The role of c-jun N-terminal kinase (JNK) in human T cell ... · resulting from engagement of the T cell receptor (TCR) (i.e. Tetanus Toxoid), down-regulates Th1 cytokine production

The role of c-jun N-terminal kinase

(JNK) in human T cell function

Michelle Melino

(B. Biotech. with Hons)

Thesis submitted for the degree of Doctor of Philosophy

Department of Microbiology & Immunology

School of Molecular & Biomedical Science

The University of Adelaide February 2009

http://www.pdfonline.com/easypdf/?gad=CLjUiqcCEgjbNejkqKEugRjG27j-AyCw_-AP

TABLE OF CONTENTS

Summary..................................................................................................................................... i

Declaration................................................................................................................................ iii

Acknowledgements................................................................................................................... iv

Publications and presentations....................................................................................................v

Abbreviations........................................................................................................................... vii

Index of Figures ........................................................................................................................ xi

Index of Tables ....................................................................................................................... xvi

1 Chapter One ........................................................................................................................1

Introduction.................................................................................................................................1

1.1 General Introduction ...................................................................................................2

1.2 T cell development......................................................................................................3

1.3 CD4+ T cell classification ...........................................................................................4

1.4 Th1 and Th2 differentiation........................................................................................7

1.5 Th1 and Th2 cytokine patterns ...................................................................................7

1.6 Cytokines which impact on helper T cells................................................................11

1.7 T cells in allergy .......................................................................................................11

1.8 T cells in autoimmunity ............................................................................................13

1.9 Mechanism of T cell activation ................................................................................16

1.10 The MAPK pathways in T cell proliferation and cytokine production ....................20

1.11 Role of ERK in T cell proliferation and cytokine production ..................................21

1.12 Role of p38 in T cell proliferation and cytokine production ....................................25

1.13 Role of JNK in T cell proliferation and cytokine production ...................................30

1.14 The TAT-JIP peptide ................................................................................................37


1.15 Concluding remarks..................................................................................................43

1.16 Aims, hypotheses and significance...........................................................................43

2 Chapter Two .....................................................................................................................45

Materials and Methods..............................................................................................................45

2.1 Materials ...................................................................................................................46

2.2 Buffers ......................................................................................................................48

2.3 Purification of human PBMC ...................................................................................51

2.4 Purification of human T cells....................................................................................52

2.5 Purification of murine splenic T cells.......................................................................54

2.6 Determination of cell purity......................................................................................54

2.7 PHA-PMA and anti-CD3-anti-CD28 induced activation .........................................57

2.8 Tetanus Toxoid induced lymphocyte responses .......................................................57

2.9 Mixed Lymphocyte Reaction....................................................................................58

2.10 Allergen induced activation ......................................................................................58

2.11 Cytokine determination.............................................................................................59

2.12 Measurement of phosphorylated JNK and phosphorylated jun by western blotting61

2.12.1 Sample preparation ...........................................................................................61

2.12.2 Lowry’s Protein assay.......................................................................................61

2.12.3 Western Blot .....................................................................................................62

2.13 siRNA .......................................................................................................................62

2.14 Kinase profiler assays ...............................................................................................63

2.15 Statistical Analysis....................................................................................................64

3 Chapter Three ...................................................................................................................65

Role of JNK in T cell responses induced by PHA-PMA..........................................................65

3.1 Introduction...............................................................................................................66

3.2 PHA-PMA induced JNK activation in human T cells ..............................................68


3.3 Effect of TAT-JIP153-163 on the JNK pathway in human T cells ...............................72

3.4 Effect of the TAT-JIP153-163 peptide on human T cell function ................................74

3.5 Effect of the TAT-JIP153-163 peptide on murine T cell function................................78

3.6 Effect of the pharmacological JNK inhibitor, SP600125 on human T cell function80

3.7 Summary...................................................................................................................83

4 Chapter Four .....................................................................................................................84

Role of JNK in T cell responses induced via the TCR .............................................................84

4.1 Introduction...............................................................................................................85

4.2 Effect of the TAT-JIP153-163 peptide on the JNK pathway in TCR-induced T cells .87

4.3 Effect on human T cell function in response to anti-CD3-anti-CD28 antibodies ....90

4.4 Effect on T cell responses in the mixed lymphocyte reaction ..................................96

4.5 Effect on antigen-induced T cell responses ..............................................................99

4.6 Effect on allergen-induced T cell responses ...........................................................102

4.7 Summary.................................................................................................................105

5 Chapter Five....................................................................................................................108

Relationship between JNK, ERK and p38 in T cell function .................................................108

5.1 Introduction.............................................................................................................109

5.2 Role of ERK and p38 in PHA-PMA-induced T cell responses..............................110

5.3 The effect of ERK, p38 and JNK inhibition on PHA-PMA-induced T cell responses

116

5.4 Role of ERK and p38 in anti-CD3-anti-CD28-induced T cell responses...............120

5.5 The effect of ERK, p38 and JNK inhibition on anti-CD3-anti-CD28-induced T cell

responses .............................................................................................................................125

5.6 Summary.................................................................................................................129

6 Chapter Six .....................................................................................................................132

Specificity of the TAT-JIP153-163 peptide ................................................................................132


6.1 Introduction.............................................................................................................133

6.2 Effect of JIP-1-derived peptides on CDK2, CK1, p70S6K, Rsk1, SGK and DYRK

activity ................................................................................................................................134

6.3 Effect of the TAT-JIP153-172 peptide on PHA-PMA and anti-CD3-anti-CD28-

induced T cell responses. ....................................................................................................147

6.4 Investigating the role of JNK using RNA interference...........................................157

6.5 Summary.................................................................................................................161

7 Chapter Seven .................................................................................................................163

Discussion...............................................................................................................................163

7.1 Introductory remark ................................................................................................164

7.2 Targeting the JNK signalling pathway with the TAT-JIP peptides........................165

7.3 Role of JNK in T cell proliferation.........................................................................168

7.4 Role of JNK in T cell cytokine production.............................................................170

7.5 Interaction between members of the MAPK family in T cell function ..................173

7.6 The relationship between Th1, Th2, Th17 and Tregs.............................................179

7.7 Infection and immunity, allergy and autoimmunity ...............................................180

7.8 Concluding remarks................................................................................................181

References...............................................................................................................................184


i

SUMMARY

T cells are involved in cellular pathways which enable the immune system to protect us

against infection and cancer. However, the same mechanisms also allow T cells to generate

chronic inflammatory conditions, including autoimmunity and allergy. Thus a concerted effort

has been made to try to understand how the immune system functions in order to inhibit

responses which may have harmful effects on tissues and organs. There is a continued search

for new immunosuppressants which can only be accomplished through a better understanding

of the pathways that regulate T cell function. This includes the intracellular signalling

pathways which modulate T cell proliferation and cytokine production.

While the Mitogen-Activated Protein Kinases (MAPK), extracellular signal-regulated protein

kinases (ERK) and p38 have received attention, the role of the stress-activated protein kinases

or c-jun N-terminal kinases (JNK) remains controversial. To overcome some of the

limitations in studying the role of JNK, a new approach was taken in this thesis. The

investigations used recently described peptides (TAT-JIP153-163 and TAT-JIP153-172) derived

from the scaffold protein, JIP-1, which have previously been demonstrated to act as JNK

pathway inhibitors. The research characterised the specificity of these inhibitors to enable the

appropriate interpretation of data.

Using these inhibitors, we were able to show that JNK regulated human T cell proliferation

and cytokine production in T cell responses induced independently of TCR ligation (PHA-

PMA) or via the TCR (anti-CD3-anti-CD28 antibodies, Mixed Lymphocyte Reaction (MLR),

Tetanus Toxoid and Der p 2). The data demonstrated that JNK primarily regulated the Th1

cytokine patterns (IFNγ, IL2 and LT) with minimal effect on Th2 cytokine production (IL4,

IL10) in response to all stimulatory models. However, while the JNK signalling pathway


ii

promoted T cell proliferation and cytokine production in response to PHA-PMA, the pathway

depressed these responses following stimulation with anti-CD3-anti-CD28 antibodies and

Tetanus Toxoid. Thus activation of JNK with microbial pathogens such as Pseudomonas

aeruginosa (PA), which non-specifically activate T cells, may promote lymphocyte

proliferation and the release of Th1 cytokines, such as IFNγ. In contrast, JNK activation

resulting from engagement of the T cell receptor (TCR) (i.e. Tetanus Toxoid), down-regulates

Th1 cytokine production. Therefore, it is likely that the JNK signalling pathway may dampen

the development of chronic inflammatory conditions resulting from infection with

intracellular parasites and autoimmune diseases. In contrast to Tetanus Toxoid, responses to

the recombinant house dust mite allergen, Dermatophagoides pteronyssinus (Der p 2) were

promoted by JNK, leading to an increase in Th1 cytokine production. Thus the results suggest

that the use of JNK inhibitors could exacerbate both inflammatory conditions (autoimmunity

and allergy) and this may also apply to p38 but not the ERK signalling pathway.


iii

DECLARATION

This work contains no material which has been accepted for the award of any other degree or

diploma in any university or other tertiary institution and, to the best of my knowledge and

belief, contains no material previously published or written by another person, except where

due reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being made

available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

………………………. …………………...

Michelle Melino Date


iv

ACKNOWLEDGEMENTS

Firstly, I would like to thank my supervisors, Professor Tony Ferrante and Professor Shaun

McColl for all your patience, guidance and support. Thank you for helping me to achieve a

goal I never thought would be possible and to Associate Professor Charles Hii, thank you for

answering all my questions and teaching me the challenging world of cell signalling.

A special thank you to Professor W.R. Thomas at the University of Western Australia for

kindly donating the recombinant allergen and to Kathie Carman for assisting me with all the

cytokine work. I am truly thankful for all your time and effort and I could not have completed

this study without you.

To all of my friends in the Immunopathology department, the diagnostic staff: Tricia, Kathie,

Lily, Tuyen, Monica, Jess and Renee, who welcomed me with open arms from the very first

day. Thank you for all your assistance, kindness and support.

Thank you to all of my friends who have shared the research lab with me over the years. To

everyone who was there at the very beginning, Laura, Amy, Mel, Christos and James, I would

have been lost without your support. To everyone who was with me until the very end, Alex

and Yong, I will miss our little corner of the lab and to Dr. Mukaro (Villey), Bernadette (BM)

and Mei (Mei, Mei), thank you for always being there, for making me laugh even when I felt

like crying and for sharing your morning tea time with me. It was always my favourite part of

the day. I am going to miss you all very much.

Finally, to my parents, Didi and all of my family and friends, thank you for all your patience,

love and support. This would not have been possible without you.


v

PUBLICATIONS AND PRESENTATIONS

Publications

Melino, M., C. S. Hii, S. R. McColl and A. Ferrante (2008). "The effect of the JNK inhibitor,

JIP peptide, on human T lymphocyte proliferation and cytokine production." J Immunol

181(10): 7300-6.

Costabile, M., C. S. Hii, M. Melino, C. Easton and A. Ferrante (2005). "The

immunomodulatory effects of novel beta-oxa, beta-thia, and gamma-thia polyunsaturated fatty

acids on human T lymphocyte proliferation, cytokine production, and activation of protein

kinase C and MAPKs." J Immunol 174(1): 233-43.

Presentations

“The role of c-jun N-terminal kinase (JNK) in human T cell proliferation and cytokine

production.”

San Raffaele Scientific Institute (2008)

Milan, Italy

“Regulation of human T lymphocyte proliferation and cytokine production by c-jun N-

terminal kinase (JNK).”

Australasian Society for Immunology (ASI) 37th Annual Conference (2007)

Sydney, Australia


vi

“Regulation of cytokine production by Mitogen-Activated Protein kinases in human T

lymphocytes.”

University of Adelaide (2006)

Adelaide, Australia


vii

ABBREVIATIONS

AICD activation-induced cell death

AP-1 activator of transcription 1

APC antigen presenting cells

APS ammonium persulfate

ASK1 apoptosis signal-regulated kinase 1

ATF2 activating transcription factor 2

ATP adenosine tri-phosphate

BD Becton Dickinson

BSA bovine serum albumin

CaMK calcium/calmodulin-dependent kinase

CARMA-1 caspase recruitment domain containing membrane-

associated guanylate kinase protein-1

CBA cytometric bead array

CDK2 cyclin dependent kinase 2

CDR complementarity determining regions

CHK2 checkpoint kinase 2

CIA collagen-induced arthritis

CK1 casein kinase 1

Con A concanavalin A

COX cyclooxygenase

DAG diacylglycerol

DMARD disease modifying antirheumatic drug

DMSO dimethyl sulfoxide

DTT dithiothreitol


viii

DYRK dual-specificity tyrosine phosphorylated and regulated

kinase

EDTA ethylenediaminetetraacetic acid

ERK extracellular signal-regulated kinase

FBS foetal bovine serum

FITC fluorescein isothiocynate

GAPDH glyceraldehyde-3-phosphate dehydrogenase

GM-CSF granulocyte monocyte-colony stimulating factor

HDM house dust mite

HIPK2 homeodomain interacting protein kinase 2

HIV human immunodeficiency virus

HPK1 hematopoietic progenitor kinase 1

HPLC high-performance liquid chromatography

HRP horse radish peroxidase

IFN interferon

Ig immunoglobulin

IKK IκB kinase

IL interleukin

IP3 inositol 1,4,5-trisphosphate

ITAM immunoreceptor tyrosine-based activation motif

iTreg induced regulatory T cells

IκB inhibitor of NFκB

JAK Janus kinase

JBD JNK binding domain

JIP-1 JNK interacting protein 1

JNK c-jun N-terminal kinase


ix

LAT linker of activated T cells

LT lymphotoxin

MAPK mitogen-activated protein kinase

MELK maternal embryonic leucine zipper kinase

MHC major histocompatibility complex

MLK3 mixed lineage kinase 3

MLR mixed lymphocyte reaction

NFAT nuclear factor of activated T cells

NFκB nuclear factor of κ-light-chain-enhancer of activated B

cells

NK natural killer cells

NP40 Nonidet-40

NSAID non steroidal anti-rheumatic drug

p70S6K p70 ribosomal protein S6 kinase

PA pseudomonas aeruginosa

PBMC peripheral blood mononuclear cells

PDK 3’ phosphoinositide-dependent kinase

PE phycoerythrin

PG prostaglandin

PHA phytohaemagglutinin

PI3K phosphatidylinositol 3 kinase

PIP2 phosphatidylinositol-4,5-bisphosphate

PKC protein kinase C

PLCγ1 phospholipase Cγ1

PMA 12-myristate-13-acetate

PMSF phenylmethylsulfonyl fluoride


x

PTK protein tyrosine kinase

RA rheumatoid arthritis

Rag1 recombination activating gene 1

RPMI Roswell Park Memorial Institute

RPMI/ΔAB RPMI 1640 containing 5 % heat-inactivated blood group

AB serum

RPMI/ΔFBS RPMI 1640 containing 5 % heat-inactivated foetal

bovine serum

Rsk1 ribosomal S6 protein kinase 1

SDS sodium dodecyl sulphate

SGK serum and glucocorticoid-regulated kinase

siRNA small interfering RNA

SLE systemic lupus erythematosus

SLP-76 SH2 domain-containing leukocyte protein of 76 kDa

SOCS suppressor of cytokine signalling

SOS son of sevenless

STAT signal transducer and activator of transcription

TAK1 transforming growth factor β-activated kinase 1

TAT transactivator of transcription

TCR T cell receptor

Th helper T cell

TNF tumour necrosis factor

Treg regulatory T cell

ZAP-70 ζ-associated protein-70


xi

INDEX OF FIGURES

Fig. 1.1. Summary of CD4+ helper T cell subsets. .....................................................................6

Fig. 1.2. Mechanism of T cell activation. .................................................................................19

Fig. 1.3. The ERK1/ERK2 cascade. .........................................................................................23

Fig. 1.4. The p38 cascade. ........................................................................................................28

Fig. 1.5. The JNK cascade. .......................................................................................................34

Fig. 1.6. The chemical structure of SP600125..........................................................................35

Fig. 1.7. JIP-1 is a scaffold protein for the JNK signalling pathway........................................40

Fig. 2.1. Flow chart of experimental procedure........................................................................53

Fig. 2.2. Dot plot of T cell analysis by flow cytometry. ...........................................................56

Fig. 2.3. Examples of standard curves for human cytokine production. ..................................60

Fig. 3.1. JNK is phosphorylated in human T cells in response to PHA-PMA stimulation. .....70

Fig. 3.2. Jun is phosphorylated in human T cells in response to PHA-PMA stimulation. .......71

Fig. 3.3. Inhibition of jun phosphorylation by TAT-JIP153-163 in intact human T cells in

response to PHA-PMA stimulation. .........................................................................................73

Fig. 3.4. Inhibition of human T cell proliferation by the TAT-JIP153-163 peptide. ....................75

Fig. 3.5. The control peptide did not inhibit T cell proliferation in response to PHA-PMA

stimulation. ...............................................................................................................................76

Fig. 3.6. Inhibition of human T cell cytokine production by the TAT-JIP153-163 peptide. ........77

Fig. 3.7. Inhibition of T cell proliferation by TAT-JIP153-163 in mouse splenic T cells. ...........79

Fig. 3.8. SP600125 does not inhibit human T cell proliferation in response to PHA-PMA

stimulation. ...............................................................................................................................81

Fig. 3.9. SP600125 did not inhibit jun phosphorylation in human T cells. ..............................82


xii

Fig. 4.1. Jun is phosphorylated in human T cells in response to anti-CD3-anti-CD28

antibodies. .................................................................................................................................88

Fig. 4.2. Inhibition of JunB phosphorylation by the TAT-JIP153-163 peptide in human T cells in

response to anti-CD3-anti-CD28 antibodies.............................................................................89

Fig. 4.3. Enhancement of T cell proliferation by the TAT-JIP153-163 peptide in response to anti-

CD3-anti-CD28 antibody stimulation.......................................................................................92

Fig. 4.4. Enhancement of cytokine production by the TAT-JIP153-163 peptide in response to

anti-CD3-anti-CD28 antibody stimulation. ..............................................................................93

Fig. 4.5. Inhibition of T cell proliferation by SP600125 in response to anti-CD3-anti-CD28

antibodies. .................................................................................................................................94

Fig. 4.6. The effect of SP600125 on cytokine production in response to anti-CD3-anti-CD28

stimulation. ...............................................................................................................................95

Fig. 4.7. Enhancement of cell proliferation by the TAT-JIP153-163 peptide in the MLR...........97

Fig. 4.8. Enhancement of IFNγ production by the TAT-JIP153-163 peptide in the mixed

lymphocyte reaction..................................................................................................................98

Fig. 4.9. Enhancement of lymphocyte proliferation by the TAT-JIP153-163 peptide in response

to Tetanus Toxoid. ..................................................................................................................100

Fig. 4.10. Enhancement of cytokine production by the TAT-JIP153-163 in response to antigen

stimulation. .............................................................................................................................101

Fig. 4.11. Inhibition of lymphoproliferation by the TAT-JIP153-163 peptide in response to Der p

2. .............................................................................................................................................103

Fig. 4.12. Inhibition of cytokine production by TAT-JIP153-163 peptide in response to Der p 2.

................................................................................................................................................104

Fig. 5.1. Enhancement of T cell proliferation by PD98059 in response to PHA-PMA

stimulation. .............................................................................................................................112


xiii

Fig. 5.2. Effect of the ERK pathway inhibitor, PD98059 on T cell cytokine production in

response to PHA-PMA stimulation. .......................................................................................113

Fig. 5.3. Inhibition of T cell proliferation by the p38 pathway inhibitor, SB203580 in response

to PHA-PMA stimulation. ......................................................................................................114

Fig. 5.4. Inhibition of T cell cytokine production by SB203580 in response to PHA-PMA

stimulation. .............................................................................................................................115

Fig. 5.5. Inhibition of T cell proliferation by a combination of ERK, p38 and JNK inhibitors

in response to PHA-PMA stimulation. ...................................................................................117

Fig. 5.6. Inhibition of T cell cytokine production by p38 and JNK inhibitors in response to

PHA-PMA stimulation. ..........................................................................................................118

Fig. 5.7. Inhibition of T cell cytokine production by a combination of ERK, p38 and JNK

inhibitors in response to PHA-PMA stimulation....................................................................119

Fig. 5.8. Inhibition of T cell proliferation by PD98059 in response to anti-CD3-anti-CD28

antibodies. ...............................................................................................................................121

Fig. 5.9. Inhibition of T cell cytokine production by PD98059 in response to anti-CD3-anti-

CD28 antibodies. ....................................................................................................................122

Fig. 5.10. Enhancement of T cell proliferation by SB203580 in response to anti-CD3-anti-

CD28 antibodies. ....................................................................................................................123

Fig. 5.11. Enhancement of IL2 production by SB203580 in response to anti-CD3-anti-CD28

antibodies. ...............................................................................................................................124

Fig. 5.12. The effect of combining ERK, p38 and JNK inhibitors on T cell proliferation in

response to anti-CD3-anti-CD28 antibodies...........................................................................126

Fig. 5.13. The effect of combining p38 and JNK inhibitors on T cell proliferation in response

to anti-CD3-anti-CD28 antibodies..........................................................................................127


inhibitors in response to CD3-CD28 stimulation. ..................................................................128


xiv

Fig. 6.1. TAT-JIP153-163 inhibits CDK2/cyclin A activity.......................................................135

Fig. 6.2. TAT-JIP153-163 inhibits p70S6K activity...................................................................136

Fig. 6.3. TAT-JIP153-163 inhibits SGK activity. .......................................................................137

Fig. 6.4. TAT-JIP153-163 does not inhibit CK1 activity............................................................138

Fig. 6.5. TAT-JIP153-163 does not inhibit DYRK activity........................................................139

Fig. 6.6. TAT-JIP153-163 does not inhibit Rsk1 activity...........................................................140

Fig. 6.7. TAT-JIP153-172 does not inhibit CDK2/cyclin A activity..........................................141

Fig. 6.8. TAT-JIP153-172 does not inhibit p70S6K activity. .....................................................142

Fig. 6.9. TAT-JIP153-172 does not inhibit SGK activity. ..........................................................143

Fig. 6.10. TAT-JIP153-172 does not inhibit CK1 activity..........................................................144

Fig. 6.11. TAT-JIP153-172 does not inhibit DYRK activity......................................................145

Fig. 6.12. TAT-JIP153-172 inhibits Rsk1 activity......................................................................146

Fig. 6.13. Inhibition of human T cell proliferation by the TAT-JIP153-172 peptide in response to

PHA-PMA. .............................................................................................................................149

Fig. 6.14. Inhibition of human T cell cytokine production by the TAT-JIP153-172 peptide in

response to PHA-PMA. ..........................................................................................................150

6.15. Enhancement of human T cell proliferation by the TAT-JIP153-172 peptide in response to

anti-CD3-anti-CD28 antibodies. .............................................................................................151


CD3-CD28 stimulation. ..........................................................................................................152

6.17. Enhancement of human T cell proliferation by the TAT-JIP153-172 peptide in response to

Tetanus Toxoid. ......................................................................................................................153


Tetanus Toxoid stimulation. ...................................................................................................154

Fig. 6.19. Inhibition of lymphoproliferation by the TAT-JIP153-172 peptide in response to Der p

2. .............................................................................................................................................155


xv

Fig. 6.20. Inhibition of cytokine production in TAT-JIP153-172 treated PBMC in response to

Der p 2. ...................................................................................................................................156

Fig. 6.21. The effect of siRNA on JNK1 and GAPDH expression. .......................................159

Fig. 7.1. Summary of the role of the MAPK in human T cell function in response to PHA-

PMA (A) and anti-CD3-anti-CD28 antibodies (B).................................................................176

Fig. 7.2. Summary of the role of the MAPK in human T cell function in response to Tetanus

Toxoid. ....................................................................................................................................177

Fig. 7.3. Summary of the role of the MAPK in human T cell function in response to Der p 2

allergen....................................................................................................................................178


xvi

INDEX OF TABLES

Table 1.1: Effect of ERK inhibition on T cell function. ...........................................................24

Table 1.2. Effect of p38 inhibition on T cell function. .............................................................29

Table 1.3. Effect of JNK inhibition on T cell function.............................................................36

Table 1.4. The amino acid sequences for the TAT peptide and the long and short JIP-1-

derived peptides. .......................................................................................................................41

Table 1.5. Recent studies involving the use of JIP-derived peptides. ......................................42

Table 4.1. Summary of the effect of the TAT-JIP153-163 peptide on T cell function in TCR-

induced models. ......................................................................................................................107

Table 5.1. Comparison of the effect of MAPK inhibition on T cell proliferation in response to

PHA-PMA and CD3-CD28 stimulation. ................................................................................130

Table 5.2. Comparison of the effect of MAPK inhibition on T cell cytokine production in

response to PHA-PMA and CD3-CD28 stimulation. .............................................................131

Table 6.1 Comparison of the effect of the JIP-1 derived peptides on human T cell function in

response to PHA-PMA, anti-CD3-anti-CD28 antibodies, Tetanus Toxoid and Der p 2........162

Table 7.1. Comparison between the effect of SP600125, TAT-JIP153-163 and TAT-JIP153-172 on

CDK2/cyclin A, CK1, p70S6K, Rsk1, SGK and DYRK activity. .........................................167


1

1Chapter One

Introduction


2

1.1 General Introduction

The main lymphocyte populations, T cells and B cells, originate from the same precursors in

the bone marrow but have quite distinct roles in the immune response. T cells develop into

antigen-responding cells in the thymus and can mature into cytotoxic T cells, which attack

and lyse virus-infected cells. They may also develop into helper T (Th) cells which are

required for the development of effector T cells such as cytotoxic T cells, B cell responses and

antibody production. While this enables the immune system to produce antibodies against

foreign materials and fight infection, in autoimmunity antibodies are produced in response to

auto-antigen, resulting in tissue destruction. T cells are also responsible for the activation of

macrophages which eliminate intracellular bacteria and viruses, the suppression of the

immune response and the regulation of tolerance to auto-antigens.

T cells are divided into two main populations, the CD8+ cytotoxic T cells and the CD4+ Th

cells. Furthermore, the CD4+ Th cells can be sub-categorised into naïve and memory T cells

which respond to new antigens and previously encountered pathogens respectively. There are

also four subpopulations of effector T cells: Th1, Th2, Th17 and regulatory T cells (Treg).

These cells are responsible for mediating inflammatory responses through the production of

distinct sets of cytokines.

In the case of autoimmunity, these cells and their products have become targets of

immunosuppressive therapies such as anti-CD3 antibodies, which are responsible for the

depletion of T cells and cyclosporine, which targets the calcium-calcineurin intracellular

signalling pathway. While some benefits are provided by current therapies, there are also

numerous side effects and therefore the search continues for appropriate therapeutics which

more selectively target T cell inflammatory pathways. This challenge has attracted studies


3

into the role of T cell intracellular signalling pathways such as the Mitogen-Activated Protein

Kinases (MAPK). The MAPK superfamily consists of the extracellular signal-regulated

protein kinases (ERK), the stress-activated protein kinases or c-Jun N-terminal kinases (JNK)

and p38. These kinases have been implicated in cell proliferation, differentiation, survival and

apoptosis and therefore may provide a potential target for therapeutic intervention.

