediator of protection in a work on chronic Con A hepatitis after several
weeks of repeated restimulation (158), thereby supporting the interpretation of IL-
10 as important tolerance factor. Nevertheless, a more focused investigation of
several mouse strains might be necessary in order to represent the human genetic
diversity and to exclude different influencing mechanisms regarding tolerance
establishment.
In conclusion, considering the above mentioned aspects, the aim might be the
development and enrichment of highly potent Tregs for the therapy of autoimmune
diseases. CD4+CD25+FoxP3+ IL-10-producing regulatory T cells identified in the
present study might actually represent an interesting and promising population in
the treatment of such immune-pathologies.
SUMMARY________________________________________________________
98
5 SUMMARY
Concanavalin A (Con A)-induced liver failure serves as model for immune-
mediated hepatic injury, in particular for human autoimmune hepatitis, since the
murine model and the human disease have some features in common such as
good responsiveness to immunosuppressive drugs, genetic prevalence with
respect to susceptibility, prevalence of CD4+ T cells, and immunosuppression in
state of remission. This study describes new aspects of mechanisms of
immunosuppression following Con A restimulation and especially elucidates the
role of IL-10-producing CD4+CD25+FoxP3+ regulatory T cells and Kupffer cells in
development of Con A tolerance.
The following results were obtained:
1. Con A restimulation induced tolerance against Con A within one week. The
typical pro-inflammatory Th1/Th17 cytokine response detected in Con A
hepatitis shifted to a Th2 response with downregulation of IFNγ, TNFα, IL-2,
IL-6 and IL-17 and a simultaneous augmentation of IL-10 expression.
Moreover, transaminase activities and liver necrosis were clearly
diminished indicating an attenuated liver damage.
2. Con A tolerance developed within 8 days after the first Con A administration
and persisted for several weeks suggesting a long-lasting process.
3. In vivo depletion of both CD4+CD25+ Tregs by anti-CD25 mAb and KCs by
clodronate-liposomes significantly reduced the tolerization-induced IL-10
release suggesting these two populations as main source of IL-10. Con A
pretreatment appears to convert CD4+ T cells into IL-10-producing FoxP3+
regulatory T cells and KCs from type I macrophages into IL-10-secreting
type II macrophages, respectively.
SUMMARY
99
4. CD4+CD25+ Tregs from Con A-tolerized mice significantly ameliorated Con A-
induced hepatitis in wt mice in an IL-10-dependent manner, since tolerized
Tregs from IL10-/- mice failed to reduce Con A liver injury. Interestingly, the
adoptively transferred CD4+CD25+ Tregs were FoxP3-positive in contrast to
peripherally induced ‘IL-10 Tregs’, which have recently been identified to
exert therapeutic effects in other mouse models of autoimmune diseases.
5. The necessity of IL-10 during establishment of Con A tolerance seems to
depend on gender, since female IL10-/- mice were still able to develop Con
A tolerance in contrast to male IL10-/- mice, correlating with gender-related
differences in humans regarding the incidence of autoimmune diseases.
6. CD4+CD25+ regulatory T cells from tolerized mice also exhibited a higher
suppressive activity in vitro, since they inhibited cytokine-production of co-
cultured CD4+CD25- responder cells more efficiently than naïve Tregs.
Moreover, in vitro neutralization or lack of IL-10 failed to reverse the
immunosuppressive capacity of Tregs whereas IL-10 was indispensable in
vivo in the present study.
7. NKT cells are essential for Con A hepatitis due to their pronounced IFNγ
release. However, NKT-cell deficient CD1d-/- mice developed Con A
tolerance, thereby excluding NKT cells as tolerance-mediating cell
population, although the frequency of intrahepatic NKT cells is significantly
elevated in Con A-treated mice at day 8 following intervention.
In summary, Con A pretreatment caused an immunosuppressive milieu followed
by induction of Con A tolerance upon restimulation. The tolerogenic state was
characterized by an anti-inflammatory cytokine profile with elevated IL-10 release,
which was mainly produced by Kupffer cells and CD4+CD25+FoxP3+ Tregs. The
latter population primed by the first Con A challenge ameliorated fulminant Con A-
induced hepatitis in an IL-10-dependent manner. In contrast, in vitro Treg-mediated
suppression was mediated cytokine-independent.
