Tolerance induction in the liver after T and NKT cell ... fileTolerance induction in the liver after...

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

INTRODUCTION

19

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

INTRODUCTION

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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23

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.

INTRODUCTION____________________________________________________

28

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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29

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.


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.


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.


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


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.


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.


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.


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.


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


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)


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


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).

RESULTS

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,

RESULTS_________________________________________________________

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-

RESULTS

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).

RESULTS

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).

RESULTS

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).

RESULTS_________________________________________________________

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).

RESULTS

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)

RESULTS_________________________________________________________

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

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

115

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

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