1.2 T cell development

During fetal and early postnatal life, lymphoid precursor cells derived from the bone marrow,

enter the thymic cortex and undergo cell expansion and differentiation (Mowat et al. 2005).

At this time, the “triple negative” cells have no T cell receptor (TCR), CD3 or co-receptor

molecules (CD4, CD8), however, following the development of a pre-TCR, these “double

negative” cells become “double positive” by expressing both CD4 and CD8 molecules

(Mowat et al. 2005). The mature αβ TCR then replaces the pre-TCR (Mowat et al. 2005).

TCR genes are assembled from separate V, D and J gene segments by genetic recombination

(Goldrath et al. 1999). The TCRβ chain is assembled at the “double negative” stage, whereby

a short D gene segment is juxtaposed to a short J segment prior to rearrangement with a V

gene segment, while TCRα chains are arranged at the “double positive” stage whereby there

are no D segments, only the rearrangement of the V and J segments (Goldrath et al. 1999).

The complementarity determining regions (CDR), CDR1 and CDR2 are encoded by the V

gene segment, while CDR3 is created by the VJ segments, thus providing greater diversity

(Goldrath et al. 1999). Since the TCR alone is unable to transduce signals after antigen

binding, T cells also possess a signalling CD3 complex which is first expressed at low levels

in the “double negative” stage of development (Mowat et al. 2005).


4

Following the expression of a mature CD3-TCR complex and both the CD4 and CD8 co-

receptor molecules, T cells undergo positive and negative selection in the thymic cortex (Starr

et al. 2003). Cells that recognise self major histocompatibility complexes (MHC) and antigens

are positively selected (Starr et al. 2003; Jiang et al. 2005). Furthermore, T cells expressing

high affinity/avidity for MHC and self-antigens are eliminated during negative selection (Starr

et al. 2003; Jiang et al. 2005). This ensures the survival of T cells with a low affinity/avidity

for MHC and self-antigen complexes (Starr et al. 2003; Jiang et al. 2005). The surviving cells

which have a TCR that recognises MHC class I retain the CD8 co-receptor molecule, while

those that recognise MHC class II retain the CD4 co-receptor molecule (Starr et al. 2003).

These “single positive” cells undergo further maturation in the medulla before exiting the

thymus whereby they recirculate from the blood to the secondary lymphoid organs (Starr et al.

2003).

1.3 CD4+ T cell classification

Upon activation, helper T cells can be subdivided into Th1, Th2, Th17 or Treg effector cells

which specialize in producing distinct cytokines (Fig. 1.1) (Zhu et al. 2008). Th17 cells

regulate responses to extracellular bacteria and fungi through the production of IL17, IL21

and IL22, while Treg cells play an important role in self-tolerance and suppression of the

immune response through the production of TGF-β, IL10 and IL35 (Zhu et al. 2008). TGFβ is

also important for inducing regulatory T cells (iTreg) and Th17 differentiation (Zhu et al.

2008).

Th1 and Th2 cells, the focus of this study, were first classified by Mosmann et al. (1986).

These experiments identified two distinct subsets of helper T cells in murine clones. Those

clones that produced IL2, Interferon γ (IFNγ), and Granulocyte Monocyte-Colony Stimulating


5

Factor (GM-CSF) in response to antigen or Concanavalin A (Con A) were classified as Th1

cells, while those that produced IL4 and IL10 were classified as Th2 cells (Mosmann et al.

1986).

Human Th1 and Th2 cells similar to those described in mice were later discovered by Del

Prete et al. (1991). T cell clones specific for the bacterial antigen, Mycobacterium

tuberculosis secreted predominantly IL2 and IFNγ (Th1 profile), while T cell clones specific

for the nematode Toxocara canis produced IL4 and IL5 (Th2 profile) (Del Prete et al. 1991).

Similarly, T cell clones specific for house dust mite (HDM) (Dermatophagoides

pteronyssinus) or grass pollen allergens (Lolium perenne) were shown to produce high levels

of IL4, IL5 and minimal IFNγ (Parronchi et al. 1991).


6

Fig. 1.1. Summary of CD4+ helper T cell subsets. Th cells can be divided into four

subpopulations: Th1, Th2, Th17 and Treg, which all have unique cytokine patterns. Adapted

from Zhu et al. (2008).

IFNγ, IL2, LT

IL4, IL5, IL10, IL13

TGFβ, IL10, IL35

IL17, IL21, IL22

TGFβ, IL6,

IL21, IL23

IFNγ,

IL12

TGFβ IL4

Th1

Th2

Th17

iTreg

Naïve CD4+

Extracellular bacteria, fungi, autoimmunity

Self-tolerance, suppression of the immune response

Extracellular parasites, allergy

Intracellular pathogens, autoimmunity

IFNγ, IL2, LT


TGFβ, IL10, IL35

IL17, IL21, IL22

TGFβ, IL6,

IL21, IL23

IFNγ,

IL12

TGFβ IL4

Th1

Th2

Th17

iTreg

Naïve CD4+

IFNγ, IL2, LT


TGFβ, IL10, IL35

IL17, IL21, IL22

TGFβ, IL6,

IL21, IL23

IFNγ,

IL12

TGFβ IL4

Th1

Th2

Th17

iTreg

Naïve CD4+

Extracellular bacteria, fungi, autoimmunity

Self-tolerance, suppression of the immune response

Extracellular parasites, allergy

Intracellular pathogens, autoimmunity


7

1.4 Th1 and Th2 differentiation

Th1 differentiation is initiated by TCR signalling in combination with signalling through

IFNγ and IL27 cytokine receptors which are associated with signal transduction and activator

of transcription 1 (STAT1) (Hibbert et al. 2003; Lucas et al. 2003). STAT1 signalling up

regulates the transcription factor, T-bet, which is the main factor involved in Th1 commitment

(Szabo et al. 2000). This is followed by an increase in IFN gene expression and the up-

regulation of the IL12 receptor, while Th2 factors are suppressed (Robinson et al. 1997;

Mullen et al. 2001).

Th2 differentiation is initiated by TCR signalling in combination with IL4 receptor signalling

through STAT6 (Ouyang et al. 2000; Harrington et al. 2006). This leads to an increase in the

transcription factor, GATA3 which enhances Th2 gene expression while suppressing Th1

factors (Zheng et al. 1997). In addition, GATA3 auto activation provides an IL4-independent

mechanism for Th2 differentiation (Ouyang et al. 1998; Ouyang et al. 2000).

1.5 Th1 and Th2 cytokine patterns

Immune cells produce many cytokines which have single and overlapping properties. Some

cytokines are produced predominantly by one cell type, while others may be secreted by

several cells of the immune system. T cells play a unique role in immunological responses by

releasing cell-specific cytokines. In addition, T cells release cytokines which are common to

other cell types, thus dominating the immune response.


8

IL2 production is induced by antigens and mitogens including phytohaemagglutinin (PHA)-

phorbol 12-myristate 13-acetate (PMA) and anti-CD3 and anti-CD28 antibodies. The

cytokine binds the receptor, IL2R, which consists of an chain involved in ligand binding,

and a and chain which are responsible for signal transduction (Arai et al. 1990; Curfs et al.

1997; Feghali et al. 1997). Receptor engagement leads to the activation, growth and

differentiation of T cells and promotes B cell growth and differentiation, Natural Killer (NK)

cell growth and activity, enhances expression of MHC class II molecules and increases

production of IFNγ and lymphotoxin (LT) (Arai et al. 1990; Curfs et al. 1997; Feghali et al.

1997). IL2 may also promote innate immunity by stimulating neutrophil cell migration,

oxygen radical production and degranulation (Kowanko et al. 1987a).

IL3 is a Th2 cytokine which induces the differentiation of granulocytes and macrophages,

expression of MHC class II molecules on neutrophils and the differentiation and growth of

thymocytes (Curfs et al. 1997; de Groot et al. 1998; Guthridge et al. 1998). The IL3 receptor

contains a specific ligand-binding subunit, IL3Rα, and a β subunit which is common to IL3,

IL5 and GM-CSF (de Groot et al. 1998; Guthridge et al. 1998). IL3 receptors are expressed on

early hematopoietic progenitor cells in addition to eosinophils and basophils (de Groot et al.

1998; Guthridge et al. 1998).

IL4 promotes Th2 cytokine production while inhibiting the Th1 response (Kumaratilake et al.

1992; Curfs et al. 1997; Feghali et al. 1997). In addition, the cytokine stimulates

immunoglobulin E (IgE) production by B cells and promotes Th2 differentiation while

suppressing the development of Th1 cells and IL1 and tumour necrosis factor (TNF)

production by monocytes/macrophages (Curfs et al. 1997; Feghali et al. 1997).


9

IL5, a Th2 cytokine activates eosinophils, basophils and stimulates B cell isotype switching

towards IgA (Curfs et al. 1997; Feghali et al. 1997). In addition, IL5 also increases B cell

proliferation and T cell cytotoxicity (Feghali et al. 1997).

IL9 is readily produced by Th2 cells and functions through the IL9 receptor (IL9R) (Curfs et

al. 1997; Feghali et al. 1997). IL9 enhances mast cell activity and T cell survival and acts in

combination with IL4, to promote the production of IgG and IgE (Curfs et al. 1997; Feghali et

al. 1997).

IL10, produced by Th2 cells, Tregs and monocytes, inhibits cell-mediated immunity while

promoting humoral responses (Commins et al. 2008). IL10 reduces IFNγ and IL2 production

by Th1 cells, IL4 and IL5 production by Th2 cells, IL12 and TNF production by macrophages

and IFNγ and TNF production by NK cells (Curfs et al. 1997; Feghali et al. 1997; Commins

et al. 2008). In addition, IL10 inhibits the expression of the co-stimulatory molecule, CD28

and stimulates proliferation and immunoglobulin secretion by B cells (Curfs et al. 1997;

Feghali et al. 1997; Commins et al. 2008).

IL13, like IL4 and IL10, also promotes Th2 and suppresses Th1 responses (Curfs et al. 1997;

Feghali et al. 1997). IL13, predominantly secreted by Th2 cells, inhibits the production of

inflammatory cytokines such as IL1, TNF, IL6 and IL8 while enhancing B cell proliferation,

differentiation and IgG and IgE class switching (Curfs et al. 1997; Feghali et al. 1997).

IFNγ, secreted predominantly by Th1 cells, binds a heterodimeric receptor consisting of

IFNR1 and IFNR2 chains, resulting in the activation of the Janus Kinase (JAK)-STAT

pathway (Pestka et al. 1997). IFNγ promotes the Th1 response while suppressing Th2

cytokine production, enhances MHC class II expression on APC and stimulates the priming,


10

activation and function of neutrophils and macrophages leading to the production of pro-

inflammatory cytokines (Kowanko et al. 1987b; Kumaratilake et al. 1990; Kowanko et al.

1992; Curfs et al. 1997).

TNF (TNF) binds the receptors, TNF-RI and TNF-RII and is predominantly secreted by

macrophages (Schottelius et al. 2004). The second species of TNF, LT (TNF) exists as

secreted LT or membrane-associated LT and is readily produced by Th1 cells (Schneider et

al. 2004; Schottelius et al. 2004). While LT also binds TNF-RI and TNF-RII, LT binds the

specific LT receptor (Schneider et al. 2004). Together, TNF and LT prime and activate a

wide variety of immune cells including macrophages, lymphocytes, neutrophils, eosinophils

and endothelial cells (Ferrante et al. 1988; Kowanko et al. 1996; Curfs et al. 1997).

GM-CSF is produced by a wide variety of immune cells including T cells, B cells,

macrophages, mast cells, eosinophils and neutrophils (Curfs et al. 1997; Barreda et al. 2004;

Hamilton 2008). The GM-CSF receptor is composed of an 85 kDa α chain and a 130 kDa β

chain, primarily expressed on macrophages, neutrophils and eosinophils (Curfs et al. 1997;

Barreda et al. 2004; Hamilton 2008). GM-CSF binding activates three pathways including

JAK-STAT, MAPK and PI3K which in turn promote the proliferation, differentiation,

activation and survival of macrophages, neutrophils and eosinophils (Barreda et al. 2004;

Hamilton 2008). GM-CSF also activates haematopoiesis, enhances antigen presentation,

histamine release, antibody-dependent cell killing and phagocytosis and has been implicated

in the pathogenesis of rheumatoid arthritis, psoriasis, asthma and cancer (Barreda et al. 2004;

Hamilton 2008).


11

1.6 Cytokines which impact on helper T cells

IL12 consists of two disulfide-linked subunits including p40 and p35 and is secreted by

dendritic cells, monocytes, macrophages, neutrophils and B cells (Langrish et al. 2004;

Paunovic et al. 2008). IL12 binds a receptor complex consisting of IL12β1 and IL12β2,

which is expressed on T cells, NK cells and dendritic cells (Langrish et al. 2004; Paunovic et

al. 2008). The JAK-STAT pathway is activated by IL12β2, while IL12β1 is required for high

affinity binding of the cytokine (Langrish et al. 2004; Paunovic et al. 2008). IL12 stimulates

IFNγ production by Th1 cells while suppressing IL10 and IL13 production by Th2 cells

(Langrish et al. 2004; Paunovic et al. 2008). In addition, IL12 enhances the cytolytic activity

of NK cells and is negatively regulated by suppressor of cytokine signalling (SOCS)-1

(Langrish et al. 2004; Paunovic et al. 2008).

IL27 is a member of the IL6 family and is readily produced by antigen-presenting cells

including dendritic cells and macrophages (Stumhofer et al. 2008). IL27 binds a receptor

complex consisting of a ligand binding subunit, IL27ra and glycoprotein 130, a 130 kDa

signal transducing subunit (Stumhofer et al. 2008). IL27 acts in a pro-inflammatory manner to

increase IFNγ production by CD4+ T cells, CD8+ T cells and NK cells and in an anti-

inflammatory manner to enhance IL10 production, thereby reducing the release of IFNγ by

CD4+ T cells and the production of IL6 and TNF by monocytes (Villarino et al. 2004a;

Villarino et al. 2004b; Paunovic et al. 2008; Stumhofer et al. 2008).

1.7 T cells in allergy

T cells are the main regulators of allergic diseases such as asthma and hayfever. Upon

exposure to environmental allergens including the HDM, Dermatophagoides pteronyssinus


12

and Dermatophagoides farinae, non-allergic (non-atopic) individuals develop an

immunological response which involves the production of allergen-specific IgG1 and IgG4

antibodies and modest T cell responses (Galli 2000; Kay 2000). However, atopic individuals

have a genetic predisposition to produce IgE antibodies in response to environmental

allergens and thus have elevated IgE serum levels (Kay 2000; 2001a; Holt 2004). T cells from

atopic individuals produce high levels of Th2 cytokines including IL4, IL5 and IL13 in vitro

(Galli 2000; Kay 2000; 2001a).

In an allergic response, IL4 and IL13 enhance IgE antibody production which requires both

the NFκB pathway and IL4-induced STAT6 activation and is suppressed by IFNγ production

(Kay 2001a). IgE antibodies bind to FcεR1 receptors on tissue mast cells, blood basophils and

eosinophils. Subsequent allergen exposure stimulates cross-linking of the membrane-bound

IgE, causing degranulation (Kay 2001a; Akdis 2006a; Akdis 2006b). Granules containing

inflammatory mediators such as histamine, proteolytic enzymes (tryptase), prostaglandins,

leukotrienes, cytokines and chemokines are released into the surrounding tissue inducing the

symptoms associated with an acute allergic reaction such as wheezing, sneezing and

rhinorrhea (Kay 2001a; Akdis 2006a; Akdis 2006b).

Chronic allergy is controlled by IgE antibodies which bind FcεRI receptors on dendritic cells

and monocytes and FcεRII receptors on B cells, thereby enhancing allergen uptake and

presentation to T cells (Akdis 2006a; Akdis 2006b). Th2 cytokines such as IL4, IL5 and IL13

are all involved in chronic allergic inflammation (Kay 2001a). IL4 and IL13 stimulate the

continual production of IgE, IL5 and IL9 are involved in eosinophil development, IL4 and

IL9 stimulate mast cell development and IL4, IL9 and IL13 enhance mucus production which

results in the symptoms associated with chronic inflammation (Kay 2001a).


13

Current treatment for allergy includes anti-allergic medication and specific immunotherapy

(Kay 2001b). Anti-allergic medications such as histamine H1-receptor antagonists (anti-

histamines) and anti-cholinergic agents aim to relieve the symptoms associated with allergy

(Kay 2001b). In addition to H1-receptor antagonism, anti-histamines also regulate the

production of pro-inflammatory cytokines such as TNF, IL1 and IL6, in addition to the Th2

cytokines IL4 and IL13, while anti-cholinergic agents prevent the contraction of bronchial

smooth muscle and are thus used to relieve asthma (Marshall 2000; Inagaki et al. 2001).

Specific immunotherapy involves the administration of increasing concentrations of allergen

extract over a long period, resulting in an increase in Th1 cytokines and a reduction in Th2

cytokines (Kay 2001b). The enhanced IFNγ and IL12 production induces a reduction in IgE

production, thus suppressing allergic inflammation (Kay 2001b). However, unfortunately, this

treatment has been associated with numerous side effects (Winther et al. 2006).

1.8 T cells in autoimmunity

CD4+ T cells including Th1 and Th17 cells are important in the pathogenesis of autoimmune

diseases, particularly rheumatoid arthritis (RA). In 1975, a predominance of CD4+ T cells

was observed in the synovium of RA patients (Van Boxel et al. 1975). Furthermore, mice

lacking the IFN receptor were shown to develop collagen-induced arthritis (CIA)

significantly earlier and more severely than the wildtype thus providing evidence for the role

of Th1 cells in the disease (Manoury-Schwartz et al. 1997; Vermeire et al. 1997).

More recently, Th17 cells were implicated in the pathogenesis of RA as they were first

discovered following experiments in CIA mice (Harrington et al. 2005). However, recent

reports show that Th1 and not Th17 cells are abundant in the joints of RA patients (Yamada et


14

al. 2008), suggesting that both cells may play a role in autoimmune disease. Furthermore,

evidence has emerged to suggest that Tregs may suppress Th1 and Th17 responses to auto-

antigens (Romagnani 2006).

Consequently, the treatment of autoimmune disease involves targeting T cells however, not

all therapy functions in this manner. Some of the current treatments for autoimmunity include

non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen and meloxicam

which aim to relieve pain and inflammation associated with RA and systemic lupus

erythematosus (SLE) (Suleyman et al. 2007). NSAIDs have been demonstrated to inhibit the

synthesis of cyclooxygenase (COX) and lipoxygenase products, prevent neutrophil

aggregation, adhesion and chemotaxis in addition to the release of toxic oxygen radicals

(Warner et al. 1999; Suleyman et al. 2007). Furthermore, disease modifying antirheumatic

drugs (DMARDs), including methotrexate, are also commonly used in the treatment of RA.

Methotrexate has been demonstrated to inhibit pro-inflammatory cytokine production,

lymphocyte proliferation, neutrophil chemotaxis and adherence (Kremer et al. 1994;

Constantin et al. 1998). However, like NSAIDs, prolonged use of DMARDs commonly

produce gastrointestinal discomfort such as nausea, diarrhoea and constipation, while rare side

effects include liver disease, leukopenia and lymphoma (Borchers et al. 2004).

As an alternative or in conjunction with NSAIDs and DMARDs, the TNF antagonists,

infliximab, adalimumab and etanercept are frequently used for the treatment of RA and

psoriasis (Fan et al. 2007). Infliximab and adalimumab are IgG1 monoclonal antibodies,

which bind both soluble and membrane-bound TNF, fix complement and induce cytotoxicity

(Graves et al. 2007). Etanercept, however, is a human soluble TNF receptor fusion protein

which binds predominantly soluble TNF and LT, thus preventing TNF receptor binding


15

(Graves et al. 2007). Unfortunately, the adverse effects associated with TNF antagonists

include serious infection and lymphoma (Fan et al. 2007; Graves et al. 2007).

The T cell signalling pathways are specifically targeted by Cyclosporine A, FK506 and

rapamycin which are widely used in the treatment of autoimmune diseases and are produced

by Tolypocladium inflatumgams, Streptomyces tsukubaenis and Streptomyces hygroscopicus

respectively (Kunz et al. 1993). Cyclosporine A acts by binding to the enzyme, cyclophilin,

which inhibits calcineurin thus preventing NFAT translocation and IL2 gene transcription (Ho

et al. 1996; Almawi et al. 2000). While Cyclosporine A initially binds cyclophilin, FK506

and rapamycin bind FK506-binding protein-12 (Ho et al. 1996; Almawi et al. 2000;

Lindenfeld et al. 2004). Unfortunately, these immunosuppressive drugs are associated with

side effects including nephrotoxicity (tremor, headache, seizures, insomnia, mental status

changes and visual problems), hypertension, hyperlipidemia, nausea, vomiting, development

of osteoporosis and increased risk of type II diabetes (Lindenfeld et al. 2004).

Anti-CD3 monoclonal antibodies act to deplete T cells by inducing apoptosis (Janssen et al.

1992; Wesselborg et al. 1993) or cellular cytotoxicity (Jung et al. 1987). Treatment with anti-

CD3 antibody has not only been shown to reverse the rejection of heart (Gilbert et al. 1987)

and liver transplantations (Farges et al. 1994) but also to improve autoimmune diseases. In

clinical trials involving type I diabetes patients, treatment with the humanized CD3

monoclonal antibody hOKT31 (Ala-Ala) resulted in improved insulin production (Herold et

al. 2002) and furthermore this antibody also reduced joint inflammation in psoriatic arthritis

patients (Utset et al. 2002). However, common side effects include fever, rash and anaemia

(Herold et al. 2002).


16

1.9 Mechanism of T cell activation

While some benefits are derived from current autoimmune disease therapy, there are serious

concerns with the associated side effects. It is therefore not surprising that we have sought

alternatives. Protein kinases have now become the second largest group of drug targets, after

the G-protein-coupled receptors (Cohen 2002). A number of kinase inhibitors have recently

been approved for clinical use including Imatinib, which is a tyrosine kinase inhibitor that is

used for the treatment of chronic myeloid leukaemia (Deininger et al. 2003), Sorafenib, which

targets the Raf-MEK-ERK pathway and is currently administered for the treatment of primary

liver cancer (Wilhelm et al. 2008) and Sunitinib, a receptor tyrosine kinase inhibitor which is

used for the treatment of gastrointestinal stromal tumour (Demetri et al. 2006). The interaction

between the protein kinases during T cell activation is described below.

The TCR complex contains a ligand-binding subunit which consists of a αβ heterodimer and a

signal transducing subunit which includes CD3γ-CD3ε, CD3ε-CD3δ and a ζ-ζ homodimer

(Qian et al. 1997; Kane et al. 2000) (Fig. 1.2). Each CD3 chain contains immunoreceptor

tyrosine-based activation motifs (ITAMs), which upon phosphorylation create binding sites

for the protein tyrosine kinases (PTKs) (Fig. 1.1) (Qian et al. 1997; Kane et al. 2000).

The four families of PTKs include Src, Csk, Tec and Syk (Qian et al. 1997; Kane et al. 2000).

Prior to TCR engagement, the Src family PTK, Lck is maintained in an inactive state by Csk.

Following stimulation there is an increased distribution of the TCR to the lipid rafts,

heterogeneous lipid microdomains enriched in sphingomyelin, glycosphingolipids and

cholesterol (Huang et al. 2004). Lck becomes activated by decreased exposure to Csk and

increased exposure to CD45, a transmembrane phosphatase which removes the inhibitory


17

phosphate group from the tyrosine kinase, resulting in ITAM phosphorylation (Huang et al.

2004).

Following phosphorylation, the ITAMs then serve as binding sites for -associated protein-70

(ZAP-70), a Syk family PTK (Fig. 1.2). After ZAP-70 activation, adaptor proteins including

linker of activated T cells (LAT) and SH2 domain-containing leucocyte protein of 76 kDa

(SLP-76) are phosphorylated, thus enabling the formation of the signalosome (Fig. 1.2) (Qian

et al. 1997; Kane et al. 2000; Huang et al. 2004). LAT binds the linker protein Grb2 which

forms a complex with son of sevenless (SOS), inducing the conversion of GDP-bound Ras to

the active form (Roose et al. 2000). SLP-76, however, binds Vav, Nck and Itk which serve as

an integrator of signals arising from the signalosome and from phosphatidylinositol 3-kinase

(PI3K) (Fig. 1.2) (Huang et al. 2004). These adaptor proteins also regulate the activation of

Phospholipase Cγ1 (PLCγ1) and the subsequent hydrolysis of phosphatidylinositol-4,5-

bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3),

second messengers in protein kinase C (PKC) activation and calcium mobilization (via IP3)

respectively (Fig. 1.2) (Kane et al. 2000; Huang et al. 2004; Matthews et al. 2006; Mondino et

al. 2007).

A sustained increase in calcium concentration leads to the activation of the phosphatase,

calcineurin, which regulates transcription factors such as the Nuclear Factor of Activated T-

cells (NFAT) (Loh et al. 1996). Upon dephosphorylation by calcineurin, NFAT translocates

to the nucleus and induces the transcription of T cell cytokines such as interleukin 2 (IL2)

(Loh et al. 1996). PKC, especially the novel PKC isoform, PKCθ, also plays an important role

in activation of the NFAT, nuclear factor of κ-light-chain-enhancer of activated B cells

(NFκB) and MAPK pathways (Isakov et al. 2002). Upon TCR stimulation, the scaffold

molecule, caspase recruitment domain containing membrane-associated guanylate kinase


18

protein-1 (CARMA-1) is phosphorylated by PKCθ, thus enabling the formation of a

CARMA-1/ B-cell CLL/lymphoma 10 (Bcl-10)/ mucosa associated lymphoid tissue

lymphoma translocation gene 1 (MALT-1) complex (Fig. 1.2) (Matthews et al. 2006; Weil et

al. 2006; Mondino et al. 2007). Bcl-10 and MALT-1 then regulate the ubiquitination of

inhibitor of NFκB (IκB) kinase (IKK), leading to NFκB activation (Fig. 1.2) (Matthews et al.

2006; Weil et al. 2006). NFκB is also induced by PI3K, which phosphorylates PIP2 to

generate PIP3, enabling the binding of the serine/ threonine kinase, 3'-phosphoinositide-

dependent kinase-1 (PDK-1) and its downstream target, Akt (Fig. 1.2) (Huang et al. 2004;

Weil et al. 2006; Mondino et al. 2007).

The cell surface glycoproteins, CD4 and CD8 are also important for T cell activation. CD4 is

expressed in mature helper T cells and interacts directly with MHC class II-restricted

molecules, while CD8 is expressed in mature cytotoxic T cells and interacts with MHC class

I-restricted molecules. Upon TCR stimulation, these co-receptors are believed to play a

critical role through their association with the Src family PTK, Lck which is essential for

signal transduction in T cells as mentioned earlier (Fig. 1.2) (Veillette et al. 1988; Miceli et al.