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100
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DEUTSCHSPRACHIGE ZUSAMMENFASSUNG___________________________
114
Deutschsprachige Zusammenfassung
Die Concanavalin A (Con A)-induzierbare, T- und NKT-Zell-vermittelte
Leberschädigung bei der Maus dient als Modell für immunvermittelte
Lebererkrankungen beim Menschen. Es spiegelt vor allem das Krankheitsbild der
Autoimmunhepatitis sehr gut wider, da das Mausmodell und die humane
Erkrankung einige Gemeinsamkeiten aufweisen wie zum Beispiel die gute
Ansprechbarkeit auf Immunsuppressiva, die genetische Prävalenz, die
Abhängigkeit von CD4+ T-Zellen und Immunsuppression in der Remissionsphase.
Diese Arbeit beschreibt neue Aspekte immunsuppressiver Mechanismen nach
Con A-Restimulation und untersucht im Besonderen die Rolle von IL-10-
produzierenden CD4+CD25+FoxP3+ regulatorischen T-Zellen und Kupffer-Zellen
bei der Entwicklung der Con A-Toleranz.
Folgende Ergebnisse wurden erzielt:
1. Con A-Injektion induzierte innerhalb einer Woche Toleranz gegenüber Con
A-Restimulation. Die bei der Con A-vermittelten Hepatitis ausgeprägte pro-
inflammatorische Th1/Th17-Antwort verschob sich zugunsten einer Th2-
Antwort. Eine verminderte IFNγ−, TNFα−, IL-2-, IL-6- und IL-17-Produktion
ging mit einem Anstieg des immunosuppressiven Zytokins IL-10 einher.
2. Con A-Toleranz entwickelte sich ab Tag 8 nach der ersten Con A-Gabe und
hielt über mehrere Wochen an, was für einen langfristigen Prozess spricht.
3. In vivo-Depletion regulatorischer T-Zellen mittels anti-CD25-Antikörpern
und Depletion der Kupffer-Zellen mit Hilfe von Clodronat-Liposomen führte
zu einem signifikanten Rückgang der IL-10-Freisetzung in Con A-
vorbehandelten Tieren. Somit stellten diese beiden Zellpopulationen die
Hauptproduzenten von IL-10 dar. Es wird postuliert, dass die Con A-
Vorbehandlung eine FoxP3-Expression und gesteigerte IL-10-Freisetzung
in herkömmlichen CD4+ T-Zellen induziert hat. Zusätzlich veränderten
DEUTSCHSPRACHIGE ZUSAMMENFASSUNG
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Kupffer-Zellen ihren Phänotyp von Typ I-Makrophagen zu IL-10-
produzierenden Typ II-Makrophagen.
4. CD4+CD25+ regulatorische T-Zellen aus Con A-toleranten Mäusen
schützten signifikant vor einem Con A-induzierten Leberschaden. Der
therapeutische Effekt hing von IL-10 ab, da regulatorische T-Zellen aus Con
A-vorbehandelten IL-10 KO-Mäusen nicht in der Lage waren, den
Leberschaden zu reduzieren. Interessanterweise exprimierten die injizierten
Zellen den Transkriptionsfaktor FoxP3, was den spezifischen Marker
natürlich-vorkommender, im Thymus gereifter regulatorischer T-Zellen
darstellt. Normalerweise sind in der Peripherie induzierte regulatorische T-
Zellen, die in anderen Mausmodellen für Autoimmunerkrankungen
therapeutische Effekte mit Hilfe von IL-10 erzielen, FoxP3-negativ.
5. Die Notwendigkeit von IL-10 für die Ausprägung der Con A Toleranz hängt
möglicherweise vom Geschlecht ab, da weibliche IL-10 KO-Mäuse trotz
fehlendem IL-10 Toleranz entwickelten im Gegensatz zu männlichen IL-10
KO-Mäusen. Solche geschlechtsspezifischen Unterschiede kann man auch
bei humanen Autoimmunerkrankungen beobachten, da in vielen Fällen
Frauen eine stärkere Prävalenz aufweisen.