1991). Another T cell surface molecule, CD28, provides a co-stimulatory signal which is

required for T cell activation (June et al. 1990). CD28 recruits Grb2, which as mentioned

earlier is important for the activation of Ras, and PI3K which leads to Akt activation (Fig.

1.2) (August et al. 1994; Okkenhaug et al. 1998).


19

Fig. 1.2. Mechanism of T cell activation. T cell activation involves many pathways including PI3K, Ca++/calmodulin, MAPK, PKCθ and NFκB.

ε γ ε δ

CD4 L

A

T

Lck

SLP-76

Nck Vav Itk

PI3K

PLC-γ

Grb2

P

ζ ζ

CD3

ss

ss

ss

ss

α ß

TCR

ss

ss s

sss

ITAMSAKT PKCθ

B

c

l

MALT-1

CARMA -1 ZAP 70 P ZAP-70

CD28 PIP2 → PIP3

IP 3

Ca ++

NFAT

NF - κB I

κ

B P

Cell Membrane

Gene Transcription

Nuclear Membrane

SOS

DAG

PKC

Ras

IKK

PDK-1

Calcineurin MAPK


20

1.10 The MAPK pathways in T cell proliferation and cytokine production

MAPK amplify and integrate signals from a wide variety of extracellular stimuli thereby

allowing cells to adapt and respond to changes in their environment. The MAPK superfamily

comprises the extracellular signal-regulated protein kinases (ERK), the stress-activated

protein kinases or c-Jun N-terminal kinases (JNK) and p38 which all play a role in cell

proliferation, differentiation and motility (Chang et al. 2001; Pearson et al. 2001). In addition,

these MAPK have also been implicated in the regulation of peripheral immune tolerance

(DeSilva et al. 1996; Li et al. 1996; Mondino et al. 1996; Zhang et al. 2000). In particular,

ERK and JNK have been demonstrated to play a critical role in T cell anergy (DeSilva et al.

1996; Li et al. 1996; Mondino et al. 1996), while JNK and p38 are important in the regulation

of activation-induced cell death (AICD) (Zhang et al. 2000). Therefore these kinases are an

attractive target for therapeutic intervention.

The MAPK are regulated by a phosphorylation cascade and each module consists of the

serine/threonine specificity kinases, MAPK kinase kinases (MAPKKK), dual specific kinases,

MAPK kinases (MAPKK) and MAPK (Pearson et al. 2001; Dong et al. 2002). Each MAPK

module consists of a different set of MAPKKK which are activated following receptor

occupancy by an appropriate ligand (Boldt et al. 2004; Ashwell 2006). MAPKKK in turn

phosphorylate MAPKK which then phosphorylate MAPK on conserved Threonine-X-

Tyrosine (TxY) motifs (Chang et al. 2001; Boldt et al. 2004). Upon activation, MAPK can

consequently phosphorylate cytosolic targets or translocate to the nucleus and activate various

transcription factors thus altering gene expression (Chang et al. 2001; Boldt et al. 2004).


21

1.11 Role of ERK in T cell proliferation and cytokine production

ERK1 and ERK2, 44kDa and 42kDa respectively, are the best characterised isoforms of the

ERK family (Pearson et al. 2001). While both isoforms are ubiquitously expressed, ERK2

exists predominantly in immune cells (Pearson et al. 2001). The ERK cascade is triggered in

response to mitogenic signals and commences with the activation of the MAPKKK, raf-1 by

the G protein, Ras or PKC (Fig. 1.3) (Pearson et al. 2001; Boldt et al. 2004). ERK is

subsequently activated by the MAPKK, MEK1 and MEK2 upon phosphorylation of the

Threonine-Glutamic Acid-Tyrosine (TEY) motif. Following activation, ERK can translocate

to the nucleus and regulate various transcription factors including Elk-1, c-Myc and Fos

which in turn regulate cell proliferation, differentiation, apoptosis and metabolism (Boldt et

al. 2004).

In the last decade, the role of ERK in T cell function has been extensively studied (Table 1.1).

The chemical inhibitor, PD98059 has been widely utilised in the examination of the role of

ERK in T cell function. PD98059 has been demonstrated to block the activation of

MEK1/MEK2 by the upstream regulator, raf, thus inhibiting ERK phosphorylation (Alessi et

al. 1995). In support of previous findings in murine T cells transfected with constitutively

active MEK1, Egerton et al. (1996) observed reduced IL3, IL4, IL5, IL10 and IFNγ

production by murine T cells in the presence of the PD98059 (Egerton et al. 1998).

Similar results were also demonstrated in anti-CD3-PMA-activated human T cells which

displayed reduced lymphoproliferation, IL2 (mRNA and protein), IFNγ (mRNA and protein)

and TNF in the presence of the chemical inhibitor (Dumont et al. 1998). Interestingly,

however, at the same concentration of PD98059, IL4 (mRNA and protein), IL5 and IL13

production were all enhanced, while IL10 and IL6 production were reduced (Dumont et al.


22

1998). Thus ERK1/ERK2 may not only differentially regulate Th1 and Th2 cytokine patterns

but may also control individual cytokines within the Th1 and Th2 subsets.

A different approach was used in a previous study which examined the role of ERK in PHA-

PMA-induced cytokine production (Li et al. 1999a; Li et al. 1999b). Following transient

transfection with a dominant negative mutant of ERK1, IL2 production was significantly

reduced in Jurkat T cells (Li et al. 1999b). Further studies using dominant negative mutants

of all members of the ERK pathway including Ras, raf and ERK1 also demonstrated a

reduction in LT production (Li et al. 1999a). In support of this result, PD98059 suppressed

lymphoproliferation, IL2 and LT production in Jurkat and purified human T cells thus

suggesting the ERK1/ERK2 module plays a significant role in Th1 cytokine production. (Li et

al. 1999a; Li et al. 1999b).

Recent investigations on the ERK pathway have focused on distinguishing between the ERK1

and ERK2 isoforms in T cell function. ERK1 and ERK2 short hairpin RNA (shRNA) was

demonstrated to inhibit the MAPK isoforms in 1B6 T cell hybridoma (Wille et al. 2007).

Interestingly, IL2 production was demonstrated to be dependent on both the ERK1 and ERK2

isoforms.

In summary, the role of ERK1/ERK2 in T cell proliferation and cytokine production has been

studied extensively in previous years. The results suggest that ERK1/ERK2 plays a significant

role in both Th1 and Th2 cytokine patterns and poses the question that the MAPK may

differentially regulate individual cytokines within these subsets.


23

Fig. 1.3. The ERK1/ERK2 cascade. Mitogenic signals initiate the activation of Ras or PKC,

which phosphorylate the MAPKKK, raf-1. Upon activation, MEK1 and MEK2 phosphorylate

the MAPK, ERK1 and ERK2, which target various transcription factors including Elk-1 and

Myc.

MAPKKK

Transcription Factors

MAPKK

MAPK

Stimuli

Nuclear membrane

Cell membrane

Mitogen

Elk-1, Myc

MEK1, MEK2

ERK1, ERK2

raf-1

Ras or PKC

MAPKKK


MAPKK

MAPK

Stimuli

Nuclear membrane

Cell membrane

Mitogen

Elk-1, Myc

MEK1, MEK2

ERK1, ERK2

raf-1

Ras or PKC


24

Table 1.1: Effect of ERK inhibition on T cell function.

Mouse T cells

(Egerton et al. 1998)

Mouse T cell line (Wille et al. 2007)

Human T cells

(Dumont et al. 1998)

Human T cells/

human T cell line(Li et al. 1999a)

Human T cell line(Li et al. 1999b)

Proliferation ↓ ↓

IFNγ ↓* ↓

TNF ↓

LT ↓

IL2 ↓ ↓ ↓

IL3 ↓

IL4 ↓ ↑

IL5 ↓ ↑

IL6 ↓

IL10 ↓ ↓

IL13 ↑

*Arrows indicates whether inhibition of ERK enhanced (↑) or inhibited (↓) T cell proliferation

and cytokine production and (-) signifies ERK inhibition had no affect on T cell function.

Spaces indicate that proliferation or cytokine production was not measured.


25

1.12 Role of p38 in T cell proliferation and cytokine production

The MAPK, p38, is stimulated in response to cellular stress, pro-inflammatory cytokines and

endotoxin (Boldt et al. 2004; Ashwell 2006). While five isoforms of p38 MAPK have been

identified, p38α/β/β2/γ and δ, in T cells, p38α is the major isoform activated (Herlaar et al.

1999). The p38 cascade may initiate with the activation of the serine/threonine kinases

MAPKKK4 (MEKK4), Mixed Lineage Kinase 3 (MLK3), Transforming growth factor -

activated kinase 1 (TAK1) or Apoptosis signal-regulating kinase 1 (ASK1) by GTPases (Fig.

1.4) (Ashwell 2006). The dual specific kinases, MKK3 and MKK6 are then activated leading

to the phosphorylation of Thr 180 and Tyr 182 residues in the Threonine-Glycine-Tyrosine

(TGY) p38 activation loop (Ashwell 2006). Upon activation, p38 phosphorylates many

substrates including activating transcription factor 2 (ATF2) and MAPKAPK2, 3 and 5 which

are involved in the synthesis of inflammatory mediators and activation of inflammatory

pathways (Herlaar et al. 1999; Boldt et al. 2004; Ashwell 2006).

The majority of research investigating the role of p38 in previous years has involved the use

of the specific p38 chemical inhibitor, SB203580 (Table 1.2) (Cuenda et al. 1995). Upon

stimulation with Concanavalin A (Con A), Rincon et al. (1998) observed reduced IFNγ gene

expression and cytokine production by murine splenic Th1 cells in the presence of SB203580,

while Th2 cells produced normal levels of IL4 (Rincon et al. 1998). Upon treatment with

SB203580, Zhang et al. (1999) also observed a reduction in IFN gene expression in murine

splenic T cells, in addition to IL2, IL4 and lymphoproliferation. The discrepancy observed for

IL4 production may be due to the variation in stimuli (anti-CD3-anti-CD28 antibodies

compared to Con A) and cell preparation. Irrespective of such limitations, p38 is involved in

regulating the immune response via Th1 cells.


26

In support of the results observed in murine T cells, Koprak et al. (1999) demonstrated a

reduction in IL4, IL5, IL10 (mRNA and protein), IL13, IFNγ (mRNA and protein) and TNF

production by purified human T cells in the presence of SB203580. These results are also

supported in the recent study by Kogkopoulou et al. (2006) which showed that SB203580

inhibited IL4, IL5 (mRNA and protein), IL10 and IL13 (mRNA and protein) production in the

same cell type. Thus in addition to Th1 responses, there is also strong evidence to suggest that

p38 regulates Th2 cytokine production. Interestingly, while Kogkopoulou et al. (2006)

observed an increase in IL2 (mRNA and protein) cytokine production, Koprak et al. (1999)

observed a reduction. This discrepancy in results may be due to the variation in stimuli, as the

former study used plate-bound anti-CD3 antibodies at higher concentrations, while the latter

used soluble antibodies.

In recent years, different approaches have been employed to investigate the role of p38 in

cytokine production. Retroviral vectors encoding SB203580-resistant p38α strengthened the

data obtained with the use of the chemical inhibitor. While IFNγ and IL10 were reduced in

wildtype splenic T cells, this effect was completely neutralised in SB203580-resistant p38α T

cells. (Guo et al. 2003). Since p38α-deficient mice die during embryonic development,

Berenson et al. 2006 recently generated antigen-specific p38α +/- and p38α -/- murine T cells

using RAG2-/- blastocyst complementation and retroviral expression of the TCR.

Interestingly, p38 was not required for TCR-induced cytokine production as no substantial

reduction in IFNγ was observed. Furthermore, studies using CD4+ peripheral T cells isolated

from MKK3-/- and MKK6-/- mice observed no significant difference in T cell proliferation

compared to the wild type following stimulation with anti-CD3-anti-CD28 antibodies (Tanaka

et al. 2002). Despite the controversies, with regard to human T cells, p38 may not play a role


27

in lymphoproliferation; however, the MAPK appears to promote the production of both Th1

and Th2 cytokine patterns.


28

Fig. 1.4. The p38 cascade. Cellular stress can initiate the activation of rac or cdc42, which

phosphorylate the MAPKKK, MLK3, TAK1 or ASK1. Upon activation, MKK3 and MKK6

phosphorylate the MAPK, p38 which target various transcription factors including ATF2 and

MAPKAPK2,3,5.

MAPKKK


MAPKK

MAPK

Stimuli

Nuclear membrane

Cell membrane

Stress

ATF2, MAPKAPK2, 3, 5

p38

MLK3, TAK1, ASK1

rac, cdc42

MKK3, MKK6

MAPKKK


MAPKK

MAPK

Stimuli

Nuclear membrane

Cell membrane

Stress

ATF2, MAPKAPK2, 3, 5

p38

MLK3, TAK1, ASK1

rac, cdc42

MKK3, MKK6


29

Table 1.2. Effect of p38 inhibition on T cell function.

Th1, Th2Mouse

(Rincon et al. 1998)

Splenic CD4+

Mouse (Zhang et al.

1999)

Splenic CD4+

Mouse(Guo et al.

2003)

Splenic CD4+

Mouse(Berenson et

al. 2006)

CD4+|T cells Mouse

(Tanaka et al. 2002)

CD4+ T cellsHuman

(Koprak et al. 1999)

CD4+ T cellsHuman

(Kogkopoulou et al. 2006)

Proliferation ↓ - -

IFNγ ↓* ↓ ↓ - ↓

TNF ↓

IL2 ↓ ↓ ↑

IL4 - ↓ ↓ ↓

IL5 ↓ ↓

IL10 ↓ ↓ ↓

IL13 ↓ ↓

*Arrows indicates whether inhibition of p38 enhanced (↑) or inhibited (↓) T cell proliferation and cytokine production and (-) signifies p38 inhibition

had no affect on T cell function. Spaces indicate that proliferation or cytokine production was not measured.


30

1.13 Role of JNK in T cell proliferation and cytokine production

JNK is stimulated in response to environmental stress, cytokines and growth factors (Kallunki

et al. 1994; Davis 1999; Tournier et al. 2000). The JNK pathway commences with the

activation of several MAPKKK including ASK, MEKK, MLK and TAK1 by the G-proteins

Cdc42, Rac and Ras and downstream TNF receptors which are independent of G-proteins

(Fig. 1.5) (Barr et al. 2001; Weston et al. 2002). These kinases, in turn activate MKK4 and

MKK7 which phosphorylate JNK on threonine 183 and tyrosine 185 in the Threonine-

Proline-Tyrosine (TPY) motif, respectively (Barr et al. 2001; Weston et al. 2002).

JNK is encoded by three genes: Jnk1 and Jnk2 which are ubiquitously expressed and Jnk3

which is predominantly expressed in the brain, heart and testes (Barr et al. 2001; Weston et al.

2002). These three genes are alternatively spliced to create 10 JNK isoforms (JNK11,

JNK12, JNK11, JNK12, JNK21, JNK22, JNK21, JNK22, JNK31, JNK32) which

were first identified in the adult brain (Gupta et al. 1996). While initially JNK1 and JNK2

were believed to correspond to the observed 46 and 54 kDa isoforms respectively, it was later

clarified that proteins of both molecular weights were encoded by the Jnk1 and Jnk2 genes

(Derijard et al. 1994; Kallunki et al. 1994; Gupta et al. 1996). JNK binds and phosphorylates

nuclear substrates, including c-Jun, JunB, JunD, ATF2, Elk-1, c-Myc, p53, NFATc2, FOXO4,

STAT3, STAT1, Pax2 and TCFβ1 in addition to non-nuclear substrates such as Itch, Bcl2,

Akt and paxillin (Fig. 1.5) (Bogoyevitch et al. 2006).

While there is substantial evidence to suggest that both ERK and p38 play a significant role in

Th1 and Th2 cytokine production in both murine and human T cells, the role of JNK remains

ill-defined especially in the human system. While the JNK1-/- and JNK2-/- mice have provided


31

appropriate tools to study the role of JNK in murine cells, conflicting results have emerged

(Table 1.3). Attempts to investigate the role of JNK in human T cells have relied on

pharmacological inhibitors of questionable specificities (Bain et al. 2003; Bain et al. 2007).

Using knockout mice, two groups have produced discrepant results on the role of JNK1 in IL2

cytokine production. Isolated splenic T cells from JNK1-/- mice were demonstrated to produce

normal (Dong et al. 1998) and reduced (Sabapathy et al. 2001) IL2 in response to varied

concentrations of anti-CD3 stimulation. Sabapathy et al. (1999) also observed reduced IL2 in

the JNK2-/- mouse thus suggesting that the JNK1 and JNK2 isoforms play similar roles in T

cell function. In contrast, Yang et al. (1998) observed normal IL2 production in spleen cells

isolated from JNK2-/- mice. To complicate matters further, in an attempt to overcome the fact

that JNK1 and JNK2 knockout mice are not viable (Kuan et al. 1999), Dong et al. (2000)

generated primary embryonic stem (ES) cells from blastocysts of JNK1+/- JNK2-/-

intermatings. The resulting JNK1-/-JNK2-/- ES cells were used to reconstitute Recombination

activating gene-1 (Rag1) knockout mice, which lack mature B cells and T cells (Mombaerts et

al. 1992). The reconstituted mice had normal peripheral lymphocyte and thymocyte

populations and the isolated CD4+ T cells displayed enhanced IL2 production in response to

Con A (Dong et al. 2000).

In contrast to IL2, there is evidence to suggest that JNK plays a definite role in IFNγ

production by murine T cells. Dong et al. (1998) demonstrated that purified splenic T cells

isolated from JNK1-/- mice produced less IFNγ in comparison to the wildtype. Similarly,

Sabapathy et al. (1999) observed the same result in the JNK2-/- mouse thus suggesting that the

JNK1 and JNK2 isoforms play a similar role in the production of the cytokine.


32

Like IL2, the role of JNK in IL4 cytokine production is also unclear due to conflicting results.

While Dong et al. (1998) observed enhanced IL4, IL5 and IL10 production by purified splenic

T cells isolated from JNK1-/- mice, Sabapathy et al. (1999) demonstrated reduced IL4

production in JNK2-/- mice upon stimulation with anti-CD3 and anti-CD28 antibodies. Thus

the role of JNK in Th2 cytokine production remains ill-defined.

The role of JNK in cell proliferation is also controversial. While Dong et al. (1998)

demonstrated enhanced proliferation of splenocytes isolated from JNK1-/- mice, the same

group also observed normal proliferation of spleen cells isolated from JNK2-/- mice (Yang et

al. 1998), thus suggesting that JNK1 and JNK2 play different roles in cell proliferation. In

contrast, Sabapathy et al. (1999; 2001) observed reduced proliferation of splenic T cells

isolated from both the JNK1-/- and the JNK2-/- mice. To complicate matters further, Dong et

al. (2000) bred transgenic mice that expressed dominant-negative JNK1 in T cells with JNK2-

/- mice. Peripheral CD4+ T cells isolated from the resulting dominant-negative JNK1+JNK2-/-

mice experienced enhanced proliferation following stimulation with anti-CD3-anti-CD28

antibodies (Sabapathy et al. 1999; Dong et al. 2000; Sabapathy et al. 2001).

Attempts have also been made to investigate the JNK pathway using the JNK specific

inhibitor, SP600125 (anthra[1,9-cd]pyrazole-6(2H)-one) which was first identified by Bennett

et al. (2001) (Fig.1.6). This fully reversible ATP competitive inhibitor was initially

demonstrated to selectively target all isoforms of JNK including JNK1, JNK2 and JNK3 with

IC50 values of 40, 40 and 90 nM respectively (Bennett et al. 2001). SP600125 is an

anthrapyrazolone and consists of a nitrogen-containing ring system for interaction with key

residues in the active site, however due to its structure, a major disadvantage of SP600125 is

its poor solubility (Bennett et al. 2001).


33

Initial studies with SP600125 involved human peripheral blood mononuclear cells (PBMC)

which were differentiated into Th1 and Th2 subsets. SP600125 effectively blocked IFNγ,

TNF and IL10 production by Th1 and Th2 cells (Table 1.3) (Bennett et al. 2001). However,

the specificity of the inhibitor has been questioned (Bain et al. 2003). SP600125 was found to

be a weak inhibitor of the JNK isoforms with IC50 values of 5.8 μM and 6.1 μM for JNK1 and

JNK2, respectively (Bain et al. 2003). Notably, a higher concentration of adenosine tri-

phosphate (ATP) was used in this study, which may account for the higher IC50 values

compared to those reported by Bennett et al. (2001). Importantly, SP600125 inhibited 13 of

28 protein kinases tested to a similar or greater extent than JNK1 and JNK2 (Bain et al. 2003).

Thus, any conclusions drawn from data obtained with this chemical inhibitor need to be

treated with reservation.


34

Fig. 1.5. The JNK cascade. Cellular stress can initiate the activation of Ras, cdc42 or rac

which phosphorylate the MAPKKK, ASK1, MEKK, MLK or TAK1. Upon activation, MKK4

and MKK7 phosphorylate the MAPK, JNK1 and JNK2 which target various nuclear and non-

nuclear substrates.

MAPKKK

Transcription

Factors

MAPKK

MAPK

Stimuli

Nuclear membrane

Cell membrane

Stress

c-Jun, ATF2, Elk-1, c-Myc, p53, NFATc2, STAT1, STAT3, FOXO4,

Pax 2, TCFβ1

JNK1, JNK2

MEKK, MLK, ASK1, TAK1

cdc42, rac, ras

MKK4, MKK7

Itch, Bcl2, Akt,

paxillin

MAPKKK

Transcription

Factors

MAPKK

MAPK

Stimuli

Nuclear membrane

Cell membrane

Stress

c-Jun, ATF2, Elk-1, c-Myc, p53, NFATc2, STAT1, STAT3, FOXO4,

Pax 2, TCFβ1

JNK1, JNK2

MEKK, MLK, ASK1, TAK1

cdc42, rac, ras

MKK4, MKK7

Itch, Bcl2, Akt,

paxillin


35

Fig. 1.6. The chemical structure of SP600125. The chemical inhibitor, SP600125 is an

anthrapyrazolone with the chemical structure anthra[1,9-cd]pyrazol-6(2H)-one.

N NH

O

N NH

O


36

Table 1.3. Effect of JNK inhibition on T cell function.

JNK1-/-

Splenic T cells Mouse

(Dong et al. 1998)

JNK1-/-


(Sabapathy et al. 2001)

JNK2-/-


(Sabapathy et al. 1999)

JNK2-/-

Spleen cellsMouse

(Yang et al. 1998)

Impaired JNK1/JNK2

T cellsMouse

(Dong et al. 2000)

Impaired JNK1/JNK2

T cellsHuman

(Bennett et al. 2001)

Proliferation ↑*

(splenocytes)

↓ ↓ - ↑

IFNγ ↓ ↓ ↓

TNF ↓

IL2 - ↓ ↓ - ↑

IL4 ↑ ↓

IL5 ↑

IL10 ↑ ↓

*Arrows indicates whether inhibition of JNK enhanced (↑) or inhibited (↓) T cell lymphoproliferation and cytokine production and (-) signifies JNK

inhibition had no affect on T cell function. Spaces indicate that proliferation or cytokine production was not measured.


37

1.14 The TAT-JIP peptide

There has been serious concern over the ability to effectively study the role of JNK in

biological functions using the methods described above. This has led to the development of

other approaches such as the construction of peptides which interfere with the ability of JNK

to phosphorylate its substrates. These peptides are described below.

JNK-interacting protein 1 (JIP-1) was first identified by a yeast two-hybrid system (Dickens

et al. 1997), however since then three mammalian members have been observed (JIP-1, JIP-2,

JIP-3) (Whitmarsh et al. 1998; Yasuda et al. 1999; Kelkar et al. 2000). JIP-1 contains a N-

terminal JNK binding domain (JBD) (residues 1-282) and a SRC homology 3 (SH3) domain

in the COOH terminus (residues 283-660) (Dickens et al. 1997). Initial studies demonstrated

that JNK1 was present in JIP-1 immunoprecipitates isolated from COS-1 cells transfected

with vectors encoding the two proteins (Dickens et al. 1997). Dickens et al. (1997) also

identified four critical residues of JIP-1 (Lys 155, Thr 159, Leu 160 and Leu 162) that were

important for JNK binding. Importantly, ERK2 and p38 did not co-immunoprecipitate with

JIP-1. These data demonstrate that JIP-1 specifically interacts with JNK (Dickens et al. 1997).

Further work discovered that JIP-1 acts as a scaffold protein which brings specific

components of the signalling pathway together to enhance signal transduction (Fig.1.7)

(Whitmarsh et al. 1998). JIP-1 was found to bind MKK7, in addition to the MAPKKK,

MLK3 and the upstream signalling molecule, Hematopoieitc progenitor kinase 1 (HPK1)

(Whitmarsh et al. 1998). The combined interaction of these signalling components with JIP-1

substantially enhanced JNK activation (Whitmarsh et al. 1998)


38

Interestingly, Dickens et al. (1997) demonstrated that over-expression of JIP-1 inhibited JNK

signalling. It has been proposed that under these circumstances JIP-1 blocks nuclear

translocation of JNK by containing the MAPK in the cytoplasm and excess scaffold may also

isolate JNK pathway signalling components into separate JIP-1 complexes (Dickens et al.

1997; Whitmarsh et al. 1998). Thus JIP-1 is an effective tool to selectively modulate JNK

activity.

JIP-derived peptides can act as JNK inhibitors (Bonny et al. 2001; Barr et al. 2002). Initially,

Bonny et al. (2001) identified minimal conserved domains of islet-brain-1 (IB-1)/JIP-1 and

IB-2/JIP-2 which blocked cell apoptosis. When linked to the human immunodeficiency

virus-transactivator of transcription (HIV-TAT) peptide, IB-1/JIP-1 (TAT-JIP153-172) and IB-

2/JIP-2 were shown to penetrate β cells and prevent IL1--induced apoptosis (Bonny et al.

2001). Furthermore, following the identification of four critical residues of JIP-1 that were

important for JNK binding (Lys 155, Thr 159, Leu 160 and Leu 162), Barr et al. (2002)

chemically engineered a shortened 11 amino acid peptide, Truncated inhibitory region of JIP

(TI-JIP) consisting of residues 153-163 of JIP-1 (Table 1.4).

Future studies with the JIP-derived peptide revealed that in contrast to SP600125, TI-JIP was

ATP non-competitive and acted as an allosteric modulator of JNK (Barr et al. 2004b). A

model of TI-JIP bound to JNK3 demonstrated that the JIP-derived peptide acts at a location

distant from the active site (Barr et al. 2004b). Further investigations by Heo et al. (2004)

established that TI-JIP binds to the docking groove of JNK1, away from the ATP and

substrate recognition site. In addition, binding of the peptide to JNK1 distorts the ATP

binding cleft and reduces the affinity of JNK1 for ATP (Heo et al. 2004).


39

When coupled to the TAT peptide (Bogoyevitch et al. 2002), JIP-derived peptides can be

studied both in vitro and in in vivo animal models of disease. Table 1.5 adapted from

Bogoyevitch et al. (2005), briefly summaries recent studies involving the use of these

peptides. Inhibition of the JNK pathway via the JIP-derived peptides has been demonstrated

to prevent apoptosis of pancreatic β-cells and improve insulin resistance and glucose tolerance

for the treatment of diabetes, prevent permanent hearing loss in response to acoustic trauma

and inhibit cell death induced by oxygen and glucose deprivation in the brain (Bonny et al.

2001; Wang et al. 2003; Hirt et al. 2004; Kaneto et al. 2004).


40

Fig. 1.7. JIP-1 is a scaffold protein for the JNK signalling pathway. Adapted from

Whitmarsh et al. (1998). JIP-1 is a mammalian scaffold protein which acts to enhance JNK

signalling by placing each component of the JNK pathway in close proximity to one another.


a1172507

Text Box

NOTE: This figure is included on page 40 of the print copy of the thesis held in the University of Adelaide Library.

41

Table 1.4. The amino acid sequences for the TAT peptide and the long and short JIP-1-

derived peptides.

Three letter amino acid code

Single letter amino acid code

TAT 48-57 Gly-Arg-Lys-Lys-

Arg-Arg-Gln-Arg-

Arg-Arg

GRKKRRQRRR

JIP153-172 Arg-Pro-Lys-Arg-

Pro-Thr-Thr-Leu-

Asn-Leu-Phe-Pro-

Gln-Val-Pro-Arg-Ser-

Gln-Asp-Thr

RPKRPTTLNLFPQVPRSQDT

JIP153-163 Arg-Pro-Lys-Arg-

Pro-Thr-Thr-Leu-

Asn-Leu-Phe

RPKRPTTLNLF


42

Table 1.5. Recent studies involving the use of JIP-derived peptides.

Model Findings

Pancreatic -cell apoptosis in vitro

(Bonny et al. 2001; Nikulina et al. 2003;

Roehrich et al. 2003)

Protect pancreatic -cells from apoptosis

induced by IL1- and lipoproteins

Obese diabetic mice in vivo

(Kaneto et al. 2004)

Improved insulin resistance and glucose

tolerance

Middle cerebral artery occlusion in vivo

(Borsello et al. 2003)

Reduced size of brain lesion

Cell death in hippocampal slices in vitro

(Hirt et al. 2004)

Prevented cell death induced by oxygen

and glucose deprivation

Optic nerve crush injury in vivo

(Tezel et al. 2004)

Prevented degeneration of retinal

ganglion cells

Amyloid- peptide effects in vivo and in

vitro

(Minogue et al. 2003)

Prevented inhibition of long term

potentiation by Amyloid- peptide

Auditory hair-cell death in vitro and in

vivo

(Wang et al. 2003; Wang et al. 2004)

Prevented acoustic-trauma-induced

permanent hearing loss and increased

sensitivity of cochlear hair cells to

damage by cisplatin

Islets isolated from pig pancreata in vitro

(Noguchi et al. 2005)

Prevented islet apoptosis and improved

islet graft function

Bovine aortic and human umbilical vein

endothelial cells in vitro

(Miho et al. 2005)

Inhibited thrombin-induced ICAM-1

expression


43

1.15 Concluding remarks

The JNK signalling pathway has been implicated in the pathogenesis of chronic inflammatory

diseases in which T cells play an important role (Bogoyevitch 2006), thus making the JNK

pathway a potential target for therapeutics. However, its role in T cell responses remains ill-

defined. Not only have studies in human T cells been limited but the more extensive studies

with mice have generated conflicting results with regard to the role of the JNK signalling

pathway in immune responses. Here we have addressed this issue, with a new approach to

analyse the role of JNK in human T cell function. This involved the use of the recently

described peptide inhibitors of JNK, JIP153-163 and JIP153-172 , which when coupled to the short

cell-permeable HIV-TAT sequence are able to cross the plasma membrane and selectively

inhibit JNK activity (Barr et al. 2002). Using these TAT-JIP peptides, this study has

addressed the role of JNK in human T cell function in several models of T cell activation

including PHA-PMA, anti-CD3 and anti-CD28 antibodies, Tetanus Toxoid and HDM.

1.16 Aims, hypotheses and significance

General Aim:

To examine the role of the JNK signalling pathway in human T cell function in vitro.

Specific aims:

1. To examine the role of JNK in proliferation and cytokine production (Th1 versus Th2)

in T cells stimulated with PHA-PMA.


44

2. To examine the role of JNK in proliferation and cytokine production in T cells

stimulated via the TCR; anti-CD3-anti-CD28 antibodies, Tetanus Toxoid and HDM.

3. To study the relationship between JNK and other MAPK (ERK, p38) in T cell

function.

4. To assess the usefulness of the TAT-JIP peptides in studying the JNK signalling

pathway.

Hypotheses

JNK plays a critical role in the regulation of human T cell proliferation and cytokine

production and differentially regulates Th1 and Th2 cytokine patterns.

Significance

Current therapy for autoimmune diseases involves depletion of T cells and the suppression of

their responses. Pharmaceuticals such as Cyclosporine A and FK506 inhibit T cell activation,

proliferation and cell function by targeting intracellular signalling pathways (Bierer et al.

1990; Szamel et al. 1993; Kuwano et al. 1994). Unfortunately however, there are numerous

side effects associated with these treatments (Lindenfeld et al. 2004). By gaining a clearer

understanding of the intracellular signalling pathways which control T cell function it is likely

that more selective inhibitors can be developed to improve the treatment of inflammatory

disorders.


45

2Chapter Two

Materials and Methods


46

2.1 Materials

Media: Roswell Park Memorial Institute (RPMI) 1640 tissue culture medium, foetal bovine

serum (FBS) and L-glutamine were purchased from SAFC Biosciences, Lenexa, KS.

Penicillin and streptomycin were obtained from Sigma-Aldrich, St. Louis, MO.

Gradients: Ficoll 400 was purchased from Pharmacia Biotech, Uppsala, Sweden, sodium

diatrizoate was acquired from Sigma-Aldrich and angiografin was obtained from Schering

AG, Berlin, Germany. Ficoll-Paque PLUS was purchased from GE Healthcare, Uppsala,

Sweden.

Peptide and kinase inhibitors: The TAT-JIP153-163 peptide (GRKKRRQRRRRPKRPTTLNLF)

was synthesised by GenScript Corporation (Piscataway, NJ) and the control peptide

(GRKKRRQRRRRPKAATTLNLF) by Mimotopes Pty Ltd (Clayton, Australia). The TAT-

JIP153-172 peptide (GRKKRRQRRRPPRPKRPTTLNLFPQVPRSQDT) was purchased from

Calbiochem (Bad Soden, Germany). All peptides were purified by high-performance liquid

chromatography (HPLC) and analysed by mass spectrometry to be > 80% pure. Peptides were

stored at -20°C and prepared in RPMI 1640 prior to use. The MEK1/MEK2 inhibitor,

PD98059, was purchased from Cell Signalling Technology, Danvers, MA. The p38 inhibitor,

SB203580 and the JNK inhibitor, SP600125, were purchased from Sigma-Aldrich. The

chemical inhibitors were solubilised in dimethyl sulfoxide (DMSO) obtained from Merck,

Darmstadt, Germany and stored at -20°C prior to use.

Mitogens and antigens for lymphocyte stimulation: PHA and PMA were purchased from

Murex Diagnostics, Dartford, U.K. and Sigma-Aldrich respectively and Tetanus Toxoid from


47

Calbiochem. Recombinant Der p 2 was provided by Professor W.R. Thomas at the University

of Western Australia (Perth, Australia) (Thomas et al. 2004).

Antibodies: Antibodies against phosphorylated JNK (G-7), phosphorylated c-jun (serine

63/serine 73), JNK1 (C-17) and GAPDH (0411) were purchased from Santa Cruz

Biotechnology, Santa Cruz, CA. Horseradish peroxidise (HRP) conjugated rabbit anti-mouse

IgG and sheep anti-rabbit IgG were from Dakocytomation, Glostrup, Denmark and Chemicon

Australia, Boronia, Australia respectively. Soluble anti-CD3 and anti-CD28 antibodies were

purchased from eBioscience, San Diego, CA.

Protease inhibitors: Benzamidine, leupeptin, pepstatin A and phenylmethylsulfonyl fluoride

(PMSF) were purchased from Sigma-Aldrich and aprotinin from Calbiochem.

Radiochemicals: Methyl-3H Thymidine was purchased from Amersham Life Sciences,

Buckinghamshire, England.

General chemicals/biochemicals: NaCl, Na2CO3, NaOH, CuSO4, Na/K tartrate and TWEEN-

20 were purchased from Ajax Chemicals, NSW, Australia while HCl and methanol were

obtained from Merck. -mercaptoethanol, Bromophenol blue, Trypan blue, Folin and

Ciocalteau’s Phenol Reagent, ponseau S, Trizma base, Nonidet-P40 (NP40),

ethylenediaminetetraacetic acid (EDTA), HEPES, glycine, TEMED, Mitomycin C, DL-

dithiothreitol (DTT) and Sigma 104 were acquired from Sigma-Aldrich. Bovine serum

albumin (BSA) was purchased from Bovogen Biologicals, Essendon, Australia and sodium

dodecyl sulphate (SDS), 30% Acrylamide/Bis Solution, 29:1 mixture and ammonium

persulfate (APS) were from Bio-Rad, Hercules, CA. Isoton II was purchased from Beckman

Coulter Australia, Gladesville, Australia.


48

2.2 Buffers

Buffer A

Materials:

1.5 M Trizma base

MilliQ water

Method:

Trizma base (90.825g) was added to 500 ml of MilliQ water and mixed until dissolved. The

pH of the buffer was adjusted to 8.8 with HCl and stored at 4 °C until use.

Buffer B

Materials:

0.5 M Trizma base

MilliQ water

Method:

Trizma base (30.275g) was added to 500 ml of MilliQ water and mixed until dissolved. The

pH of the buffer was adjusted to 6.8 with HCl and stored at 4°C until use.

Running Buffer (5X)

Materials:

384 mM Glycine

50 mM Trizma base

MilliQ water

0.1% SDS


49

Method:

Glycine (144.1g), Trizma base (30.28g) and SDS (5g) were added to 1L of MilliQ water and

mixed until dissolved. The buffer was stored at room temperature and diluted to 1X prior to

running a gel by adding 200 ml of buffer to 800 ml of MilliQ water.

Blocking solution

Materials:

25 mM Trizma base

100 mM NaCl

MilliQ water

Skim milk powder

Method:

Trizma base (3.02g) and NaCl (5.84g) were added to 1L of MilliQ water. The solution was

adjusted to a pH of 7.4 with HCl prior to the addition of 50g of skim milk powder and mixed

until dissolved. Blocking solution was stored at 4°C until use.

Transfer Buffer

Materials:

25mM Trizma base

20% v/v Methanol

152 mM Glycine

MilliQ water


50

Method:

To make 5L of transfer buffer, 15.14g of Trizma base and 57.05g of glycine were dissolved in

4L of MilliQ water prior to the addition of 1L of methanol. The buffer was stored at 4°C.

Lysis Buffer

Materials:

0.5% NP40

20 mM HEPES

100 mM NaCl

1 mM EDTA

MilliQ water

Inhibitors:

1 mM DTT

1 mM PMSF

Leupeptin (10 mg/ml of lysis buffer)

Aprotinin (10 μg/ml of lysis buffer)

Pepstatin A (10 μg/ml of lysis buffer)

Benzamidine (10 mg/ml of lysis buffer)

Sigma 104 (1 g/ml of lysis buffer)

Method:

For 500 ml of lysis buffer, NP40 (2.5 ml), HEPES (2.383g), NaCl (2.922g) and EDTA

(0.1861g) were added to 497.5 ml of MilliQ water. The buffer was then adjusted to a pH of

7.8 and stored in 10 ml aliquots at -20°C. Prior to use, the inhibitors (DTT, PMSF, leupeptin,


51

aprotinin, pepstatin A, benzamidine and sigma 104) were added to thawed lysis buffer which

was then mixed and kept on ice during the experiment.

Laemmli Buffer

Materials:

20 mM Trizma base

40% sucrose

6% SDS

0.5% w/v bromophenol blue

β-mercaptoethanol

MilliQ water

Method:

Trizma Base (0.121g), sucrose (20g) and SDS (3g) were added to 50 ml of MilliQ water and

adjusted to a pH of 6.8 (Solution 1). Bromophenol blue (0.05 ml) was added to 1 ml of

Solution 1 and immediately before use; 0.1 ml of β-mercaptoethanol was added.

2.3 Purification of human PBMC

PBMC were purified as described previously (Ferrante et al. 1982) (Fig. 2.1). Venous blood

from healthy adult donors was collected in 9 ml tubes containing lithium heparin. The blood

(6 ml) was layered onto 4 ml of Hypaque-Ficoll gradient (8% Ficoll 400, adjusted to a density

of 1.114 with sodium diatrizoate and angiografin). After centrifugation at 600 g for 35 min at

room temperature, the leukocytes resolved into two distinct bands while erythrocytes

accumulated at the bottom of the tube. The PBMC, consisting of monocytes and lymphocytes


52

in the top band, were harvested, washed twice with RPMI 1640 by repeated centrifugation at

600 g for 5 min and resuspension of the cells. The viability of the PBMC was > 99% as

determined by their ability to exclude trypan blue.

2.4 Purification of human T cells

T cells were purified from PBMC as described previously (Zhang et al. 1992) (Fig. 2.1).

Tissue culture plates were coated with autologous plasma for 30 min and incubated at 37°C

and 5% CO2. PBMC were resuspended in RPMI 1640 supplemented with 10% heat-

inactivated FBS (RPMI/FBS). The cells were dispensed into tissue culture plates ( 4 x 107

cells per plate in 10 ml of media) (Techno Plastic Products AG, Trasadingen, Switzerland)

and incubated for 30 min at 37°C and 5% CO2, to enable the monocytes to adhere. The non-

adherent cells were harvested and washed in 10 ml of RPMI/FBS. After centrifugation at

600g for 5 min, the pellet, containing T and B lymphocytes, was resuspended in 600 μl of

RPMI/FBS and applied to a 1 ml syringe packed with sterile nylon wool (column)

(Geneworks, Adelaide, Australia). The column, which had been pre-equilibrated with

RPMI/FBS for 30 min at 37C and 5% CO2, was incubated under the same conditions for a

further 30 min. The non-adherent T cells were eluted by passing 10 ml of RPMI/FBS

through the column. The cells were then layered onto Ficoll-Paque PLUS and centrifuged for

15 min at 600 g, T cells aspirated, washed twice and resuspended in RPMI 1640

supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml) and L-glutamine (4

mM/ml). T cell purity was > 90 % (Section 2.6) and cell viability was > 99% as determined

by their ability to exclude trypan blue.


53

Fig. 2.1. Flow chart of experimental procedure. PBMC were isolated from whole blood

and utilised in Tetanus Toxoid, MLR and Der p 2 experiments. T cells were purified from

PBMC by adhesion to plasma-coated plates and nylon wool. T cells were utilised in PHA-

PMA and anti-CD3-anti-CD28 antibody experiments.

Whole Blood

Plate andnylon wool adhesion

PBMC

Purified T cellsTetanus Toxoid, MLR, Der p 2

PHA-PMA,anti-CD3-anti-CD28

antibodies

Hypaque-Ficoll gradient


54

2.5 Purification of murine splenic T cells

Spleens were removed from 6-7 week old Balb/c mice. The T cells were prepared essentially

as described previously (Julius et al. 1973). Briefly, each spleen was placed in RPMI 1640,

cut finely and transferred into a homogeniser. After homogenisation, the spleen cell solution

was transferred into a sterile 5 ml syringe containing cotton wool, which had been pre-

equilibrated with RPMI 1640. The eluted single cell suspension was then layered onto Ficoll-

Paque PLUS and centrifuged for 15 min at 600 g. Splenocytes were aspirated, washed twice

and resuspended in RPMI/ΔFBS. Cells were added to ΔFBS-coated tissue culture plates ( 4

x 107 cells per plate in 10 ml of media) and incubated for 30 min at 37°C and 5% CO2. Non-

adherent cells were washed with 10 ml of RPMI/FBS and centrifuged at 600 g for 5 min.

The pellet was resuspended in 600 μl of RPMI/FBS and applied to a sterile nylon wool

column. The column, which had been pre-equilibrated with RPMI/FBS for 30 min at 37C

and 5% CO2, was then incubated under the same conditions for a further 30 min. The non-

adherent splenic T cells were eluted by passing 10 ml of RPMI/FBS through the column,

washed twice and resuspended in RPMI 1640 supplemented with penicillin (100 units/ml),

streptomycin (100 μg/ml) and L-glutamine (4 mM/ml). T cell purity was > 90 % (Section 2.6)

and cell viability was > 99% as determined by their ability to exclude trypan blue.

2.6 Determination of cell purity

T cell purity was determined using a Simultest™ IMK-Lymphocyte Kit (Becton Dickinson

(BD), Franklin Lakes, NJ). Cells (1x 106) were washed twice at 4C and resuspended in 100

l of Isoton II, prior to the addition of 2 l of each antibody pair (CD3/CD4, CD3/CD8,

CD3/CD19, CD45/CD14. CD3 and CD45 were fluorescein isothiocyanate (FITC) labelled

while CD4, CD8, CD14 and CD19 were phycoerythrin (PE) labelled. While CD3+/CD4+ and


55

CD3+/CD8+ determined the number of helper T cells and cytotoxic T cells, CD3+/CD19+ and

CD45+/CD14+ measured B cells and monocytes respectively. Following the addition of

antibodies, cells were incubated on ice for 30 min in the dark and then washed twice at 4C

with Isoton II. Fluorescence was measured by flow cytometry. T cell purity was > 90% as

illustrated in the dot plot in Fig 2.2.


56

Fig. 2.2. Dot plot of T cell analysis by flow cytometry. T cell purity was determined using a

Simultest™ IMK-Lymphocyte Kit (BD).


57

2.7 PHA-PMA and anti-CD3-anti-CD28 induced activation

Fifty microlitres of T cells (4 x 106 cells /ml) were added to 50 l of TAT-JIP153-163 (1-50

M), TAT-JIP153-172 (20 M), PD98059 (12.5-50 M) , SB203580 (1-20 M), SP600125 (1-

20 M) or control peptide (50 M) in 96-well U bottom plates (Nunc, Roskilde, Denmark)

and incubated at 37oC in a 5% CO2 humidified atmosphere for 30 min (Costabile et al. 2001).

In the PHA-PMA induced responses, T cells were then incubated for 48 h in the presence of

100 l PHA (2 μg/ml) and PMA (10 ng/ml) diluted in RPMI 1640 containing 5% heat-

inactivated blood group AB serum (RPMI/AB). For the anti-CD3-anti-CD28 induced

responses, T cells were stimulated for 72 h at 37oC in a 5% CO2 humidified atmosphere with

100 l soluble anti-CD3 (25 ng/ml) and anti-CD28 (1 ng/ml) diluted in RPMI/AB. Optimal

concentrations of PHA-PMA and anti-CD3-anti-CD28 antibodies were established for T cells

prior to the initiation of TAT-JIP investigations and cell viability was confirmed for all

experiments at the conclusion of the culture period. Six hours before harvesting, 50 l of 1μCi

[methyl-3H] Thymidine (25 Ci/mmol) diluted in RPMI/AB was added to the cells. Cell

culture fluids were collected for cytokine measurements (Section 2.11) and stored at -70 oC.

Cells were harvested using the FilterMate Harvester (Perkin Elmer, Waltham, MA) and the

incorporated radioactivity measured in a Wallac MicroBeta JET (Perkin Elmer).

2.8 Tetanus Toxoid induced lymphocyte responses

Fifty microlitres of PBMC (4 x 106 cells /ml) were incubated with 50 l of TAT-JIP153-163

(0.5-20 M) or TAT-JIP153-172 (10 M) in 96-well U bottom plates for 30 min. Tetanus

Toxoid (100 l, 1ng/ml) diluted in RPMI/AB, was added to the cells and incubated for 5

days at 37oC in a 5% CO2 humidified atmosphere. The optimal concentration of Tetanus

Toxoid for PBMC was established prior to the initiation of TAT-JIP experiments. Six hours


58

before harvesting, 50 l of 1μCi [methyl-3H]-Thymidine diluted in RPMI/AB was added.

Cell culture fluids were collected for cytokine measurements (Section 2.11) and stored at -70

oC. Cells were harvested using the FilterMate Harvester and the incorporated radioactivity

was measured in a Wallac MicroBeta JET.

2.9 Mixed Lymphocyte Reaction

The PBMC (50 μl, 4 x 106 cells /ml) from one blood donor (responder cells) were pre-treated

with 50 μl of TAT-JIP153-163 (20 μM) in 96-well U bottom plates for 30 min. PBMC (2 ml, 2 x

106 cells/ml) from a second blood donor (stimulating cells) were treated with 40 l of

Mitomycin C (20 μg/ml) for 30 min at 37oC in a 5% CO2 humidified atmosphere and washed

four times with RPMI 1640 by centrifugation for 5 min at 600 g. The stimulating cells (100

l, 2 x 106 cells/ml in RPMI/AB) were added to responder PBMC and cultured for 6 days at

37oC and 5% CO2. Six hours before harvesting, 50 l of 1μCi [methyl-3H]-Thymidine diluted

in RPMI/AB was added. Cell culture fluids were collected for cytokine measurements

(Section 2.11) and stored at -70 oC. Cells were harvested and the incorporated radioactivity

was measured as described above.

2.10 Allergen induced activation

PBMC (50 l, 4 x 106 cells/ml) from atopic donors were incubated with 50 l of TAT-JIP153-

163 (20 M) or TAT-JIP153-172 (20 M) in 96-well U bottom plates for 30 min. Cells were

stimulated with 100 l of recombinant Der p 2 (20 μg/ml) diluted in RPMI/AB at 37oC in a

5% CO2 humidified atmosphere for 5 days. The optimal concentration of Der p 2 for PBMC

was established prior to the initiation of TAT-JIP experiments. Six hours before harvesting, 50


59

l of 1μCi [methyl-3H]-Thymidine diluted in RPMI/AB was added. Cell culture fluids were

collected for cytokine measurements (Section 2.11) and stored at -70oC. Cells were harvested

and the incorporated radioactivity was measured as described above.

2.11 Cytokine determination

The amount of IFN, LT, IL2, IL4 and IL10 present in cell culture fluids was measured by

fluorescent cytokine capturing beads using the BD Cytometric Bead Array (CBA) Flex Set

System. Standards from each BD CBA Human Soluble Flex Set were reconstituted with 2 ml

of assay diluent and equilibrated for 15 min. Each standard was then diluted 1:2, 1:4, 1:8,

1:16, 1:32, 1:64, 1:128, 1:256, 1:512 and 1:1024 in assay diluent. Cell culture fluids were also

diluted 1:20, if necessary with assay diluent. To start the assay, 50 l of cytokine capture bead

suspension was placed in a 96 well U-bottom plate. Each standard dilution (50 l, 1:1024,

1:256, 1:64, 1:16, 1:4, 1:1) was added to the appropriate wells to give a standard curve of 0, 5,

20, 80, 312.5, 1250 pg/ml. Each sample (50 l) was added to the wells and incubated for 1 h

in the dark at room temperature. The PE-conjugated anti-human cytokine antibody detection

reagent (PE Detection Reagent) (50 l) was then added to the wells and incubated for a

further 2 h in the dark at room temperature. Each well was washed with 150 l of wash buffer

and the plate was centrifuged at 200 g for 5 min. The supernatant was aspirated and 150 l of

wash buffer was added to each well. The samples were analysed by flow cytometry on the BD

FACSArray System. Quality control beads were used prior to the reading of samples to

ensure the instrument was working properly. Examples of the standard curves are shown in

Fig. 2.3.


60

Fig. 2.3. Examples of standard curves for human cytokine production. Standard curves

were generated for IFNγ, LT, IL2 and IL10 using the BD CBA Flex Set System.

IL10 LT

pg/ml pg/ml

IL2 IFNγ

pg/ml pg/ml

IL10 LT

pg/ml pg/ml

IL10 LT

pg/ml pg/mlpg/ml pg/ml

IL2 IFNγ

pg/ml pg/ml

IL2 IFNγ

pg/ml pg/mlpg/ml pg/ml


61

2.12 Measurement of phosphorylated JNK and phosphorylated jun by western blotting

2.12.1 Sample preparation

T cells (1 x 107; 1 x 106/ml) were resuspended in RPMI 1640 and pre-treated with or without

TAT-JIP153-163 (1-20 M) or SP600125 (10-20 M) for 30 min at 37oC and 5% CO2. Samples

were stimulated with 2 μg/ml PHA and 10 ng/ml PMA or 25 ng/ml anti-CD3 and 1 ng/ml

anti-CD28 antibodies at 37oC and 5% CO2 essentially as described previously (Costabile et al.

2001). Following stimulation, cells were centrifuged at 4C for 5 min at 1200 g and then

resuspended in 100 μl of lysis buffer (Section 2.2) and placed on a rocking platform for 2 h at

room temperature or overnight at 4C. The samples were centrifuged at 4C for 5 min at 1200

g and soluble fractions were collected. A protein assay was then performed (Section 2.12.2),

prior to the addition of Laemmli buffer (Section 2.2). Samples were boiled at 100C for 5

min and stored at -20C until use.

2.12.2 Lowry’s Protein assay

Protein standards (0, 3.125, 6.25, 12.5, 25 and 50 g) were prepared for each assay by serially

diluting 1% BSA with H2O (Lowry et al. 1951). Lowry’s solution containing 2% Na2CO3, 1%

SDS, 0.4% NaOH and 0.16% Na/K tartrate was diluted 100:1 with CuSO4 (Solution 1). A

total of 150 l of Solution 1 was then added to 50 l of standards and samples (diluted 1:10).

After 15 min of incubation at room temperature, 15 l of Solution 2 (H2O: Folin and

Ciocalteau’s Phenol Reagent, 1:1) was added. Following 20 min of incubation at room

temperature, 180 l of each standard and sample was transferred into a 96 well flat-bottomed

plate (Nunc, Roskilde, Denmark) and the optical density at 540 nm was measured using a

plate reader (Dynatech MR 5000, Dynatech Laboratories, Alexandria, VA). A standard curve


62

was generated from the protein standards, enabling the concentration of protein in each

sample to be determined.

2.12.3 Western Blot

Each sample (40 μg of protein) was separated by a 12% SDS-PAGE at 175 V for

approximately 1 h using the Bio-Rad Mini-PROTEAN 3 system (BioRad) (Costabile et al.

2001). The samples were electrophoretically transferred to nitrocellulose membrane (Pierce,

Illinois, USA) at 100 V for 1h. To monitor the extent of protein transfer, the membrane was

stained with Ponceau S (0.1% in 5% acetic acid). The membrane was immersed in blocking

solution (Section 2.2) for 1 hr at room temperature or overnight at 4C. The membrane was

then incubated with primary antibody for 1 h at room temperature. Following washing (3 x 10

min, 10 ml blocking solution), the membrane was treated with secondary antibody, HRP-

conjugated rabbit anti-mouse IgG or HRP-conjugated sheep anti-rabbit IgG, depending on the

primary antibody, for 1 h at room temperature. Immunoreactive material was detected by

enhanced chemiluminescence (Western Lightning Chemiluminescence, Perkin Elmer,

Waltham, MA) according to the manufacturers instructions. The blots were quantitated using

Image QuantTM software.