6. Zusätzlich zeichneten sich CD4+CD25+ regulatorische T-Zellen aus
toleranten Mäusen durch erhöhte Suppressionskapazität in vitro aus. Sie
inhibierten die Zytokinantwort von CD4+CD25- Effektor-T-Zellen stärker als
naive regulatorische T-Zellen. In vitro-Neutralisation von IL-10 hob die
Suppressionseigenschaften der regulatorischen T-Zellen nicht auf,
wohingegen IL-10 für immunmodulatorische Effekte in vivo unabdinglich
war.
7. NKT-Zellen sind für einen Con A-vermittelten Leberschaden aufgrund der
IFNγ-Freisetzung notwendig. NKT-Zell-defiziente CD1d KO-Mäuse waren
jedoch in der Lage, Con A-Toleranz zu entwickeln. Deshalb konnte man
NKT-Zellen als eine mögliche Toleranz-vermittelnde Zellpopulation
DEUTSCHSPRACHIGE ZUSAMMENFASSUNG___________________________
116
ausschließen, obwohl sich die Anzahl der NKT-Zellen in Con A-
behandelten Mäusen an Tag 8 nahezu verdoppelt hatte.
Zusammenfassend kann man feststellen, dass eine Vorbehandlung mit Con A
eine Immunaktivierung hervorrief, die bei Restimulation Toleranz gegenüber Con
A induziert hat. Das Toleranzstadium war durch ein anti-inflammatorisches
Zytokinprofil und erhöhte IL-10-Produktion gekennzeichnet. IL-10 wurde
größtenteils von Kupffer-Zellen und CD4+CD25+FoxP3+ regulatorischen T-Zellen
freigesetzt. Die durch die erste Con A-Gabe polarisierten regulatorischen T-Zellen
zeigten in vivo therapeutische Effekte, da sie einen Con A-vermittelten
Leberschaden mit Hilfe von IL-10 verbessern konnten, wohingegen die
suppressive Aktivität dieser Zellpopulation in vitro nicht von IL-10 abhing.
DANKSAGUNG
117
DANKSAGUNG
Zunächst möchte ich mich herzlich bei der Betreuerin meiner Dissertation, Frau
Prof. Dr. Gisa Tiegs, bedanken, die mir dieses interessante Forschungsthema
überlassen hat. Sie hat mich stets durch kompetente und hilfreiche
Diskussionsbeiträge unterstützt, aber auch zu eigenständigem Arbeiten motiviert.
Sie hat zudem hervorragende Arbeitsbedingungen und ein angenehmes,
freundliches Klima innerhalb der Arbeitsgruppe geschaffen, was den Fortschritt
dieser Arbeit sehr erleichtert hat.
Weiterhin möchte ich meinen besonderen Dank an Dr. Markus Biburger
aussprechen, der mich in die Thematik der Doktorarbeit und die Methodik der
Durchflusszytometrie hervorragend eingewiesen hat und mir anschließend zu
jedem Zeitpunkt meiner Arbeit mit interessanten Diskussionen und
weiterführenden Vorschlägen und Ideen zur Seite stand. Seine Vorarbeiten, aber
auch seine Ausdauer und Unterstützung haben entscheidend zum Gelingen der
hier vorliegenden Arbeit beigetragen.
Ferner danke ich Prof. Dr. Manfred Lutz und PD Dr. Reinhard Voll, den Mitgliedern
meiner Betreuungskommission im Rahmen des Graduiertenkollegs 592
„Lymphozyten“, für positive Diskussionsbeiträge und weiterführende
experimentelle Vorschläge. PD Dr. Robert Slany danke ich für die Übernahme des
Zweitgutachtens der vorliegenden Arbeit. Allen weiteren Mitgliedern des
Graduiertenkollegs, vor allem dem Sprecher Prof. Dr. Hans-Martin Jäck, danke ich
für die Möglichkeit, an Seminaren, Workshops, Symposien und Kongressen
teilzunehmen und somit zusätzliche Fähigkeiten zu erlangen. Besonders zu
erwähnen im Rahmen des Graduiertenkollegs sind die Kollegiaten Ruzi, Sandra,
Mitch, Benni, Stöpsel, Alex, Jens und Damian, die mich sehr herzlich in die bereits
bestehende 2. Förderperiode des GK 592 aufgenommen haben und dem
Färberhof 13 schließlich zu seinem jetzigen Image verholfen haben (☺) und immer
für Aufmunterung gesorgt haben.