2.13 siRNA

The supplied Accell non-targeting siRNA #1, green non-targeting siRNA #1, human

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) control siRNA and human MAPK8

(JNK1) siRNA (Dharmacon, Lafayette, CO) were resuspended in 1X siRNA buffer

(Dharmacon) to a stock concentration of 100 μM. The siRNA was stored at -80°C prior to

use. T cells (2 x 106 cells /ml) were resuspended in Accell siRNA delivery media


63

(Dharmacon) containing 0.1 or 1% ΔFBS. T cells (2 ml) were treated with 1, 2, or 5 μM

siRNA and mixed gently. Samples were placed in a 6 well plate (Nunc) and incubated at 37°C

and 5% CO2 for 4, 5 or 6 days. Following incubation, cells were collected, washed twice with

Accell siRNA delivery media and placed in 40 μl of lysis buffer prior to western blot (Section

2.11). Transfection efficiency was determined using green non-targeting siRNA #1. The cells

were viewed using the Dialux EB-20 fluorescent microscope (Leitz, Germany) after a 24 h

incubation period.

2.14 Kinase profiler assays

To determine the effect of TAT-JIP153-163 and TAT-JIP153-172 on cyclin dependent kinase 2

(CDK2)/cyclin A, casein kinase 1 (CK1), p70 ribosomal protein S6 kinase (p70S6K),

ribosomal S6 protein kinase 1 (Rsk1), serum and glucocorticoid-regulated kinase (SGK) and

dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) activity, kinase

profiler assays were performed by Millipore. In these assays, each kinase (5-10 mU) was

incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA and the respective substrate in a final

reaction volume of 25 μl. CDK2/cyclin A was incubated with 0.1 mg/ml histone H1, while

CK1, p70S6K, Rsk1, SGK and DYRK were incubated with kinase-specific peptide

KRRRALS(p)VASLPGL (200 μM), KKRNRTLTV (100 μM), KKKNRTLSVA (30 μM),

GRPRTSSFAEGKK (30 μM) or casein (2mg/ml) respectively. The reaction was initiated by

the addition of 10 mM magnesium acetate and [32p-ATP] and after 40 min incubation at

room temperature, it was stopped by the addition of 5 μl of 3% phosphoric acid solution. The

reaction (10 μl) was then spotted onto a P30 filter mat and washed (3 x for 5 min) in 75 mM

phosphoric acid and in methanol prior to drying and scintillation counting.


64

2.15 Statistical Analysis

Statistical significance was calculated using GraphPad Prism 5.0. To compare the control

response to multiple groups, a two-way ANOVA followed by Bonferroni’s Multiple

Comparison test or a one-way ANOVA followed by Dunnett’s Multiple Comparison test was

performed. A paired or unpaired two-tailed Student’s t test was used to compare the means of

two groups with matched or unmatched responses respectively. A value of p < 0.05 was

considered significant.


65

3Chapter Three

Role of JNK in T cell responses induced by PHA-

PMA


66

3.1 Introduction

The role of JNK in T cell responses remains ill-defined. Using the JNK chemical inhibitor,

SP600125, and human peripheral blood lymphocytes, differentiated into Th1 and Th2 subsets,

JNK was demonstrated to play a role in regulating cytokine production (Bennett et al. 2001).

Unfortunately, this agent has been recognised to lack specificity (Bain et al. 2003).

Furthermore, studies involving mice lacking either JNK1 or JNK2 isoforms have not been

able to overcome this lack of understanding as contradictory results have been reported.

Isolated splenic T cells from JNK1-/- mice were demonstrated to exhibit either normal (Dong

et al. 1998) or reduced IL2 production (Sabapathy et al. 2001). In addition, while splenic T

cells from JNK2-/- mice showed a reduction in lymphoproliferation in studies by Sabapathy et

al. (1999), no change was reported by Yang et al. (1998). To complicate matters further while

one group showed that splenic T cells from either JNK1-/- or JNK2-/- mice had depressed

lymphoproliferation and IL2 production (Sabapathy et al. 1999; Sabapathy et al. 2001), Dong

et al. (2000) showed that splenic T cells lacking JNK had increased T cell function as

discussed in (Section 1.13).

Thus, to address this issue, a new approach was used to analyse the role of JNK in human T

cell function. This involved the application of a recently described peptide inhibitor of JNK,

TAT-JIP153-163 derived from the minimal JBD of the scaffold protein JNK-Interacting Protein

1 (JIP1) (Barr et al. 2001; Barr et al. 2002; Barr et al. 2004a).

PHA-PMA, was the first model of T cell activation used in this study as it has been

demonstrated to stimulate proliferation, cytokine production and MAPK activation in purified

human and Jurkat leukemic T cells (Li et al. 1999a; Li et al. 1999b; Costabile et al. 2001).

While the role of JNK has not been investigated using this model, ERK has been


67

demonstrated to regulate the production of IL2 and LT in PHA-PMA-induced Jurkat T cells

(Li et al. 1999a; Li et al. 1999b).

PHA, derived from the kidney bean phaseolus vulgaris, is a sugar binding protein (lectin)

which binds specifically to N-acetyl galactosamine molecules on the surface of lymphocytes

(Fisher et al. 1968; Crumpton et al. 1975). Physiologically, PHA acts in a similar manner to

lectins isolated from the human pathogen, pseudomonas aeruginosa (PA). The lectins, PA-I

and PA-II, specific for D-galactose and L-fucose, D-mannose and L-galactose respectively,

have been demonstrated to be mitogenic for human PBMC (Avichezer et al. 1987).

Specifically, PHA binds to the sheep erythrocyte binding glycoprotein CD2, a T cell surface

molecule distinct from CD3 (Leca et al. 1986). PHA binding results in increased PIP2

turnover, generating DAG (Kern et al. 1986). The consequential rise in cytosolic Ca++ and

PKC activation by DAG leads to increased gene expression. DAG can be substituted by

phorbol esters such as PMA in the activation of conventional (Ca++-dependent) and novel

(Ca++-independent) isozymes of PKC (Ashendel 1985; Kazanietz et al. 1993). DAG binds to

the C1 domain of PKC and aids in its recruitment to the plasma membrane (Wilkinson et al.

1998). This high affinity interaction results in a conformational change which removes the

pseudo substrate region from the active site of PKC, thus allowing substrate binding and

catalysis (Wilkinson et al. 1998). PMA treatment is sufficient for the induction of IL2Rα, an

early response seen in activated T cells. However, synthesis and secretion of IL2 as well as

IL2-dependent T cell proliferation require a second signal (Altman et al. 1992), which can be

provided by PHA (Klein et al. 1983).

The objective of this research was to examine the role of JNK in human T cell cytokine

production and proliferation in response to PHA-PMA stimulation. The PHA-PMA model


68

was adopted initially as this mode of T cell stimulation was successfully used in the

investigation of other MAPK in our laboratory (Li et al. 1999a; Li et al. 1999b).

3.2 PHA-PMA induced JNK activation in human T cells

The TAT-JIP153-163 peptide was first standardised in enzymatic assays performed in our

laboratory. Active JNK was extracted from TNF-stimulated HL-60 cells (human

promyelocytic leukaemia cell line) and used to phosphorylate the substrate, GST-jun (1-79).

The TAT-JIP153-163 peptide significantly blocked JNK activity in these assays (unpublished

data).

Initially, it was important to establish if JNK was activated in the PHA-PMA-stimulated

human T cells and to characterise the time and magnitude of the response. This was examined

in two ways, by assessing the phosphorylation of JNK and by examining the phosphorylation

of the JNK substrate, jun. Freshly isolated human PBMC were applied to plasma-coated

plates and nylon wool columns to remove monocytes and B cells. The preparation, containing

highly purified T cells was then incubated in the presence of PHA-PMA at 37°C in a 5% CO2

humidified atmosphere over a 60 min period. Samples were then examined for JNK

phosphorylation essentially as described previously (Costabile et al. 2001). Cell lysates were

prepared and subjected to western blotting using an antibody directed against phosphorylated

forms of JNK. The observed p46 and p55 kDa isoforms of JNK are both encoded by the Jnk1

and Jnk2 genes (Derijard et al. 1994; Kallunki et al. 1994; Gupta et al. 1996). To standardise

for loading variation, blots were re-probed with -actin antibody and initial studies examining

total protein expression also confirmed these results.


69

The results demonstrate that there was minimal phosphorylation of the p46 kDa isoform over

the 60 min period (Fig. 3.1). However, in the presence of PHA-PMA, T cells displayed a

transient increase in JNK phosphorylation. While phosphorylation of the p46 and p54 kDa

isoforms followed a similar pattern over the 60 min period, phosphorylation of the p54 kDa

isoform was more pronounced relative to the basal phosphorylation levels compared to the

p46 kDa isoform. Maximum phosphorylation was observed at 30-45 min after stimulation

(Fig. 3.1).

Jun is the main substrate for JNK (Pulverer et al. 1991; Derijard et al. 1994). To ensure that

JNK is able to activate the transcription factor in human T cells stimulated with PHA-PMA,

we examined the phosphorylation of this substrate in intact T cells. T cells were activated in

the presence of PHA-PMA over a 24 h period. At various times, samples were examined for

jun phosphorylation by western blot. The antibody used detected phosphorylated forms of jun,

including c-jun, junB and junD. In the presence of PHA-PMA, jun phosphorylation was

significantly increased by 2 h, maximal by 4 h and then declined over the 24 h period (Fig.

3.2). Densitometric analysis of the most prominently phosphorylated band, junB was

performed and the pooled data are presented in Fig. 3.2 (lower panel).


70

Fig. 3.1. JNK is phosphorylated in human T cells in response to PHA-PMA stimulation.

T cells were stimulated with PHA-PMA at 37C in a 5% CO2 humidified atmosphere over a

60 min period. Samples were taken at the indicated time and examined for phosphorylated

JNK by western blot using an anti-phosphorylated-JNK (G-7) antibody. A representative

western blot is displayed in the top panel. Blots were re-probed with -actin antibody to

standardise for loading variation. The bands were quantitated using Image QuantTM software

(lower panel). Data are presented as a percentage of the control response and are expressed as

mean ± SEM of three experiments. Significance of difference from time 0 min: *** p< 0.001,

Bonferroni’s Multiple Comparison Test.

p54p46p54p46

actin

0 10 20 30 45 60 min

p54p46p54p46

actin

0 10 20 30 45 60 min

***

0

1000

2000

3000

0 10 20 30 40 50 60

Time (min)

JNK

ph

osph

oryl

atio

n (%

of

con

trol

)

p46

p54***

p54p46p54p46

actin

0 10 20 30 45 60 min

p54p46p54p46

actin

0 10 20 30 45 60 min

***

0

1000

2000

3000

0

1000

2000

3000

0 10 20 30 40 50 60

Time (min)

JNK

ph

osph

oryl

atio

n (%

of

con

trol

)

p46

p54

p46

p54***


71

Fig. 3.2. Jun is phosphorylated in human T cells in response to PHA-PMA stimulation. T

cells were stimulated with PHA-PMA over a 24 h period. Samples were examined for

phosphorylated jun by western blot using an anti-phosphorylated-c-jun antibody. A

representative western blot is displayed in the top panel. The observed jun forms include c-

jun, JunB and JunD. Densitometric data of the phosphorylation of the most dominant band,

junB are represented in the histogram above. Blots were re-probed with -actin antibody to

standardise for loading variation. Data are presented as a percentage of the control response

and are expressed as mean ± SEM of three experiments. Significance of difference from time

0 min: * p< 0.05, ** p<0.01, *** p<0.001, Dunnett's Multiple Comparison Test.

0

100

200

300

0 1 2 4 8 24Time (h)

Jun

Bph

osp

hor

ylat

ion

(%

of

con

trol

)

**

******

*

phospho-c-jun phospho-junBphospho-junC

actin

0

100

200

300

0

100

200

300

0 1 2 4 8 24Time (h)

*

0 1 2 4 8 24 h8 24 h

0

100

200

300

0

100

200

300

0 1 2 4 8 24Time (h)

Jun

Bph

osp

hor

ylat

ion

(%

of

con

trol

)

**

******

*



actin

0

100

200

300

0

100

200

300

0 1 2 4 8 24Time (h)

*

0 1 2 4 8 24 h8 24 h0 1 2 4 8 24 h8 24 h


72

3.3 Effect of TAT-JIP153-163 on the JNK pathway in human T cells

The TAT-JIP153-163 peptide has been shown to inhibit the ability of JNK to phosphorylate its

substrates in cell-free systems (Barr et al. 2004a), however an effect has not been

demonstrated in intact cells. Therefore, it was important to establish that the peptide inhibited

the phosphorylation of endogenous jun, particularly in human T cells in response to PHA-

PMA. This was investigated by pre-treating the cells with the peptide (1-20 μM) prior to

stimulation with mitogen. After 4 h of PHA-PMA stimulation, samples were examined for jun

phosphorylation by western blot (Fig. 3.3). The TAT-JIP153-163 peptide inhibited the

phosphorylation of junB (dominant band) in a concentration-dependent manner, with

significant inhibition observed at a concentration of 10 and 20 μM.


73

Fig. 3.3. Inhibition of jun phosphorylation by TAT-JIP153-163 in intact human T cells in

response to PHA-PMA stimulation. T cells were pre-treated with TAT-JIP153-163 prior to

stimulation with PHA-PMA at 37C in a 5% CO2 humidified atmosphere for 4 h. Samples

were examined for phosphorylated jun by western blot using an anti-phosphorylated-c-jun

antibody. A representative western blot is displayed in the top panel. Blots were re-probed

with -actin antibody to standardise for loading variation. The bands were quantitated using

Image QuantTM software. Data are presented as a percentage of the control response and are

expressed as mean ± SEM of three experiments. Significance of difference compared to the

stimulated control: * p< 0.05, *** p< 0.001, Dunnett's Multiple Comparison Test.

0

25

50

75

100

0 5 10 15 20

% I

nh

ibit

ion

of

JunB

ph

osph

oryl

atio

n

*

***

actin

0

25

50

75

100

0

25

50

75

100

0 5 10 15 20

TAT-JIP153-163 peptide (μM)

*

actin

0 1 5 10 20 μM

phospho-c-junphospho-junBphospho-junC

0

25

50

75

100

0

25

50

75

100

0 5 10 15 20

% I

nh

ibit

ion

of

JunB

ph

osph

oryl

atio

n

*

***

actin

0

25

50

75

100

0

25

50

75

100

0 5 10 15 20


*

actin

0 1 5 10 20 μM




74

3.4 Effect of the TAT-JIP153-163 peptide on human T cell function

After establishing that TAT-JIP153-163 inhibited the ability of JNK to phosphorylate jun, the

peptide was used to investigate the role of JNK in T cell responses. Cells were pre-treated

with TAT-JIP153-163 (1-20 μM) for 30 min prior to stimulation with PHA-PMA. The

incorporation of 3H-Thymidine was measured to assess the degree of lymphoproliferation

after 48 h of culture. Consistent with the inhibition of jun phosphorylation, (Fig. 3.3), the

TAT-JIP153-163 peptide inhibited T cell proliferation at 10 μM and to a greater degree as the

concentration was increased to 20 μM (Fig. 3.4). In contrast, the control peptide, containing

alanine substitution of two critical residues in the minimal JBD (Barr et al. 2004b), did not

inhibit T cell proliferation (Fig. 3.5). A peptide concentration of 50 μM was used to ensure

the control peptide did not affect T cell proliferation even at very high doses.

To gain further insights into the role of JNK in human T cell function, we investigated the

effect of the peptide on T cell cytokine production. Cells were pre-treated with peptide (10

μM) for 30 min and then stimulated with PHA-PMA for 48 h. The cell culture fluids were

collected and used for the quantification of cytokine by the cytometric bead array method.

Notably, cytokine values differed considerably between donors; nevertheless, TAT-JIP153-163

inhibited the production of LT, IFN, IL2 and IL10 production by 42%, 64%, 57% and 83%

respectively (Fig. 3.6).


75

Fig. 3.4. Inhibition of human T cell proliferation by the TAT-JIP153-163 peptide. T cells

were pre-treated with TAT-JIP153-163 (1-20 μM) for 30 min and stimulated with PHA-PMA at

37 C in a 5% CO2 humidified atmosphere for 48 h. Six hours prior to harvesting, cells were

pulsed with 1 μCi of methyl-[3H]-Thymidine and incorporated radioactivity was measured.

The dpm for the basal T cell cultures and PHA-PMA stimulated cells were 1973 ± 985 and

172078 ± 18771 respectively. Data are presented as the percentage of inhibition compared to

the stimulated control response and are expressed as mean ± SEM of three experiments

performed in triplicate. Significance of difference compared to the stimulated control: ** p<

0.01, *** p<0.001, Dunnett's Multiple Comparison Test.

0

25

50

75

100

0 5 10 15 20

**

***

***

0

25

50

75

100

0

25

50

75

100

0 5 10 15 20


% I

nh

ibit

ion

of

lym

ph

opro

lifer

atio

n

0

25

50

75

100

0

25

50

75

100

0 5 10 15 20

**

***

***

0

25

50

75

100

0

25

50

75

100

0 5 10 15 20


% I

nh

ibit

ion

of

lym

ph

opro

lifer

atio

n


76

Fig. 3.5. The control peptide did not inhibit T cell proliferation in response to PHA-

PMA stimulation. T cells were pre-treated with TAT-JIP153-163 (50 μM) or control peptide

(50 μM) for 30 min and stimulated with PHA-PMA at 37 C and in a 5% CO2 humidified

atmosphere for 48 h. Six hours prior to harvesting, cells were pulsed with 1 μCi of methyl-

[3H]-Thymidine and incorporated radioactivity was measured. Data are presented as mean ±

SEM of three experiments performed in triplicate. Significance of difference compared to the

untreated group: *** p< 0.001, Dunnett’s Multiple Comparison Test.

0

25000

50000

75000

Untreated Control peptide

TAT-JIP

Treatment

***

Lym

ph

opro

life

rati

on (

dp

m)

0

25000

50000

75000

0

25000

50000

75000

Untreated Control peptide

TAT-JIP

Treatment

***

Lym

ph

opro

life

rati

on (

dp

m)


77

Fig. 3.6. Inhibition of human T cell cytokine production by the TAT-JIP153-163 peptide. T

cells were pre-treated with TAT-JIP153-163 (10 μM) for 30 min and stimulated with PHA-PMA

at 37 C in a 5% CO2 humidified atmosphere. Cell culture fluids were harvested after 48 h

and cytokine levels were determined by the cytometric bead array method. Cytokine

production by control cells stimulated with PHA-PMA was as follows: IFN: 12726 ± 5181

pg/ml, IL2: 42277 ± 14311 pg/ml, LT: 1279 ± 376 pg/ml and IL10: 414 ± 296 pg/ml. Data are

presented as the percentage of inhibition compared to the stimulated control response and are

expressed as mean ± SEM of three experiments performed in triplicate. Significance of

difference compared to the stimulated control: * p< 0.05, ** p<0.01, *** p<0.001, Dunnett's

Multiple Comparison Test.

0

25

50

75

100

IFN LT IL2 IL10

Cytokine

% I

nh

ibit

ion

of

cyto

kin

e p

rod

uct

ion

*

****

**

0

25

50

75

100

0

25

50

75

100

IFNγ LT IL2 IL10

Cytokine

*

****

***

0

25

50

75

100

0

25

50

75

100

IFN LT IL2 IL10

Cytokine

% I

nh

ibit

ion

of

cyto

kin

e p

rod

uct

ion

*

****

**

0

25

50

75

100

0

25

50

75

100

IFNγ LT IL2 IL10

Cytokine

*

****

***


78

3.5 Effect of the TAT-JIP153-163 peptide on murine T cell function

Since the results we obtained were different from those using JNK1 and JNK2 knockout mice

(Dong et al. 1998; Yang et al. 1998; Dong et al. 2000), we examined whether this was due to

the variation in species. Mouse splenic T cells were prepared and treated with TAT-JIP153-163

(5-50 μM) for 30 min prior to stimulation with PHA-PMA. The results demonstrate that like

human T cells, mouse splenic T cell proliferation was also inhibited by the TAT-JIP153-163

peptide in a concentration dependent manner (Fig. 3.7).


79

Fig. 3.7. Inhibition of T cell proliferation by TAT-JIP153-163 in mouse splenic T cells.

Mouse splenic T cells were pre-treated with TAT-JIP153-163 (5-50 μM) for 30 min and

stimulated with PHA-PMA at 37 C in a 5% CO2 humidified atmosphere for 48 h. Six hours

prior to harvesting, cells were pulsed with 1 μCi of methyl-[3H]-Thymidine and incorporated

radioactivity measured. The dpm for the basal T cell cultures and PHA-PMA stimulated cells

were 520 ± 124 and 204558 ± 5021 respectively. Data are presented as the percentage of

inhibition compared to the stimulated control response and are expressed as mean ± SEM of

three experiments performed in triplicate. Significance of difference compared to the

stimulated control: * p< 0.05, ** p<0.01, *** p<0.001, Dunnett's Multiple Comparison Test.

0

25

50

75

100

125

0 10 20 30 40 50

*

**

***

0

25

50

75

100

125

0

25

50

75

100

125

0 10 20 30 40 500 10 20 30 40 50

*

**

***

% I

nh

ibit

ion

of

lym

ph

opro

lifer

atio

n


0

25

50

75

100

125

0 10 20 30 40 50

*

**

***

0

25

50

75

100

125

0

25

50

75

100

125

0 10 20 30 40 500 10 20 30 40 50

*

**

***

% I

nh

ibit

ion

of

lym

ph

opro

lifer

atio

n

0

25

50

75

100

125

0

25

50

75

100

125

0 10 20 30 40 50

*

**

***

0

25

50

75

100

125

0

25

50

75

100

125

0 10 20 30 40 500 10 20 30 40 50

*

**

***

% I

nh

ibit

ion

of

lym

ph

opro

lifer

atio

n



80

3.6 Effect of the pharmacological JNK inhibitor, SP600125 on human T cell function

In view of previous findings with the classical pharmacological JNK inhibitor, SP600125

(Bennett et al. 2001), it was necessary to confirm these results under our experimental

conditions. At present, there has been only one study which has reported the effects of this

inhibitor on T cells (Bennett et al. 2001). In our studies human T cells were pre-treated with

SP600125 (1-20 μM) for 30 min prior to stimulation with PHA-PMA. In contrast to TAT-

JIP153-163, SP600125 had no effect on PHA-PMA-induced T cell proliferation (Fig. 3.8). This

was not surprising since the examination of the ability of SP600125 to inhibit jun

phosphorylation under these conditions failed to show any effect (Fig. 3.9).


81

Fig. 3.8. SP600125 does not inhibit human T cell proliferation in response to PHA-PMA

stimulation. T cells were pre-treated with TAT-JIP153-163 or SP600125 (1-20 μM) for 30 min

and stimulated with PHA-PMA at 37 C in a 5% CO2 humidified atmosphere for 48 h. Six

hours prior to harvesting, cells were pulsed with 1 μCi of methyl-[3H]-Thymidine and

incorporated radioactivity was measured. Data are presented as a percentage of the stimulated

control response and are expressed as mean ± SEM of three experiments. Significance of

difference compared to the stimulated control: * p< 0.05, *** p< 0.001, Bonferroni's Multiple

Comparison Test.

0

25

50

75

100

125

150

0 5 10 15 20

*

*** ***

0

25

50

75

100

125

150

0 5 10 15 20

Inhibitor (μM)

0

25

50

75

100

125

150

0

25

50

75

100

125

150

0 5 10 15 20

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol) TAT-JIP

SP600125

TAT-JIP

SP600125

TAT-JIP

SP600125

TAT-JIP153-163

SP600125

*

*** ***

0

25

50

75

100

125

150

0 5 10 15 20

*

*** ***

0

25

50

75

100

125

150

0 5 10 15 20

Inhibitor (μM)

0

25

50

75

100

125

150

0

25

50

75

100

125

150

0 5 10 15 20

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol) TAT-JIP

SP600125

TAT-JIP

SP600125

TAT-JIP

SP600125

TAT-JIP153-163

SP600125

*

*** ***


82

Fig. 3.9. SP600125 did not inhibit jun phosphorylation in human T cells. T cells were pre-

treated with SP600125 prior to stimulation with PHA-PMA at 37C in a 5% CO2 humidified

atmosphere for 4 h. Samples were examined for phosphorylated jun by western blot using as

described in the legend of Fig. 3.2. A representative western blot is displayed in the top panel.

The blots were quantitated using Image QuantTM software. Data are presented as a percentage

of the stimulated control response and are expressed as mean ± SEM of three experiments.

Significance of difference compared to stimulated control: p> 0.05, Dunnett’s Multiple

Comparison Test.

0

25

50

75

100

125

0 10 20

Jun

Bph

osp

hor

ylat

ion

(%

of

con

trol

)

actin

0 10 20

0

25

50

75

100

125

0

25

50

75

100

125

0 20

SP600125 (μM)

10 20 μM


0

25

50

75

100

125

0

25

50

75

100

125

0 10 20

Jun

Bph

osp

hor

ylat

ion

(%

of

con

trol

)

actin

0 10 20

0

25

50

75

100

125

0

25

50

75

100

125

0 20

SP600125 (μM)

10 20 μM




83

3.7 Summary

To date there has been limited use of the TAT-JIP153-163 peptide in the examination of the JNK

pathway in cellular function and no studies have been reported for T cells. For the first time

we demonstrate the effectiveness of the TAT-JIP153-163 peptide in its ability to inhibit the JNK

pathway in intact cells, specifically human T cells. PHA-PMA stimulated the phosphorylation

of JNK and its substrate and significant inhibition of jun phosphorylation occurred at a TAT-

JIP153-163 concentration of 10 μM.

In association with the observed inhibition of JNK activity by the TAT-JIP153-163 peptide,

PHA-PMA-induced T cell proliferation and IL2, IFNγ, LT, IL10 cytokine production were

reduced. This inhibition also occurred with mouse splenic T cells in response to PHA-PMA

however, the pharmacological inhibitor, SP600125 failed to inhibit JNK activity. In summary,

the data suggest that JNK up-regulates human T cell responses and plays an important role in

T cell function.


84

4Chapter Four

Role of JNK in T cell responses induced via the

TCR


85

4.1 Introduction

While studies on PHA-PMA-induced T cell responses are useful, they represent only selective

aspects of the immune system and part of the innate immune response. Therefore, to

understand the role of JNK in adaptive immunity further studies using agonists which act via

the TCR were examined.

Previous studies investigating the role of JNK have complicated matters by using different

models of T cell activation. In JNK2-/- mice, Sabapathy et al. (1999) observed reduced

lymphoproliferation and IL2 production in response to anti-CD3 and anti-CD28 antibodies,

while Yang et al. (1998) showed normal T cell function in the presence of PMA alone.

The most commonly used model of T cell activation involves anti-CD3 in combination with

anti-CD28 antibodies. The anti-CD3 monoclonal antibody, OKT3 was generated in 1979

(Kung et al. 1979). In vitro, this antibody induces optimal T cell proliferation and cytokine

production in the presence of antigen presenting cells (APC) due to FcR-mediated cross-

linking (Van Lier et al. 1987). Anti-CD3 specifically binds the CD3ε portion of the TCR

inducing a conformational change which initiates early T cell signalling (Smith et al. 1997;

Kjer-Nielsen et al. 2004).

CD28 is a homodimeric glycoprotein expressed on the surface of most mature T cells (95%

CD4+) (June et al. 1987). CD28 binds the B7 protein on APC and provides the costimulation

required for TCR-induced activation of T cells (Linsley et al. 1990; Gimmi et al. 1991).

Unlike the CD3 signalling pathway, CD28 may act independently of PKC (Van Lier et al.

1991) and has been associated with PI3K activation in T cells (Truitt et al. 1994; Garcon et al.

2008). Following its discovery, the CD28 monoclonal antibody was demonstrated to increase


86

proliferation of purified T cells induced by anti-CD3 (Weiss et al. 1984; Martin et al. 1986).

Thus, the combination of anti-CD3 and anti-CD28 antibodies are frequently used for the

activation of purified T cells (Costabile et al. 2001; Costabile et al. 2005).

The mixed lymphocyte reaction (MLR) provides another model to investigate the role JNK in

human T cell function (Schwarz 1968). This assay examines the host proliferative response to

allogeneic cells which have been prevented from proliferating by mitomycin C. The MLR is

controlled by the HLA-D region of MHC in humans (Manzo et al. 1994; Bishara et al. 1998).

A disparity in the HLA-D region between two donors causes a stimulatory response (Manzo

et al. 1994; Bishara et al. 1998).

TCR modulated responses can also be initiated in vitro using antigens to which individuals

have already been sensitized. Tetanus Toxoid, from Clostridium tetani stimulates the TCR on

sensitized T cells, driving the cells through such antigenic stimulation. In the presence of APC

such as monocytes, the antigen promotes T cell proliferation and IL2, IFN and LT cytokine

production (Alpert et al. 1981; Fernandez et al. 1994; Piersma et al. 2006).

Another antigen class which gives rise to T cell responses are allergens. The discovery of the

highly specific recombinant allergens has now provided a better tool for studying T cell

responses in these models. The recombinant HDM allergen, Determatophagoides

pteronyssinus, Der p 2, can be easily produced and has been optimised to induce high levels

of proliferation and cytokine release (Thomas et al. 2004). Thus the effects of the TAT-JIP153-

163 peptide in each of these TCR-induced models were examined.


87

4.2 Effect of the TAT-JIP153-163 peptide on the JNK pathway in TCR-induced T cells

While previous studies have established that JNK is activated in the anti-CD3-anti-CD28

model in T cells (Su et al. 1994), it was important to determine the kinetics of jun

phosphorylation and consequently confirm the efficacy of the JNK pathway inhibitor TAT-

JIP153-163 in the TCR-induced model. Human peripheral blood T cells were stimulated over a

60 min period with soluble anti-CD3 and anti-CD28 antibodies and examined for

phosphorylated jun by western blot. A prominent increase in jun phosphorylation was

observed after 5 min and maintained until 15 min before declining (Fig. 4.1).

The TAT-JIP153-163 peptide inhibited the phosphorylation of jun in antibody-stimulated human

T cells. Following a 30 min pre-treatment with TAT-JIP153-163 (1-20 μM), cells were

stimulated with anti-CD3-anti-CD28 antibodies for 15 min and examined for jun

phosphorylation by western blot (Fig. 4.2). The TAT-JIP153-163 peptide inhibited the

phosphorylation of jun in a concentration-dependent manner. Significant inhibition was

observed at TAT-JIP153-163 concentrations of 10 and 20 μM.


88

Fig. 4.1. Jun is phosphorylated in human T cells in response to anti-CD3-anti-CD28

antibodies. T cells were stimulated with anti-CD3 and anti-CD28 antibodies over 60 min at

37oC in a 5% CO2 humidified atmosphere. Samples were examined for phosphorylated jun by

western blot using an anti-phosphorylated-c-jun antibody. A representative western blot

showing junB phosphorylation is displayed in the top panel. Blots were re-probed with -

actin antibody to standardise for loading variation. Phosphorylated jun was quantitated using

Image QuantTM software. Data are presented as a percentage of the stimulated control

response and are expressed as mean ± SEM of three experiments. Significance of difference

from time 0 min: * p< 0.05, Dunnett's Multiple Comparison Test.

phospho-junB

actin

0 5 15 30 60 min

0

100

200

300

0 10 20 30 40 50 60

Time (min)

Jun

Bph

osp

hor

ylat

ion

(%

of

con

trol

)

*

*

phospho-junB

actin

0 5 15 30 60 min

0

100

200

300

0

100

200

300

0 10 20 30 40 50 60

Time (min)

Jun

Bph

osp

hor

ylat

ion

(%

of

con

trol

)

*

*


89

Fig. 4.2. Inhibition of JunB phosphorylation by the TAT-JIP153-163 peptide in human T

cells in response to anti-CD3-anti-CD28 antibodies. T cells were pre-treated with TAT-

JIP153-163 for 30 min prior to stimulation with anti-CD3-anti-CD28 antibodies at 37C in a 5%

CO2 humidified atmosphere for 15 min. Samples were examined for phosphorylated jun by

western blot using an anti-phosphorylated-c-jun antibody. A representative western blot,

showing junB phosphorylation, is displayed in the top panel. Blots were re-probed with -

actin antibody to standardise for loading variation. Phosphorylated jun was quantitated using

Image QuantTM software. Data are presented as the percentage of inhibition compared to the

stimulated control response and are expressed as mean ± SEM of three experiments

performed in triplicate. Significance of difference compared to stimulated control: ** p< 0.01,

*** p< 0.001, Dunnett's Multiple Comparison Test.

phospho-junB

actin

0

25

50

75

100

0 5 10 15 20

TAT-JIP153-163 peptide (M)

% I

nh

ibit

ion

of

JunB

ph

osph

oryl

atio

n

**

***

phospho-junB

actin

0

25

50

75

100

0 5 10 15 20


% I

nh

ibit

ion

of

JunB

ph

osph

oryl

atio

n

**

***

0

25

50

75

100

0 5 10 15 20


% I

nh

ibit

ion

of

JunB

ph

osph

oryl

atio

n

**

***


90

4.3 Effect on human T cell function in response to anti-CD3-anti-CD28 antibodies

The role of JNK in T cell function was examined when both the TCR and co-stimulatory

molecules were ligated with anti-CD3 and anti-CD28 antibodies. T cells were pre-treated with

the TAT-JIP153-163 peptide (20μM) for 30 min prior to stimulation with anti-CD3 and anti-

CD28 antibodies. 3H-Thymidine incorporation was then used to measure lymphoproliferation

after 72 h. Prior to harvesting, cell culture fluids were removed and stored at -70 °C for

cytokine measurements.

The data presented in Fig. 4.3. demonstrate that in contrast to the PHA-PMA model, T cell

proliferation was significantly enhanced in the presence of the TAT-JIP153-163 peptide at 20

μM following anti-CD3-anti-CD28 stimulation. Importantly, no significant enhancement of

proliferation was observed below this concentration (data not shown). The increased

proliferation also corresponded to a rise in IL2 cytokine production; however IFNγ, LT and

IL10 production were only moderately enhanced without reaching statistically significant

levels (Fig. 4.4). The data show that the JNK signalling pathway down regulates

lymphoproliferation and cytokine production in T cells activated via the TCR.

The pharmacological inhibitor, SP600125 has been demonstrated to inhibit JNK activity in

Jurkat T cells stimulated with PMA and anti-CD3-anti-CD28 antibodies (Bennett et al. 2001).

Thus to compare the effect of SP600125 to TAT-JIP153-163 in the TCR-induced model, T cells

were pre-treated with the pharmacological inhibitor (20μM) for 30 min prior to stimulation

with anti-CD3-anti-CD28 antibodies for 72 h. In contrast, treatment of T cells with SP600125

inhibited the T cell response induced by anti-CD3-anti-CD28 antibodies (Fig. 4.5). While a

reduction in IFNγ and LT cytokine production was observed, statistical significance was not


91

achieved (Fig. 4.6). Unfortunately, the IL2 values were below detectable levels probably due

to consumption and were not included in the remainder of the Chapter 4 results.


92

Fig. 4.3. Enhancement of T cell proliferation by the TAT-JIP153-163 peptide in response to

anti-CD3-anti-CD28 antibody stimulation. T cells were pre-treated with the TAT-JIP153-163

peptide (20 μM) for 30 min prior to stimulation with anti-CD3-anti-CD28 antibodies for 72 h

at 37oC in a 5 % CO2 humidified atmosphere. Six hours prior to harvesting, cells were pulsed

with 1 μCi of methyl-[3H]-Thymidine and incorporated radioactivity measured. The dpm for

the basal T cell cultures and anti-CD3-anti-CD28-stimulated cells were 1334 ± 147 and 42082

± 10774 respectively. Data are presented as a percentage of the stimulated control response

and are expressed as mean ± SEM of four experiments each performed in triplicate.

Significance of difference compared to stimulated control: *, p< 0.05, two-tailed paired t test.

0

100

200

300

400

Control TAT-JIP

Treatment

0

100

200

300

400

0

100

200

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Treatment

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Treatment

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93

Fig. 4.4. Enhancement of cytokine production by the TAT-JIP153-163 peptide in response

to anti-CD3-anti-CD28 antibody stimulation. T cells were pre-treated with the TAT-JIP153-

163 peptide (20 μM) for 30 min prior to stimulation with anti-CD3-anti-CD28 antibodies for

72 h at 37oC in a 5 % CO2 humidified atmosphere. Cell culture fluids were harvested and

cytokine levels measured by cytometric bead array. Cytokine production by control cells

stimulated with anti-CD3-anti-CD28 antibodies was as follows: IFN: 66 ± 35 pg/ml, IL2:

4697 ± 2999 pg/ml, LT: 1334 ± 757 pg/ml and IL10: 49 ± 37 pg/ml. Data are presented as a

percentage of the stimulated control response and are expressed as mean ± SEM of three

experiments. Significance of difference compared to stimulated control: * p< 0.05, Dunnett's

Multiple Comparison Test.

0

250

500

750

Control IFNγ LT IL2 IL10

Cytokine

Cyt

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Cytokine

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94

Fig. 4.5. Inhibition of T cell proliferation by SP600125 in response to anti-CD3-anti-

CD28 antibodies. T cells were pre-treated with SP600125 (20 μM) for 30 min prior to

stimulation with anti-CD3-anti-CD28 antibodies for 72 h at 37oC in a 5 % CO2 humidified

atmosphere. Six hours prior to harvesting, cells were pulsed with 1 μCi of methyl-[3H]-

Thymidine and incorporated radioactivity was measured. The dpm for the basal T cell

cultures and anti-CD3-anti-CD28 antibody stimulated cells were 1196 ± 557 and 38478 ±

10346 respectively. Data are presented as a percentage of the stimulated control and are


difference compared to stimulated control: *, p< 0.05, two-tailed paired t test.

0

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*

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95

Fig. 4.6. The effect of SP600125 on cytokine production in response to anti-CD3-anti-

CD28 stimulation. T cells were pre-treated with the SP600125 (20 μM) for 30 min prior to

stimulation with anti-CD3-anti-CD28 for 72 h at 37oC in a 5 % CO2 humidified atmosphere.

Cell culture fluids were harvested and cytokine levels measured by cytometric bead array.

Cytokine production by control cells stimulated with anti-CD3-anti-CD28 antibodies was as

follows: IFN: 5192 ± 5104 pg/ml, LT: 768 ± 598 pg/ml, IL4: 10 ± 9 pg/ml and IL10: 1 ± 0.5

pg/ml. Data are presented as a percentage of the stimulated control response and are expressed

as mean ± SEM of three experiments. Significance of difference compared to stimulated

control: p > 0.05, Dunnett's Multiple Comparison Test.

0

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Cytokine

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96

4.4 Effect on T cell responses in the mixed lymphocyte reaction

The effect of the TAT-JIP153-163 peptide on the MLR was explored. Responder PBMC were

pre-treated with the peptide (20μM) for 30 min prior to co-culture with mitomycin C treated-

stimulators. 3H-Thymidine incorporation was then used to measure lymphoproliferation after

6 days and cell culture fluids were harvested for cytokine measurements. The PBMC

population was predominantly T cells as determined by flow cytometry and in agreement with

our findings in these cells, previous studies have confirmed that JNK is activated in a mixed

cell population (Zhou et al. 2008). As was observed in the anti-CD3-anti-CD28 antibody

model, a significant increase in lymphoproliferation was evident in cells treated with the

TAT-JIP153-163 peptide (Fig. 4.7). This also corresponded to a dramatic increase in IFNγ

production (Fig. 4.8). Unfortunately, due to cell limitations, only one concentration of TAT-

JIP153-163 was examined.


97

Fig. 4.7. Enhancement of cell proliferation by the TAT-JIP153-163 peptide in the MLR.

Responder PBMC were pre-treated with the TAT-JIP153-163 peptide (20μM) for 30 min prior

to co-culture with mitomycin C treated-stimulators for 6 days at 37oC in a 5 % CO2

humidified atmosphere. Six hours prior to harvesting, cells were pulsed with 1 μCi of methyl-

[3H]-Thymidine and incorporated radioactivity measured. The dpm for the basal T cell

cultures and stimulated cells were 6855 ± 1945 and 60365 ± 5773 respectively. Data are

presented as a percentage of the stimulated control response and are expressed as mean ±

SEM of six experiments performed in triplicate. Significance of difference compared to

stimulated control: * p< 0.05, two-tailed paired t test.

0

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98

Fig. 4.8. Enhancement of IFNγ production by the TAT-JIP153-163 peptide in the mixed

lymphocyte reaction. Responder PBMC were pre-treated with the TAT-JIP153-163 peptide

(20μM) for 30 min prior to co-culture with mitomycin C treated-stimulators for 6 days at

37oC in a 5 % CO2 humidified atmosphere. Cell culture fluids were harvested and cytokine

levels were measured by cytometric bead array. Cytokine production by stimulated control

cells were as follows: IFN: 3409 ± 978 pg/ml, LT: 5433 ± 2232 pg/ml, IL4: 5.4 ± 0.9 pg/ml

and IL10: 1.7 ± 0.4 pg/ml. Data are presented as a percentage of the stimulated control


compared to stimulated control: *** p< 0.001, Dunnett’s Multiple Comparison Test.

0

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99

4.5 Effect on antigen-induced T cell responses

Having investigated the role JNK plays in T cells in response to anti-CD3-anti-CD28

antibodies and in the MLR, it was also important to determine the role of this MAPK in

response to the antigen, Tetanus Toxoid, which has been demonstrated to induce T cell

proliferation and cytokine production in the presence of APC (Alpert et al. 1981; Fernandez et

al. 1994; Piersma et al. 2006). PBMC were pre-treated with the TAT-JIP153-163 peptide (1-20

μM) for 30 min prior to stimulation with antigen. Lymphoproliferation was determined after 5

days and cell culture fluids were harvested and stored for cytokine measurements.

The results showed that TAT-JIP153-163 (0.5-20 μM) enhanced lymphoproliferation in response

to Tetanus Toxoid with a significant increase observed at a TAT-JIP153-163 concentration of 10

μM (Fig. 4.9). This result was not observed at higher concentrations of the peptide. In

accordance with these findings, TAT-JIP153-163-treated cells also displayed enhanced IFN and

LT cytokine production (Fig. 4.10). In contrast, the peptide had minimal effect on IL4 and

IL10 responses (Fig. 4.10).


100

Fig. 4.9. Enhancement of lymphocyte proliferation by the TAT-JIP153-163 peptide in

response to Tetanus Toxoid. PBMC were pre-treated with the TAT-JIP153-163 peptide (0.5-20

μM) for 30 min prior to stimulation with Tetanus Toxoid (1ng/ml) for 5 days at 37oC in a 5 %

CO2 humidified atmosphere. Six hours prior to harvesting, cells were pulsed with 1 μCi of

methyl-[3H]-Thymidine and incorporated radioactivity measured. The dpm for the basal T cell

cultures and Tetanus Toxoid stimulated cells were 1795 ± 544 and 19491 ± 8875 respectively.

Data are presented as a percentage of the stimulated control response and are expressed as

mean ± SEM of five experiments performed in triplicate. Significance of difference compared

to stimulated control: * p< 0.05, Dunnett's Multiple Comparison Test.


0

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101

Fig. 4.10. Enhancement of cytokine production by the TAT-JIP153-163 in response to

antigen stimulation. PBMC were pre-treated with the TAT-JIP153-163 peptide (20 μM) for 30

min prior to stimulation with Tetanus Toxoid for 5 days at 37oC in a 5 % CO2 humidified

atmosphere. Cell culture fluids were harvested and cytokine levels were measured by

cytometric bead array. Cytokine production by control cells stimulated with Tetanus Toxoid

was as follows: IFN: 423 ± 334 pg/ml, LT: 274 ± 161 pg/ml, IL4: 2.2 ± 0.2 pg/ml and IL10:

1.5 ± 0.1 pg/ml. Data are presented as a percentage of the stimulated control response and are


difference compared to stimulated control: * p< 0.05, ** p<0.01, Dunnett's Multiple

Comparison Test.

0

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Cytokine

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Control IFNγ LT IL4 IL10Control IFNγ LT IL4 IL10

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102

4.6 Effect on allergen-induced T cell responses

Studies with Tetanus Toxoid showed that JNK negatively regulated IFNγ and LT while

having minimal effect on IL4 and IL10 cytokine production. This lack of effect on Th2

cytokine patterns was examined more directly by assessing the effect of the TAT-JIP153-163

peptide in an allergic reaction. PBMC were isolated from atopic donors and pre-treated with

the TAT-JIP153-163 peptide (20 μM) for 30 min prior to stimulation with the recombinant

HDM allergen, Der p 2. 3H-Thymidine incorporation was then used to measure

lymphoproliferation after 5 days and cell culture fluids were harvested for cytokine

measurements. Unlike other TCR-induced models, proliferation was substantially inhibited

following treatment with the TAT-JIP153-163 peptide (Fig. 4.11). Importantly, while non-atopic

donors produced a minimal response upon stimulation with Der p 2, the TAT-JIP153-163

peptide had no significant effect on T cell proliferation in these individuals (data not shown).

In contrast to the Tetanus Toxoid model, a reduction in IFNγ and LT cytokine production was

observed (Fig. 4.12). However, like the Tetanus Toxoid model, the TAT-JIP153-163 peptide had

minimal effect on the production of IL4 and IL10 (Fig. 4.12). Due to cell limitations, only one

concentration of TAT-JIP153-163 was examined.


103

Fig. 4.11. Inhibition of lymphoproliferation by the TAT-JIP153-163 peptide in response to

Der p 2. PBMC were isolated from atopic donors and pre-treated with the TAT-JIP153-163

peptide (20μM) for 30 min prior to stimulation with Der p 2 for 5 days at 37oC in a 5 % CO2


[3H]-Thymidine and incorporated radioactivity was measured. The dpm for the basal T cell

cultures and Der p 2 stimulated cells were 3383 ± 1137 and 20447 ± 2791 respectively. Data

are presented as a percentage of the stimulated control response and are expressed as mean ±

SEM of three experiments performed in triplicate. Significance of difference compared to

stimulated control: *** p < 0.001, two-tailed paired t test.

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104

Fig. 4.12. Inhibition of cytokine production by TAT-JIP153-163 peptide in response to Der

p 2. PBMC were isolated from atopic donors and pre-treated with the TAT-JIP153-163 peptide

(20μM) for 30 min prior to stimulation with Der p 2 for 5 days at 37oC in a 5 % CO2

humidified atmosphere. Cell culture fluids were harvested and cytokine levels were measured

by cytometric bead array. Cytokine production by control cells stimulated with Der p 2 was as

follows: IFN: 16 ± 2 pg/ml, LT: 123 ± 19 pg/ml, IL4: 3 ± 0.7 pg/ml and IL10: 1.2 ± 0.1

pg/ml. Data are presented as a percentage of the stimulated control response and are expressed

as mean ± SEM of three experiments performed in triplicate. Significance of difference

compared to stimulated control: *** p < 0.001, Dunnett’s Multiple Comparison Test.

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105

4.7 Summary

Similar to the PHA-PMA model, stimulation via the TCR also activated the JNK pathway in

human T cells. This pathway was effectively inhibited by the TAT-JIP153-163 peptide. In

contrast to the PHA-PMA model, lymphoproliferation and IL2 production were significantly

enhanced by the peptide when the cells were stimulated with anti-CD3-anti-CD28 antibodies

(Table 4.1), suggesting that JNK activation down-regulates these responses when the cells are

challenged via the TCR with CD3 and CD28 antibodies.

The negative role of JNK in T cell function was not only evident in this mitogenic response

but also when the TCR was engaged with antigen. This was observed in the MLR whereby

the addition of the TAT-JIP153-163 peptide increased IFNγ production (Table 4.1). When the

antigenic stimulation was provided by Tetanus Toxoid, a similar increase was observed for

lymphoproliferation and IFNγ and LT production were enhanced. Interestingly, the opposite

effects occurred when challenged with allergen (Table 4.1).

However, there were limitations to the experiments in this chapter including the fact that the

IL2 values obtained were outside the standard curve probably due to consumption and

therefore these could not be included in the assessment of the results. In addition, cell

limitations prevented experiments with multiple concentrations of the TAT-JIP153-163 peptide.

Nevertheless, the current results suggest that JNK primarily regulates Th1 cytokine

production without affecting Th2 cytokine patterns irrespective of the stimuli. The interesting

finding, however, was that JNK down-regulated these responses in the anti-CD3-anti-CD28

antibody, MLR and Tetanus Toxoid models but up-regulated T cell function in response to


106

allergen. Therefore the results suggest that inhibiting JNK may promote both allergic and

autoimmune inflammation, making such therapy unsuitable.


107

Table 4.1. Summary of the effect of the TAT-JIP153-163 peptide on T cell function in

TCR-induced models.

Anti-CD3-Anti-CD28

MLR Tetanus Toxoid Der p 2

Proliferation ↑ ↑ ↑ ↓

IFNγ - ↑ ↑ ↓

LT - - ↑ ↓

IL2 ↑ NI NI NI

IL4 - - - -

IL10 - - - -

*Arrows signify whether TAT-JIP153-163 enhanced (↑) or inhibited (↓) cell proliferation and

cytokine production and (-) indicates that statistical significance was not observed. “NI”

signifies that the cytokine values obtained were outside the standard curve and were not

included in the results. The concentrations of TAT-JIP153-163 peptide used for each model

were: Tetanus Toxoid 10 μM and 20 μM for anti-CD3-anti-CD28, MLR and Der p 2.


108

5Chapter Five

Relationship between JNK, ERK and p38 in T cell

function


109

5.1 Introduction

TCR binding activates not only the JNK pathway but also other MAPK such as the ERK and

p38 signalling pathways. Furthermore, ERK has been suggested to play an important role in T

cell function (Section 1.11). Human anti-CD3-PMA-induced T cells display reduced

lymphoproliferation, IL2, IFNγ, TNF and enhanced IL4, IL5 and IL13 production in the

presence of the ERK pathway inhibitor, PD98059 (Dumont et al. 1998). In addition, this

inhibitor has also been demonstrated to suppress lymphoproliferation, IL2 and LT production

in Jurkat and human T cells in response to PHA-PMA and anti-CD3-PMA respectively (Li et

al. 1999a; Li et al. 1999b).

The role of p38, however, is controversial (Section 1.12) as previous studies have reported

both an enhancement (Kogkopoulou et al. 2006) and a reduction (Koprak et al. 1999) in IL2

production in TCR-induced models. Furthermore, p38 inhibition has been demonstrated to

have minimal effect on the lymphoproliferative response in human T cells (Koprak et al.

1999).

While previous studies have examined the role of individual MAPK, few have investigated

the interaction between these pathways. A previous study by Kogkopoulou et al. (2006),

examined the role of ERK and p38 in human T cell IL2 production. The p38 inhibitor,

SB203580 enhanced IL2 production in response to anti-CD3-anti-CD28 antibodies. However,

the addition of the ERK pathway inhibitor, U0126, reduced cytokine production in a

concentration-dependent manner (Kogkopoulou et al. 2006), thus demonstrating that MAPK

interaction may be of major importance in human T cell function.


110

Accordingly, we examined the relationship between the MAPK; ERK, JNK and p38 in

cytokine production and proliferation in human T cells activated by either PHA-PMA or anti-

CD3-anti-CD28 antibodies. Since JNK signalling constitutes only one of several MAPK

pathways activated upon T cell stimulation, it was of importance to determine if the ERK and

p38 pathways played a similar or contrasting role to JNK and if indeed this interaction

influenced human T cell function.

5.2 Role of ERK and p38 in PHA-PMA-induced T cell responses

While the ERK pathway inhibitor, PD98059 has been demonstrated to inhibit anti-CD3-

PMA-induced human T cell proliferation and cytokine production (Li et al. 1999a), this

inhibitor and the p38 pathway inhibitor, SB203580, have not been used in the examination of

PHA-PMA-induced human T cell responses. Hence, in this study cells were pre-treated with

PD98059 (12.5-50μM) or SB203580 (1-20 μM) for 30 min prior to stimulation with PHA-

PMA for 48 h. Incorporation of 3H-Thymidine was used to assess lymphoproliferation and

cell culture fluids were harvested for cytokine measurements.

The data presented in Fig. 5.1 show that in contrast to previous studies with anti-CD3-induced

T cell stimulation, PD98059 significantly enhanced lymphoproliferation at 25 μM in the

PHA-PMA model. Interestingly, an increase in cytokine production particularly, LT and IL10

was also observed; although, statistical significance was not achieved (Fig. 5.2). These results

differed to that with the JNK pathway inhibitor, TAT-JIP153-163, which caused a reduction in

PHA-PMA-induced T cell proliferation and cytokine production (Section 3.4). Hence the

results suggest that in contrast to JNK, the ERK signalling pathway down-regulates T cell

responses in this model.


111

The p38 inhibitor, SB203580 decreased PHA-PMA-induced lymphoproliferation in a

concentration-dependent manner (Fig. 5.3). This also corresponded to a reduction in IFNγ,

LT, IL2 and IL4 production (Fig. 5.4), thus suggesting that p38 up-regulates the response.

Collectively the data suggest that the MAPK have similar and different roles in PHA-PMA-

induced T cell responses. While JNK and p38 up-regulate the response, ERK down-regulates

T cell function in this model.