DANKSAGUNG_____________________________________________________
118
Ein Dankeschön geht an Herrn Prof. Dr. Thomas Papadopoulos (ehemals Institut
für Pathologie, Universität Erlangen; jetzt Vivantes Klinikum Spandau, Berlin) für
die freundliche Unterstützung und die kompetente Beurteilung bei der Anfertigung
von HE-Schnitten.
Dr. Gabriele Sass möchte ich danken für eine angenehme Arbeitsatmosphäre im
gemeinsamen Büro über den Zeitraum meiner Forschungsaktivitäten hinweg.
Ganz besonderer Dank gilt auch Andrea Agli, Sonja Heinlein und Elena Tasika für
das angenehme Arbeitsklima im Labor und die hervorragende technische
Unterstützung.
Für den stets freundlichen und kollegialen Umgang sowie für den informativen
Austausch und die weiterführenden Gespräche möchte ich mich bei meinen
Mitdoktoranden Eva-Maria Vogel, Irena Kröger, Stefanie Buerbank, Mirjam
Schädle, Florian Haimerl und Dominik Abt bedanken. Sie haben es immer sehr gut
verstanden, mich zum richtigen Zeitpunkt abzulenken und bei Misserfolgen wieder
aufzubauen. Ich möchte mich auch für die zahlreichen Feierlichkeiten außerhalb
der Arbeit bei meinen lieb-gewonnenen Kollegen bedanken. Natürlich darf die
ununterbrochene und gegenseitige Versorgung mit Süßigkeiten nicht unerwähnt
blieben!
Außerhalb der Universität möchte ich meinen Dank besonders an meine Freunde
aussprechen, insbesondere an Bernd für das Lesen einiger englischer Texte,
weiterhin an Tobi B., Tobi Z., Katrin, Timo, Sonja, Thorsten, Katja, Alex, André,
Manu, Steffi, Kathrin und Susi, die immer ein offenes Ohr für mich hatten, wenn
sich mal Rückschläge bei den Forschungsarbeiten ergaben, die aber vor allem in
meinem privaten Bereich für einen äußerst guten Ausgleich gesorgt haben.
Vor allem aber möchte ich mich bei meinem verstorbenen Vater, dem diese Arbeit
auch gewidmet ist, und meiner Mutter bedanken, denn sie haben es mir erst
ermöglicht, ein Studium mit anschließender Promotion aufzunehmen. Ein Dank
geht auch an meinen Bruder Martin, der mich während der gesamten
Promotionszeit motiviert hat. Zum Schluss bedanke ich mich bei meinem Freund
René für die ununterbrochene, liebevolle und ausdauernde Unterstützung, die
DANKSAGUNG
119
leckeren Mahlzeiten (☺) und die aufmunternden und tröstenden Worte, die er stets
gefunden hat, wenn sich bei mir Schlechte-Launen-Phasen eingeschlichen haben.
Mai 2008 Annette Erhardt
LEBENSLAUF
Persönliche Daten
Name Annette Erhardt, Diplom-Biologin
Adresse Am Färberhof 13, 91052 Erlangen
Geburtsdatum 28.08.1980
Geburtsort Münchberg
Staatsangehörigkeit deutsch
Schulbildung
09/86 – 07/90 Grundschule in Helmbrechts
09/90 – 06/99 Abitur am Gymnasium Münchberg, Münchberg
Hochschulstudium
09/99 – 10/04 Diplom Biologie, Universität Bayreuth (Vordiplom) Friedrich-Schiller-Universität Jena (Diplom)
Promotion
01/05 – 03/08 Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Universität Erlangen-Nürnberg
Tolerance induction in the liver after T- and NKT-cell activation
Stipendien
03/05 – 02/08 Stipendium der Deutschen Forschungsgesellschaft im Rahmen des Graduiertenkollegs 592 („Lymphozyten“)
04/06 Stipendium „Young Investigator“, EASL, 2006, Wien
04/08 Stipendium „Young Investigator“, EASL, 2008, Mailand (Schering-Plough Unrestricted Educational Grant)
Sonstige Tätigkeiten
seit 03/05 Betreuung des Wahlpflichtpraktikums für Pharmazeuten an der Universität Erlangen-Nürnberg