112

Fig. 5.1. Enhancement of T cell proliferation by PD98059 in response to PHA-PMA

stimulation. T cells were pre-treated with PD98059 (12.5-50μM) for 30 min and then

stimulated with PHA-PMA for 48 h at 37oC in a 5 % CO2 humidified atmosphere. Six hours


radioactivity measured. The dpm for the basal T cell cultures and PHA-PMA-stimulated cells

were 2027 ± 73 and 47295 ± 10224 respectively. Data are presented as a percentage of the

stimulated control response and are expressed as mean ± SEM of three experiments

performed in triplicate. Significance of difference compared to stimulated control: * p< 0.05,

Dunnett’s Multiple Comparison Test.

0

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113

Fig. 5.2. Effect of the ERK pathway inhibitor, PD98059 on T cell cytokine production in

response to PHA-PMA stimulation. T cells were pre-treated with PD98059 (25 μM) for 30

min and then stimulated with PHA-PMA for 48 h at 37oC in a 5 % CO2 humidified

atmosphere. Cell culture fluids were harvested and cytokine levels measured by cytometric

bead array. Cytokine production by control cells stimulated with PHA-PMA was as follows:

IFN: 60251 ± 56574 pg/ml, IL2: 52178 ± 50916 pg/ml, LT: 50652 ± 50526 pg/ml, IL4: 16 ±

7 pg/ml and IL10: 16 ± 4 pg/ml. Data are presented as a percentage of the stimulated control


compared to stimulated control: p > 0.05, Dunnett's Multiple Comparison Test.

0

250

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Control IFNγ LT IL2 IL4 IL10

Cytokine

Cyt

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114

Fig. 5.3. Inhibition of T cell proliferation by the p38 pathway inhibitor, SB203580 in

response to PHA-PMA stimulation. T cells were pre-treated with SB203580 (1-20 μM) for

30 min and then stimulated with PHA-PMA for 48 h at 37oC in a 5 % CO2 humidified


Thymidine and incorporated radioactivity measured. For basal and stimulated dpm values

refer to Fig. 5.1. Data are presented as a percentage of the stimulated control response and are

expressed as mean ± SEM of three experiments. Significance of difference compared to

stimulated control: * p< 0.05, *** p< 0.001, Dunnett’s Multiple Comparison Test.

0

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***


115

Fig. 5.4. Inhibition of T cell cytokine production by SB203580 in response to PHA-PMA

stimulation. T cells were pre-treated with SB203580 (10 μM) for 30 min and then stimulated

with PHA-PMA for 48 h at 37oC in a 5 % CO2 humidified atmosphere. Cell culture fluids

were harvested and cytokine levels measured by cytometric bead array. For cytokine values

by control cells refer to Fig. 5.2. Data are presented as a percentage of the stimulated control


compared to stimulated control: * p< 0.05, Dunnett's Multiple Comparison Test.

0

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116

5.3 The effect of ERK, p38 and JNK inhibition on PHA-PMA-induced T cell responses

It is evident that agonist-induced activation of T cells leads to the stimulation of all three

MAPK but how they interact to generate a functional response is unknown. In these

experiments, T cells were treated with a combination of MAPK inhibitors to determine how

this compares to the inhibition of ERK, p38 and JNK alone.

Cells were pre-treated with PD98059 (25 μM), SB203580 (10 μM) and TAT-JIP153-163 (20

μM) in various combinations for 30 min prior to stimulation with PHA-PMA for 48 h. The

mixture of ERK and p38 or ERK and JNK inhibitors did not significantly affect PHA-PMA-

induced T cell proliferation (Fig. 5.5). In contrast however, the combination of p38 and JNK

inhibition substantially suppressed the lymphoproliferative response and IL2 production (Fig.

5.5 and 5.6). In addition, T cells pre-treated with all three inhibitors had considerably reduced

lymphoproliferation (Fig. 5.5) and while IFNγ, LT and IL2 were reduced, statistical

significance was not achieved (Fig. 5.7).

The data suggest that the interaction between the three MAPK alters the regulation of T cell

proliferation and cytokine production in response to PHA-PMA, as down-regulation by ERK

is overcome by p38 and JNK. The activation of all three pathways promotes

lymphoproliferation and cytokine production in T cells. However, if agonists preferentially

activate the ERK pathway then this may suppress the T cell response.


117

Fig. 5.5. Inhibition of T cell proliferation by a combination of ERK, p38 and JNK

inhibitors in response to PHA-PMA stimulation. T cells were pre-treated as indicated

above with PD98059 (25 μM), SB203580 (10 μM) and TAT-JIP153-163 (20 μM) for 30 min

prior to stimulation with PHA-PMA for 48 h at 37oC in a 5 % CO2 humidified atmosphere.

Six hours prior to harvesting, cells were pulsed with 1 μCi of methyl-[3H]-Thymidine and

incorporated radioactivity measured. For basal and stimulated dpm values refer to Fig. 5.1.


mean ± SEM of three experiments. Significance of difference in comparison to the stimulated

control: * p< 0.05, Dunnett’s Multiple Comparison Test.

0

25

50

75

100

125

Control ERK/p38 ERK/JNK p38/JNK ERK/p38/JNK

MAPK inhibited

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)

*

*

0

25

50

75

100

125

0

25

50

75

100

125


MAPK inhibited

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)

*

*


118

Fig. 5.6. Inhibition of T cell cytokine production by p38 and JNK inhibitors in response

to PHA-PMA stimulation. T cells were pre-treated with SB203580 (10 μM) and TAT-JIP153-

163 (20 μM) for 30 min and then stimulated with PHA-PMA for 48 h at 37oC in a 5 % CO2

humidified atmosphere. Cell culture fluids were harvested and cytokine levels measured by

cytometric bead array. For cytokine values by control cells refer to Fig. 5.2. Data are

presented as a percentage of the stimulated control and are expressed as mean ± SEM of three

experiments. Significance of difference in comparison to the stimulated control: * p< 0.05,

Dunnett's Multiple Comparison Test.

0

25

50

75

100

125


Cytokine

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)

*

0

25

50

75

100

125

0

25

50

75

100

125


Cytokine

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)

*


119


inhibitors in response to PHA-PMA stimulation. T cells were pre-treated with ERK (25

μM), SB203580 (10 μM) and TAT-JIP153-163 (20 μM) for 30 min and then stimulated with

PHA-PMA for 48 h at 37oC in a 5 % CO2 humidified atmosphere. Cell culture fluids were

harvested and cytokine levels measured by cytometric bead array. For cytokine values by

control cells refer to Fig. 5.2. Data are presented as a percentage of the stimulated control and

are expressed as mean ± SEM of three experiments. Significance of difference compared to

the stimulated control: p> 0.05, Dunnett's Multiple Comparison Test.

0

50

100

150

200

250


Cytokine

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)

0

50

100

150

200

250

0

50

100

150

200

250


Cytokine

Cyt

okin

e p

rod

uct

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(%

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trol

)


120

5.4 Role of ERK and p38 in anti-CD3-anti-CD28-induced T cell responses

The ERK pathway inhibitor, PD98059 has been demonstrated to inhibit lymphoproliferation

and cytokine production in the presence of anti-CD3-anti-CD28 antibodies (Dumont et al.

1998). However, the p38 inhibitor, SB203580 has been shown to reduce cytokine production

while having minimal effect on TCR-induced human T cell proliferation (Koprak et al. 1999).

Despite these findings, it was important to confirm these results under the same conditions as

those used for the TAT-JIP153-163 experiments. Cells were pre-treated with PD98059 (12.5-

50μM) or SB203580 (1-20 μM) for 30 min prior to stimulation with anti-CD3-anti-CD28

antibodies. 3H-Thymidine incorporation was then used to measure lymphoproliferation after

72 h and cell culture fluids were harvested for cytokine measurements.

The data presented in Fig. 5.8 illustrate that in contrast to the PHA-PMA model, PD98059

significantly inhibited the proliferative response by greater than 50 % at a concentration of 50

μM when cells were stimulated via the TCR. Interestingly, this finding also corresponded to a

significant reduction in IFNγ, LT and IL2 production (Fig. 5.9). The data suggest that in

contrast to JNK, ERK up-regulates lymphoproliferation following TCR-induced activation. In

contrast, the p38 inhibitor, SB203580 enhanced lymphoproliferation and IL2 production in

this model (Fig. 5.10 and 5.11). In summary, the data suggests that the MAPK play different

roles when T cells are activated via the TCR.


121

Fig. 5.8. Inhibition of T cell proliferation by PD98059 in response to anti-CD3-anti-

CD28 antibodies. T cells were pre-treated with PD98059 (12.5-50 μM) for 30 min prior to

stimulation with anti-CD3-anti-CD28 antibodies for 72 h at 37oC in a 5 % CO2 humidified


Thymidine and incorporated radioactivity was measured. The dpm for the basal T cell

cultures and stimulated cells were 1662 ± 152 and 68352 ± 11304 respectively. Data are

presented as a percentage of the stimulated control response and are expressed as mean ±

SEM of three experiments performed in triplicate. Significance of difference compared to the

stimulated control: * p< 0.05, Dunnett’s Multiple Comparison Test.

0

25

50

75

100

125

150

0 10 20 30 40 50

PD98059 (μM)

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)

*

0

25

50

75

100

125

150

0

25

50

75

100

125

150

0 10 20 30 40 50

PD98059 (μM)

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)

*


122

Fig. 5.9. Inhibition of T cell cytokine production by PD98059 in response to anti-CD3-

anti-CD28 antibodies. T cells were pre-treated with PD98059 (25 μM) for 30 min and then

stimulated with anti-CD3-anti-CD28 antibodies for 72 h at 37oC in a 5 % CO2 humidified


bead array. Cytokine production by control cells stimulated with anti-CD3-anti-CD28

antibodies was as follows: IFN: 1359 ± 1322 pg/ml, IL2: 1423 ± 665 pg/ml, LT: 246 ± 128

pg/ml, IL4: 8 ± 4 pg/ml and IL10: 3 ± 0.5 pg/ml. Data are presented as a percentage of the

stimulated control response and are expressed as mean ± SEM of three experiments.

Significance of difference compared to the stimulated control: ** p< 0.01, Dunnett's Multiple

Comparison Test.

0

25

50

75

100

125

150

175


Cytokine

Cyt

okin

e pr

odu

ctio

n (

% o

f co

ntro

l)

** ****

0

25

50

75

100

125

150

175

0

25

50

75

100

125

150

175


Cytokine

Cyt

okin

e pr

odu

ctio

n (

% o

f co

ntro

l)

** ****


123

Fig. 5.10. Enhancement of T cell proliferation by SB203580 in response to anti-CD3-

anti-CD28 antibodies. T cells were pre-treated with SB203580 (1-20 μM) for 30 min prior

to stimulation with anti-CD3-anti-CD28 antibodies for 72 h at 37oC in a 5 % CO2 humidified


Thymidine and incorporated radioactivity measured. For basal and stimulated dpm values

refer to Fig. 5.8. Data are presented as a percentage of the stimulated control response and are


difference compared to the stimulated control: ** p< 0.01, Dunnett’s Multiple Comparison

Test.

0

250

500

750

0 5 10 15 20

SB203580 (μM)

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)

** **

0

250

500

750

0

250

500

750

0 5 10 15 20

SB203580 (μM)

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)

** **


124

Fig. 5.11. Enhancement of IL2 production by SB203580 in response to anti-CD3-anti-

CD28 antibodies. T cells were pre-treated with SB203580 (10 μM) for 30 min and then



bead array. For cytokine values by control cells refer to Fig. 5.9. Data are presented as a


experiments. Significance of difference compared to the stimulated control: ** p< 0.01,

Dunnett's Multiple Comparison Test.

0

500

1000

1500

2000


Cytokine

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)

**

0

500

1000

1500

2000

0

500

1000

1500

2000


Cytokine

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)

**


125

5.5 The effect of ERK, p38 and JNK inhibition on anti-CD3-anti-CD28-induced T cell

responses

To further investigate the MAPK interactions, cells were pre-treated with various

combinations of PD98059 (50 μM), SB203580 (10 μM) and TAT-JIP153-163 (20 μM) for 30

min prior to stimulation with anti-CD3-anti-CD28 antibodies for 72 h. In contrast to

experiments when PHA-PMA was used as a stimulator, the combination of the p38 inhibitor,

SB203580 and TAT-JIP153-163 did not significantly affect T cell proliferation or cytokine

production in response to CD3-CD28 stimulation (Fig. 5.12 and 5.13). Similarly, the

combination of all three MAPK inhibitors did not affect lymphoproliferation (Fig. 5.12),

however a substantial reduction in IFNγ and LT production was observed with an increase in

IL10 production (Fig. 5.14). The data suggest that T cell proliferation via the TCR may be

independent of the MAPK pathways however; the MAPK may be important in the regulation

of cytokine production, particularly Th1 cytokine patterns.


126

Fig. 5.12. The effect of combining ERK, p38 and JNK inhibitors on T cell proliferation

in response to anti-CD3-anti-CD28 antibodies. T cells were pre-treated as described above

with PD98059 (50 μM), SB203580 (10 μM) and TAT-JIP153-163 (20 μM) for 30 min prior to

stimulation with anti-CD3-anti-CD28 for 72 h at 37oC in a 5 % CO2 humidified atmosphere.

Six hours prior to harvesting, cells were pulsed with 1 μCi of methyl-[3H]-Thymidine and

incorporated radioactivity measured. For the basal and stimulated dpm values refer to Fig. 5.8.


mean ± SEM of four experiments performed in triplicate. Significance of difference compared

to the stimulated control: p > 0.05, Dunnett’s Multiple Comparison Test.

0

100

200

300


MAPK inhibited

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)

0

100

200

300

0

100

200

300


MAPK inhibited

Lym

ph

opro

life

rati

on (

% o

f co

ntr

ol)


127

Fig. 5.13. The effect of combining p38 and JNK inhibitors on T cell proliferation in

response to anti-CD3-anti-CD28 antibodies. T cells were pre-treated with SB203580 (10

μM) and TAT-JIP153-163 (20 μM) for 30 min and then stimulated with anti-CD3-anti-CD28

antibodies for 72 h at 37oC in a 5 % CO2 humidified atmosphere. Cell culture fluids were

harvested and cytokine levels measured by cytometric bead array. For the cytokine values by

control cells refer to Fig. 5.9. Data are presented as a percentage of the stimulated control


compared to the stimulated control: p> 0.05, Dunnett's Multiple Comparison Test.


Cytokine

0

25

50

75

100

125

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)


Cytokine

0

25

50

75

100

125

0

25

50

75

100

125

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)


128

Fig. 5.14. Inhibition of T cell cytokine production by a combination of ERK, p38 and

JNK inhibitors in response to CD3-CD28 stimulation. T cells were pre-treated with

PD98059 (50 μM), SB203580 (10 μM) and TAT-JIP153-163 (20 μM) for 30 min and then



bead array. For cytokine values by control cells refer to Fig. 5.9. Data are presented as a


experiments. Significance of difference in comparison to the stimulated control: ** p < 0.01,

* p < 0.05, Dunnett's Multiple Comparison Test.

0

50

100

150

200


Cytokine

Cyt

okin

e p

rod

uct

ion

(%

of

con

trol

)

****

*

0

50

100

150

200

0

50

100

150

200


Cytokine

Cyt

okin

e p

rod

uct

ion

(%

of

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trol

)

****

*


129

5.6 Summary

Using the pharmacological inhibitors, PD98059, SB203580 and TAT-JIP153-163 we have been

able to identify a role for the ERK, p38 and JNK pathways in the regulation of T cell function.

When T cells were activated with PHA-PMA, inhibition of the p38 or JNK pathway reduced

T cell proliferation and cytokine production (Table 5.1 and 5.2). In contrast, ERK inhibition

enhanced PHA-PMA-induced T cell proliferation with minimal effect on cytokine production

(Table 5.1 and 5.2).

In contrast to stimulation via PHA-PMA, upon activation with anti-CD3-anti-CD28

antibodies, inhibition of the p38 and JNK pathways enhanced T cell proliferation and IL2

cytokine production and thus these pathways may act synergistically to down-regulate TCR-

induced T cell responses (Table 5.1 and 5.2). ERK inhibition however, reduced T cell

proliferation and Th1 cytokine patterns, suggesting that this MAPK acts antagonistically in

response to TCR engagement (Table 5.1 and 5.2).

In addition, the interaction between the MAPK pathways may be important in the regulation

of T cell function. This was evident in the case of PHA-PMA-induced responses, as the

activation of ERK together with p38 and JNK altered the negative regulatory role of this

MAPK with regard to T cell proliferation. In the anti-CD3-anti-CD28 model, the fact that

IL10 was enhanced, while IFNγ and LT were reduced following MAPK inhibition suggests

that these kinases may not only play an important role in cytokine regulation but also

individual cytokines may be differentially regulated.


130

Table 5.1. Comparison of the effect of MAPK inhibition on T cell proliferation in

response to PHA-PMA and CD3-CD28 stimulation.

PHA-PMA anti-CD3-anti-CD28

ERK ↑* ↓

p38 ↓ ↑

JNK ↓ ↑

ERK/p38 - -

ERK/JNK - -

p38/JNK ↓ -

ERK/p38/JNK ↓ -

*Arrows indicate whether MAPK inhibition enhanced (↑) or inhibited (↓) T cell proliferation

and (-) signifies that MAPK inhibition had no affect on T cell proliferation.


131

Table 5.2. Comparison of the effect of MAPK inhibition on T cell cytokine production in response to PHA-PMA and CD3-CD28 stimulation.

ERK p38 JNK p38/JNK ERK/p38/JNK

PHA-PMA CD3-CD28

PHA-PMA CD3-CD28

PHA-PMA CD3-CD28

PHA-PMA CD3-CD28

PHA-PMA CD3-CD28

IFNγ - ↓* ↓ - ↓ - - - - ↓

LT - ↓ ↓ - ↓ - - - - ↓

IL2 - ↓ ↓ ↑ ↓ ↑ ↓ - - -

IL4 - - ↓ - NM - - - - -

IL10 - - - - ↓ - - - - ↑

*Arrows indicate whether MAPK inhibition enhanced (↑) or inhibited (↓) T cell cytokine production and (-) signifies that MAPK inhibition did not

effect cytokine production. “NM” indicates that the corresponding cytokine was not measured.


132

6Chapter Six

Specificity of the TAT-JIP153-

163 peptide


133

6.1 Introduction

While initial reports claimed that SP600125 selectively targeted JNK1, 2, and 3 (Bennett et al.

2001), later studies questioned the specificity of the JNK inhibitor (Bain et al. 2003). Bain et

al. (2003) found SP600125 to be non-specific as 13 of the 28 protein kinases tested were

inhibited to a similar or greater extent than JNK, particularly SGK, p70S6K, CDK2, CK1δ

and DYRK1A. A more recent study by the same group revealed that SP600125 bound other

kinases including Rsk1, checkpoint kinase 2 (CHK2), maternal embryonic leucine zipper

kinase (MELK) and homeodomain interacting protein kinase 2 (HIPK2) to a similar or greater

extent than the JNK isoforms (Bain et al. 2007). Furthermore, binding assays revealed that in

addition to JNK1, 2 and 3, SP600125 bound to 36 of the 119 protein kinases that were tested

(Fabian et al. 2005).

In comparison to SP600125, the JIP-1 derived peptides are regarded as highly specific JNK

inhibitors. It was demonstrated that the 20 amino acid form of the JIP-1 derived peptide,

TAT-JIP153-172 (25 μM) blocked JNK1, 2 and 3 phosphorylation of c-jun in cell-free assays

(Bonny et al. 2001). Similarly, later studies demonstrated that the 10 amino acid form of the

JIP-1 derived peptide, TAT-JIP153-163 did not inhibit ERK or p38 activity in cell-free assays

(Barr et al. 2002). Furthermore, with the exception of JNK and the associated MKK4 and

MKK7, TAT-JIP153-172 did not inhibit ERK, p38, PKC, p34, calcium/calmodulin-dependent

protein kinase (CaMK) or protein kinase A (PKA) at a concentration of 500 μM (Borsello et

al. 2003).

Since the data generated in this thesis was based on TAT-JIP153-163, it was important to ensure

that this peptide did have selectivity for JNK. The primary aim of the following investigations


134

was to assess the specificity by examining the effects of the peptide on SGK, p70S6K, Rsk1,

CDK2/cyclin A, CK1 and DYRK.

6.2 Effect of JIP-1-derived peptides on CDK2, CK1, p70S6K, Rsk1, SGK and DYRK

activity

Kinase profiler assays were used to determine the specificity of TAT-JIP153-163. Interestingly,

the results revealed that the peptide inhibited CDK2/cyclin A (Fig. 6.1) and p70S6K activity

(Fig. 6.2) by greater than 80 %, while SGK activity was reduced by 60 % (Fig. 6.3). However,

CK1 (Fig. 6.4), DYRK (Fig. 6.5) and Rsk1 activity (Fig. 6.6) were not inhibited by the

peptide. This shows that although TAT-JIP153-163 was more selective than the pharmacological

inhibitor SP600125, it nonetheless did not have the level of specificity previously indicated.

Thus at this stage of the investigations it was also necessary to test the longer peptide upon

which TAT-JIP153-163 was derived, namely TAT-JIP153-172. This peptide was examined for

inhibition of all the above enzymes in the same assay system. The results showed that TAT-

JIP153-172 did not inhibit CDK2/cyclin A (Fig. 6.7), p70S6K (Fig. 6.8), SGK (Fig. 6.9), CK1

(Fig. 6.10) and DYRK activity (Fig. 6.11). However, the peptide did have a minimal effect on

Rsk1 as kinase activity was blocked by 25 % (Fig. 6.12). Thus, the results show that the TAT-

JIP153-172 peptide is a better tool for inhibiting the JNK signalling pathway.


135

Fig. 6.1. TAT-JIP153-163 inhibits CDK2/cyclin A activity. CDK2/cyclin A activity was

determined by kinase profiler assays performed by Millipore. Data are presented as mean ±

SD of triplicates. Significance of difference compared to control: *** p< 0.001, two-tailed

unpaired t test.

0

25

50

75

100

125

Control TAT-JIP153-163Treatment

CD

K2/

cycl

in A

act

ivit

y (%

of

con

trol

)

***

0

25

50

75

100

125

0

25

50

75

100

125


CD

K2/

cycl

in A

act

ivit

y (%

of

con

trol

)

***


136

Fig. 6.2. TAT-JIP153-163 inhibits p70S6K activity. p70S6K activity was determined by

kinase profiler assays performed by Millipore. Data are presented as mean ± SD of triplicates.

Significance of difference compared to control: *** p< 0.001, two-tailed unpaired t test.

0

25

50

75

100

125


p70

S6K

act

ivit

y (%

of

con

trol

)

***

0

25

50

75

100

125

0

25

50

75

100

125


p70

S6K

act

ivit

y (%

of

con

trol

)

***


137

Fig. 6.3. TAT-JIP153-163 inhibits SGK activity. SGK activity was determined by kinase

profiler assays performed by Millipore. Data are presented as mean ± SD of triplicates.

Significance of difference compared to control: *** p< 0.001, two-tailed unpaired t test.

0

25

50

75

100

125


SGK

act

ivit

y (%

of

con

trol

)

***

0

25

50

75

100

125

0

25

50

75

100

125


SGK

act

ivit

y (%

of

con

trol

)

***


138

Fig. 6.4. TAT-JIP153-163 does not inhibit CK1 activity. CK1 activity was determined by


0

25

50

75

100

125


CK

1 ac

tivi

ty (

% o

f co

ntr

ol)

0

25

50

75

100

125

0

25

50

75

100

125


CK

1 ac

tivi

ty (

% o

f co

ntr

ol)


139

Fig. 6.5. TAT-JIP153-163 does not inhibit DYRK activity. DYRK activity was determined by


0

25

50

75

100

125


DY

RK

act

ivit

y (%

of

con

trol

)

0

25

50

75

100

125

0

25

50

75

100

125


DY

RK

act

ivit

y (%

of

con

trol

)


140

Fig. 6.6. TAT-JIP153-163 does not inhibit Rsk1 activity. Rsk1 activity was determined by


0

25

50

75

100

125


Treatment

Rsk

1 ac

tivi

ty (

% o

f co

ntr

ol)

0

25

50

75

100

125

0

25

50

75

100

125


Treatment

Rsk

1 ac

tivi

ty (

% o

f co

ntr

ol)


141

Fig. 6.7. TAT-JIP153-172 does not inhibit CDK2/cyclin A activity. CDK2/cyclin A activity

was determined by kinase profiler assays performed by Millipore. Data are presented as mean

± SD of triplicates.

0

25

50

75

100

125


CD

K2/

cycl

in A

act

ivit

y (%

of

con

trol

)

0

25

50

75

100

125

0

25

50

75

100

125


CD

K2/

cycl

in A

act

ivit

y (%

of

con

trol

)


142

Fig. 6.8. TAT-JIP153-172 does not inhibit p70S6K activity. p70S6K activity was determined

by kinase profiler assays performed by Millipore. Data are presented as mean ± SD of

triplicates.

0

25

50

75

100

125


Treatment

p70

S6K

act

ivit

y (%

of

con

trol

)

0

25

50

75

100

125

0

25

50

75

100

125


Treatment

p70

S6K

act

ivit

y (%

of

con

trol

)


143

Fig. 6.9. TAT-JIP153-172 does not inhibit SGK activity. SGK activity was determined by


0

25

50

75

100

125


Treatment

SG

K a

ctiv

ity

(% o

f co

ntr

ol)

0

25

50

75

100

125

0

25

50

75

100

125


Treatment

SG

K a

ctiv

ity

(% o

f co

ntr

ol)


144

Fig. 6.10. TAT-JIP153-172 does not inhibit CK1 activity. CK1 activity was determined by


0

25

50

75

100

125


Treatment

CK

1 ac

tivi

ty (

% o

f co

ntr

ol)

0

25

50

75

100

125

0

25

50

75

100

125


Treatment

CK

1 ac

tivi

ty (

% o

f co

ntr

ol)


145

Fig. 6.11. TAT-JIP153-172 does not inhibit DYRK activity. DYRK activity was determined

by kinase profiler assays performed by Millipore. Data are presented as mean ± SD of

triplicates.

0

25

50

75

100

125


DY

RK

act

ivit

y (%

of

con

trol

)

0

25

50

75

100

125


DY

RK

act

ivit

y (%

of

con

trol

)


146

Fig. 6.12. TAT-JIP153-172 inhibits Rsk1 activity. Rsk1 activity was determined by kinase

profiler assays performed by Millipore. Data are presented as mean ± SD of triplicates.

Significance of difference compared to control: ** p < 0.01, two-tailed unpaired t test.

0

25

50

75

100

125


Treatment

Rsk

1 ac

tivi

ty (

% o

f co

ntr

ol)

**

0

25

50

75

100

125


Treatment

Rsk

1 ac

tivi

ty (

% o

f co

ntr

ol)

**


147

6.3 Effect of the TAT-JIP153-172 peptide on PHA-PMA and anti-CD3-anti-CD28-

induced T cell responses.

In order to make conclusions about the role of JNK in T cell responses, it was important to

confirm the results obtained with TAT-JIP153-172 as this peptide is a more selective inhibitor of

the JNK pathway. Cells were pre-treated with TAT-JIP153-172 for 30 min prior to stimulation

with PHA-PMA, anti-CD3-anti-CD28 antibodies, Tetanus Toxoid or Der p 2 allergen. The

incorporation of 3H-Thymidine was measured to assess the degree of lymphoproliferation and

cell culture fluids were collected and used for the quantification of cytokines by the

cytometric bead array method.

In agreement with the results obtained with the shorter peptide (Section 3.4), TAT-JIP153-172

inhibited PHA-PMA-induced T cell proliferation by greater than 60 % (Fig. 6.13).

Furthermore, IFNγ, LT and IL2 production were all significantly reduced (Fig. 6.14). Thus

confirming our earlier results, the data suggest that JNK promotes T cell proliferation and

cytokine production.

In the anti-CD3-anti-CD28 model, TAT-JIP153-172 also acted in a similar manner to the shorter

peptide as the lymphoproliferative response was significantly enhanced (Fig. 6.15).

Interestingly, while Th1 cytokine patterns were not affected, IL4 production was dramatically

increased following treatment with the peptide (Fig. 6.16).

Like the shorter peptide, TAT-JIP153-172 also enhanced T cell proliferation and IL2 production

in response to Tetanus Toxoid thus confirming that JNK may reduce Th1 cytokine patterns in

response to antigen (Fig. 6.17 and 6.18). However in the Der p 2 model, lymphoproliferation


148

and Th1 cytokine patterns were significantly reduced thereby confirming that JNK may up-

regulate Th1 cytokine production to prevent the associated shift in the Th1/Th2 balance

during an allergic response (Fig. 6.19 and 6.20).


149

Fig. 6.13. Inhibition of human T cell proliferation by the TAT-JIP153-172 peptide in

response to PHA-PMA. T cells were pre-treated with TAT-JIP153-172 (20 μM) for 30 min and

stimulated with PHA-PMA at 37 °C in a 5% CO2 humidified atmosphere for 48 h. Six hours


radioactivity measured. The dpm for the basal and PHA-PMA stimulated cells were 1963 ±

654 and 91777 ± 29435 respectively. Data are presented a percentage of the stimulated

control response and are expressed as mean ± SEM of three experiments performed in

triplicate. Significance of difference compared to stimulated control: * p< 0.05, two-tailed

paired t test.

0

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Fig. 6.14. Inhibition of human T cell cytokine production by the TAT-JIP153-172 peptide

in response to PHA-PMA. T cells were pre-treated with TAT-JIP153-172 (20 μM) for 30 min

and stimulated with PHA-PMA at 37 C in a 5% CO2 humidified atmosphere. Cell culture

fluids were harvested after 48 h and cytokine levels were determined by the cytometric bead

array method. Cytokine production by control cells stimulated with PHA-PMA was as

follows: IFN: 11876 ± 6440 pg/ml, IL2: 5027 ± 3612 pg/ml, LT: 409 ± 128 pg/ml, IL4: 20 ±

14 pg/ml and IL10: 8 ± 3 pg/ml. Data are presented as a percentage of the stimulated control

and are expressed as mean ± SEM of three experiments. Significance of difference compared

to stimulated control: * p< 0.05, ** p< 0.01, Dunnett's Multiple Comparison Test.

0

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Cytokine

Cyt

okin

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)

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Cytokine

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6.15. Enhancement of human T cell proliferation by the TAT-JIP153-172 peptide in

response to anti-CD3-anti-CD28 antibodies. T cells were pre-treated with TAT-JIP153-172

(20 μM) for 30 min and stimulated with anti-CD3-anti-CD28 antibodies at 37 C in a 5% CO2

humidified atmosphere for 72 h. Six hours prior to harvesting, cells were pulsed with 1 μCi of

methyl-[3H]-Thymidine and incorporated radioactivity measured. The dpm for the basal T cell

cultures and anti-CD3-anti-CD28-stimulated cells were 2839 ± 908 and 87313 ± 30993

respectively. Data are presented as a percentage of the stimulated control and are expressed as

mean ± SEM of three experiments. Significance of difference compared to stimulated control:

* p< 0.05, two-tailed paired t test.

0

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400


Treatment

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% o

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Treatment

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152


to CD3-CD28 stimulation. T cells were pre-treated with the TAT-JIP153-172 peptide (20 μM)

for 30 min prior to stimulation with anti-CD3-anti-CD28 antibodies for 72 h at 37oC in a 5 %

CO2 humidified atmosphere. Cell culture fluids were harvested and cytokine levels measured

by cytometric bead array. Cytokine production by control cells stimulated with anti-CD3-anti-

CD28 antibodies was as follows: IFN: 7620 ± 7523 pg/ml, IL2: 10465 ± 10140 pg/ml, LT:

118 ± 62 pg/ml, IL4: 30 ± 22 pg/ml and IL10: 2.7 ± 0.4 pg/ml. Data are presented as a

percentage of the stimulated control and are expressed as mean ± SEM of three experiments

Significance of difference compared to stimulated control: ** p< 0.01, Dunnett's Multiple

Comparison Test.

0

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6.17. Enhancement of human T cell proliferation by the TAT-JIP153-172 peptide in

response to Tetanus Toxoid. PBMC were pre-treated with TAT-JIP153-172 (10 μM) for 30

min and stimulated with Tetanus Toxoid at 37 C in a 5% CO2 humidified atmosphere for 5

days. Six hours prior to harvesting, cells were pulsed with 1 μCi of methyl-[3H]-Thymidine

and incorporated radioactivity measured. The dpm for the basal T cell cultures and Tetanus

Toxoid-stimulated cells were 2908 ± 891 and 43151 ± 5229 respectively. Data are presented

as a percentage of the stimulated control and are expressed as mean ± SEM of three

experiments performed in triplicate. Significance of difference compared to stimulated

control: * p< 0.05, two-tailed paired t test.

0

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Lym

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Control TAT-JIP 153-172Treatment

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154


to Tetanus Toxoid stimulation. T cells were pre-treated with the TAT-JIP153-172 peptide (10

μM) for 30 min prior to stimulation with Tetanus Toxoid at 37 C in a 5% CO2 humidifed

atmosphere for 5 days. Cell culture fluids were harvested and cytokine levels measured by

cytometric bead array. Cytokine production by control cells stimulated with Tetanus Toxoid

was as follows: IFN: 199 ± 12 pg/ml, IL2: 637 ± 300 pg/ml, LT: 119 ± 36 pg/ml, IL4: 9 ± 2

pg/ml and IL10: 3.8 ± 0.5 pg/ml. Data are presented as a percentage of the stimulated control

and are expressed as mean ± SEM of three experiments. Significance of difference compared

to stimulated control: ** p< 0.01, Dunnett's Multiple Comparison Test.

0

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Fig. 6.19. Inhibition of lymphoproliferation by the TAT-JIP153-172 peptide in response to

Der p 2. PBMC were isolated from atopic donors and pre-treated with the TAT-JIP153-172

peptide (20 μM) for 30 min prior to stimulation with Der p 2 for 5 days at 37oC in a 5 % CO2


[3H]-Thymidine and incorporated radioactivity measured. The dpm for the basal T cell

cultures and Der p 2 stimulated cells were 4146 ± 946 and 15872 ± 5649 respectively. Data

are presented as a percentage of the stimulated control and are expressed as mean ± SEM of

three experiments performed in duplicate. Significance of difference compared to stimulated

control: * p < 0.05, two-tailed paired t test.

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156

Fig. 6.20. Inhibition of cytokine production in TAT-JIP153-172 treated PBMC in response

to Der p 2. PBMC were isolated from atopic donors and pre-treated with the TAT-JIP153-172

peptide (20 μM) for 30 min prior to stimulation with Der p 2 for 5 days at 37oC in a 5 % CO2

humidified atmosphere. Cell culture fluids were harvested and cytokine levels were measured

by cytometric bead array. Cytokine production by control cells stimulated with Der p 2 was as

follows: IFN: 178 ± 9 pg/ml, IL2: 1920 ± pg/ml, LT: 96 ± 31 pg/ml, IL4: 4.5 ± 0.8 pg/ml and

IL10: 2 ± 0.4 pg/ml. Data are presented as a percentage of the stimulated control and are

expressed as mean ± SEM of three experiments. Significance of difference compared to

stimulated control: * p< 0.05, ** p<0.01, Dunnett's Multiple Comparison Test.

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6.4 Investigating the role of JNK using RNA interference

After confirming previous results with the TAT-JIP153-172 peptide, we also attempted to

investigate the role of JNK in human T cell function using RNA interference, which is a

method to cause posttranscriptional gene silencing in eukaryotic cells (Hill et al. 2003).

Specific gene silencing occurs through the use of small interfering RNA (siRNA) of

approximately 21 nucleotides in length that target the cognate mRNA sequence for

degradation (Hill et al. 2003).

Initially, human T cells were washed twice and resuspended in Accell delivery media

containing 1% ΔFBS. Non-targeting negative control, GAPDH positive control or JNK1

siRNA (1 μM) were added to the cells and incubated at 37°C and 5% CO2 for a period of 96

h. After confirming cell viability, samples were then examined for JNK1 expression as

described in Section 2.12. Cell lysates were prepared and subjected to western blotting using

an antibody directed against JNK1 and membranes were also stripped and re-probed with an

antibody directed against GAPDH.

Surprisingly, the results demonstrated that the JNK1 siRNA did not reduce JNK1 expression

(Fig. 6.21A). Furthermore, the GAPDH positive control siRNA was also ineffective as

GAPDH expression was not suppressed (Fig. 6.21B). Increasing the concentration of siRNA

to 2 μM and 5 μM, did not alter the result (Fig. 6.21C) and importantly, extending the

incubation period to 5 and 6 days did not affect JNK1 expression (Fig. 6.21D).

The next approach involved reducing the concentration of ΔFBS to 0.1% to ensure that this

did not affect siRNA uptake. Transfection efficiency was confirmed using fluorescently-

labelled negative control siRNA. However, despite the fact that siRNA was taken up by the


158

cells under these conditions, the JNK1 siRNA did not reduce the expression of the protein

(Fig. 6.21E). Therefore due to the inability of JNK1 siRNA to knockdown the protein in

primary cells, this method could not be used to further our investigation into the role of JNK

in human T cell function.

Examination of the efficacy of this system by colleagues within our institution showed that

this was also the case for primary enterocytes. Discussions with the company involved led us

to believe that the system had not been optimised for primary cells.


159

Fig. 6.21. The effect of siRNA on JNK1 and GAPDH expression. T cells were treated with

non-targeting (negative control), GAPDH (positive control) or JNK1 siRNA for 4, 5 or 6 days

at 37°C and 5% CO2. Samples were examined for JNK1 or GAPDH expression by western

Untreated Negative GAPDH JNK1

p54

p46

A

GAPDH


B

Untreated JNK1 JNK1

2 μM 5 μM

C

p54

p46

E


p54

p46

Untreated JNK1 Untreated JNK1

5 days 5 days 6 days 6 days

D

p54

p46


p54

p46

A


p54

p46

p54

p46

A

GAPDH


B

GAPDH


B

Untreated JNK1 JNK1

2 μM 5 μM

C

p54

p46

Untreated JNK1 JNK1

2 μM 5 μM

C

p54

p46

p54

p46

E


p54

p46

E


p54

p46


p54

p46

p54

p46

p54

p46



D

p54

p46



D

p54

p46

p54

p46


160

blot using anti-JNK1 and anti-GAPDH antibodies. JNK1 and GAPDH expression was

detected by enhanced chemiluminescence. A. JNK1 expression following 4 day incubation

period with 1 μM siRNA in Accell delivery media containing 1% ΔFBS. B. GAPDH

expression following 4 day incubation period with 1 μM siRNA in Accell delivery media

containing 1% ΔFBS. C. JNK1 expression following 4 day incubation period with 2 μM and 5

μM siRNA in Accell delivery media containing 1% ΔFBS. D. JNK1 expression following 5

and 6 day incubation period with 1 μM siRNA in Accell delivery media containing 1% ΔFBS.

E. JNK1 expression following 4 day incubation period with 1 μM siRNA in Accell delivery

media containing 0.1% ΔFBS.


161

6.5 Summary

The data show that while the JIP-1 derived peptides are more selective than the JNK chemical

inhibitor, SP600125, TAT-JIP153-163 also inhibited three of tested kinases including CDK2,

p70S6K and SGK. However, the longer peptide, TAT-JIP153-172 showed greater specificity as

only Rsk1 was partially inhibited. Despite the advantage over SP600125, for the first time this

study demonstrates the limitations in using the JIP-1 derived peptides, particularly TAT-

JIP153-163.

Overall, the experiments with TAT-JIP153-172 did confirm the results previously obtained with

the shorter peptide (Chapters 3, 4 and 5). Lymphoproliferation was reduced in response to

PHA-PMA and Der p 2 and enhanced following stimulation with Tetanus Toxoid and anti-

CD3-CD28 antibodies. In addition, Th1 cytokine patterns were reduced in response to PHA-

PMA and Der p 2 allergen and enhanced following activation with Tetanus Toxoid. Like the

shorter TAT-JIP153-163 peptide, Th2 cytokine patterns were not affected in the antigen or

allergen-induced models.

Therefore with a few exceptions, the findings in Chapter 3 and 4 were comparable to those of

the more selective TAT-JIP153-172 peptide and confirmed that JNK plays an important role in T

cell proliferation and cytokine production (Table 6.1). This peptide should be used in

preference to TAT-JIP153-163 and SP600125 for future JNK investigations. Unfortunately the

Accell siRNA delivery method was not successful in human T cells and thus we were not able

to gain supportive evidence for our conclusion using alternative systems.


162

Table 6.1 Comparison of the effect of the JIP-1 derived peptides on human T cell function in response to PHA-PMA, anti-CD3-anti-CD28

antibodies, Tetanus Toxoid and Der p 2.

TAT-JIP153-163 TAT-JIP153-172

PHA-PMA CD3-CD28 Tetanus Toxoid

Der p 2 PHA-PMA CD3-CD28 Tetanus Toxoid

Der p 2

Proliferation ↓ ↑ ↑ ↓ ↓ ↑ ↑ ↓

IFNγ ↓ - ↑ ↓ ↓ - - -

LT ↓ - ↑ ↓ ↓ - - ↓

IL2 ↓ ↑ NI NI ↓ - ↑ ↓

IL4 NM NM - - - ↑ - -

IL10 ↓ - - - - - - -

*Arrows indicate whether JNK inhibition enhanced (↑) or inhibited (↓) T cell lymphoproliferation and cytokine production and (-) signifies no effect

on T cell proliferation. “NM” indicates that the cytokine was not measured and “NI” indicates that the data were not included in the results.


163

7Chapter Seven

Discussion


164

7.1 Introductory remark

The data presented in this study provides evidence that JNK regulates T cell responses. This

was seen both in terms of lymphoproliferation and cytokine production. The research not only

complimented previous reports on this issue but also significantly advanced our knowledge on

the role of JNK in the regulation of human T cell responses. For example, one of the more

interesting findings was that JNK differentially regulated responses in which T cells were

stimulated via CD2 and PKC (PHA-PMA) and those that acted via the TCR, irrespective of

the mode of stimulation (CD3-CD28, Tetanus Toxoid, Der p 2, MLR). A further important

finding was that in response to Tetanus Toxoid, JNK activation down-regulated proliferation

and Th1 cytokine production, while in response to the allergen, Der p 2, JNK activation up-

regulated the lymphoproliferative response and Th1 cytokine production.

The research also provided further characterisation of the actions of the TAT-JIP peptides

which are already being used by various groups due to their selectivity of the JNK signalling

pathway. The TAT-JIP153-163 peptide inhibited several other kinases and thus was less

selective than TAT-JIP153-172. However, the TAT-JIP153-163 peptide nevertheless was more

selective than the widely used pharmacological JNK inhibitor, SP600125. These results

therefore demonstrate the care required in deriving conclusions about the role of JNK in

biological responses. There are also implications in terms of already published findings which

have used the TAT-JIP153-163 peptide.

The interrelationship between the three MAPK: ERK, p38 and JNK was also partly presented

here. The results illustrated that these kinases have overlapping as well as differing roles in

regulating proliferation and cytokine patterns in T cells. The findings suggest that JNK is


165

likely to play a regulatory role in chronic inflammation modulated via the Th1 and Th2

cytokine patterns. Therefore, therapeutic strategies for treating these conditions need to take

into consideration of the role of the JNK signalling pathway.

7.2 Targeting the JNK signalling pathway with the TAT-JIP peptides

Although it has been reported that TAT-JIP153-163 does not inhibit the p38 and ERK signalling

pathways (Barr et al. 2002), this study shows that the peptide inhibits other kinases. We

examined 6 kinases including CDK2 and p70S6K which have been demonstrated to play a

role in T cell proliferation (Firpo et al. 1994; Kawasome et al. 1998; Brennan et al. 1999;

Mohapatra et al. 2001), SGK which has been implicated in neutrophil survival (Kobayashi et

al. 2005), CK1 which is important in NFAT import in T cells (Okamura et al. 2004), DYRK

and Rsk1 both of which have been implicated in regulating apoptosis and cell survival

respectively (Carriere et al. 2008; Yoshida 2008).

Importantly, 3 of these 6 kinases were significantly inhibited by TAT-JIP153-163 (Table 7.1). In

comparison, the pharmacological inhibitor, SP600125 inhibited all 6 enzymes (Table 7.1).

The data from the kinase profiler assays also demonstrated that the 20 amino acid form of the

JIP-1 derived peptide, TAT-JIP153-172, was more selective than the shorter TAT-JIP153-163

peptide as only 1 of the 6 kinases tested was minimally inhibited (Table 7.1).

The majority of investigations in vitro and in in vivo animal models of disease have used

TAT-JIP153-172 (Bonny et al. 2001; Wang et al. 2003; Hirt et al. 2004; Kaneto et al. 2004). The

present study, however, revealed that this peptide inhibits the activity of Rsk1. Interestingly,

previous studies have reported that Rsk1 is predominantly expressed in the human kidney,


166

lung and pancreas (Zeniou et al. 2002). In addition, samples of the adult human brain also

revealed that Rsk1 is expressed abundantly in the cerebellum (Zeniou et al. 2002). Therefore,

studies which have involved the examination of these cells may need to be re-evaluated

including that of Bonny et al. (2001) which investigated the role of JNK in pancreatic β cell

apoptosis. In addition, Rsk is believed to play a critical role in T cell activation (Lin et al.

2008). In view of results from the kinase profiler assays, our data also suggests that Rsk may

be important in T cell proliferation and Th1 and Th2 cytokine production. However, further

investigations need to be conducted with specific Rsk inhibitors to confirm this hypothesis.

Furthermore, since the discovery of TAT-JIP153-163, the use of this shorter peptide has become

more frequent. This peptide had been demonstrated to prevent necrotic cell death in an in

vitro model of excitotoxic neuronal cell death and has neuroprotective effects on ischemic

brain injury in vivo (Guan et al. 2006; Arthur et al. 2007). In relation to the immune system, a

recent study in human neutrophils used TAT-JIP153-163 to demonstrate that JNK1 and JNK2

promote TNFα-induced neutrophil apoptosis (Kato et al. 2008). In our study, this peptide was

demonstrated to inhibit CDK2, p70S6K and SGK. Importantly, CDK2 (Rossi et al. 2006),

p70S6K (Gomez-Cambronero 2003) and SGK (Kobayashi et al. 2005) are all expressed in

human neutrophils and therefore the role of JNK in neutrophil apoptosis may need to be

further evaluated. Irrespective of these specificity problems, however, we have been able to

unravel the role of JNK in human T cell function using the peptide inhibitors.


167

Table 7.1. Comparison between the effect of SP600125, TAT-JIP153-163 and TAT-JIP153-

172 on CDK2/cyclin A, CK1, p70S6K, Rsk1, SGK and DYRK activity.

SP600125 TAT-JIP 153-163 TAT-JIP 153-172

CDK2/Cyclin A 20 11 ± 2 111 ± 6

p70S6K 22 18 ± 1 96 ± 3

SGK 22 44 ± 2 91 ± 7

CK1 10 109 ± 2 101 ± 4

DYRK2 19* 110 ± 3 109 ± 8

Rsk1 55* 84 ± 5 76 ± 5

The effect of SP600125 on CDK2/cyclin A, CK1, p70S6K, Rsk1, SGK and DYRK activity,

was compared to those of the JIP-1 derived peptides by the use of kinase profiler assays

(performed by Millipore). Data are presented as the mean percentage ± SD of kinase activity

remaining as compared with untreated control samples. The SP600125 results are from the

previously published work by Bain et al. (2003; 2007). All inhibitors were used at a

concentration of 10 μM unless indicated otherwise. * denotes a concentration of 1μM.


168

7.3 Role of JNK in T cell proliferation

Irrespective of the mode of stimulation (PHA-PMA, anti-CD3-anti-CD28, Tetanus Toxoid,

Der p 2), it was evident that T cell proliferation was regulated by JNK (Table 6.1). However,

substantial differences were seen in the role of JNK in these systems. In the PHA-PMA

model, both TAT-JIP153-163 and TAT-JIP153-172 inhibited the lymphoproliferative response

(Table 6.1). The mitogenic response in this model is independent of the TCR. PHA acts by

binding to N-acetyl galactosamine molecules on the T cell surface (Crumpton et al. 1975).

This mimics the action of similar lectins from bacterial pathogens such as PA (Avichezer et

al. 1987). Specifically, this interaction occurs through the CD2 glycoprotein (Leca et al. 1986)

and together with PMA co-stimulation, which activates PKC, produces a maximum response

in T cells (Klein et al. 1983). Thus non-specific activation of T cells by this system is

promoted by the JNK signalling pathway.

In comparison to JNK, activation of ERK down-regulated while p38 activation up-regulated

the lymphoproliferative response (Table 5.1). Importantly with regard to specificity, Davies et

al. (2000), revealed that PD98059 (ERK) did not inhibit any of the 24 kinases examined,

while SB203580 (p38) inhibited only 3 other kinases in the panel, however this occurred at

higher concentrations than those used in this study. Thus we can now speculate about the

relative roles of these MAPK in responses which are initiated via CD2 on T cells. Our data

also suggests that the ERK signalling pathway is not an appropriate target for the treatment of

inflammatory disorders. Previous studies have not addressed this issue from this angle,

especially when T cells are stimulated independently of the TCR.

Our work has also raised further issues regarding SP600125, as it did not inhibit the PHA-

PMA-induced proliferation of T cells and was incapable of inhibiting jun phosphorylation in

human T cells. The data questions previously reported studies which used SP600125 to


169

analyse the role of JNK in T cell function. The only other study to address this question used

the Jurkat T cell line (Bennett et al. 2001). Unfortunately limited data was presented on c-jun

phosphorylation by western blots and the data was inconclusive as the level of jun protein in

the samples were not examined. Furthermore studies on cytokine production conducted on

human CD4+ T cells differentiated towards Th1 or Th2 subsets, were restricted to one

experiment and lymphoproliferation was not examined (Bennett et al. 2001).

Of major importance was our finding that the role of JNK was different when stimulation

occurred through the TCR (Table 6.1). In the presence of TAT-JIP153-163 and TAT-JIP153-172,

lymphoproliferation was increased in response to anti-CD3-anti-CD28 antibody stimulation,

confirming that the peptides did not affect cell viability. This result is also supported by

previous findings in mice which observed enhanced T cell proliferation under the same

conditions in the absence of JNK1 and JNK2 (Dong et al. 2000). Similarly, enhanced

proliferation was also observed in the splenic T cells isolated from JNK1-/- mice (Dong et al.

1998). Therefore JNK1 may be responsible for the negative regulation of T cell proliferation.

Furthermore, a significant advancement was made by using PBMC to examine the role of

JNK in response to two different types of stimuli (antigens), Tetanus Toxoid and Der p 2. The

role of JNK was analysed in individuals who were allergic to HDM and individuals who had

been previously immunized with Tetanus Toxoid. Tetanus Toxoid-induced proliferation was

enhanced in the presence of both TAT-JIP153-163 and TAT-JIP153-172 showing that similar to

anti-CD3-anti-CD28 antibody induced stimulation, JNK activation also down-regulated this

antigenic response (Table 6.1). Interestingly however, JNK plays a positive regulatory role in

PBMC isolated from atopic individuals in response to the allergen, Der p 2. The basis for this

difference is unclear. However it is likely to have implications in the targeting of JNK for the


170

treatment of inflammatory or allergic disorders. Thus inhibition of JNK could be used to

inhibit allergen-driven responses.

7.4 Role of JNK in T cell cytokine production

The role of JNK in T cell proliferation was also reflected in cytokine production. In

accordance with the generated hypothesis, the data showed that JNK differentially regulates

individual cytokines within the Th1 and Th2 subsets. Assessment of the PHA-PMA response,

revealed that JNK regulates the production of both the Th1 (IFNγ, LT and IL2) and Th2 (IL4,

IL10) cytokine patterns (Table 6.1). Although we found that TAT-JIP153-163 inhibited the

production of IFNγ, LT, IL2 and IL10, TAT-JIP153-172 did not inhibit IL4 or IL10 production,

thus providing further confirmation that the peptides did not affect cell viability (Table 6.1).

In view of the fact that TAT-JIP153-172 more selectively targets JNK we conclude that JNK

plays a positive regulatory role in Th1 cytokine production. The data therefore suggest that in

response to pathogens acting via CD2, JNK activation induces a predominantly Th1 response.

This is also supported by Bennett et al. (2001) who demonstrated that differentiated human

CD4+ Th1 cells displayed reduced IFNγ, while Th2 cells displayed normal levels of IL4 in the

presence of the JNK pharmacological inhibitor, SP600125.

Furthermore, it is widely accepted that the polarisation of T helper cell differentiation is at

least in part determined by cytokine production (Murphy 1998). We have demonstrated that

the JNK pathway influences IFNγ production, a cytokine that promotes Th1 development

(Seder et al. 1993). Interestingly, previous studies have revealed impairment in Th1

differentiation in JNK2 -/- mice, which was due primarily to a reduction in IFNγ secretion at

the early stages of differentiation in IL12-stimulated CD4+ T cells (Yang et al. 1998).


171

Furthermore, T cells isolated from JNK1 -/- mice preferentially differentiate into Th2 cells

(Dong et al. 1998). Thus the JNK pathway may also be important in regulating the balance

between Th1 and Th2 cell type development.

Our findings with the TAT-JIP peptides suggest that JNK regulates individual cytokines

within the T cell subsets in responses induced by agonists (Table 6.1). JNK inhibition did not

affect IFNγ, LT and IL10 but substantially enhanced IL4 production in response to anti-CD3-

anti-CD28 antibody stimulation. This conclusion was primarily derived from the use of TAT-

JIP153-172. It is likely that the up-regulation of IL2 by TAT-JIP153-163 and the lack of effect of

TAT-JIP153-172 is related to the selectivity of the inhibitors. Unlike the longer peptide, TAT-

JIP153-163 inhibited CDK2 and p70S6K activity, which have both been demonstrated to play a

role in T cell function (Firpo et al. 1994; Kawasome et al. 1998; Brennan et al. 1999;

Mohapatra et al. 2001) and thus there are some reservations with regard to the results obtained

with the shorter peptide.

In response to anti-CD3-anti-CD28 stimulation, JNK plays a suppressive role in regulating

lymphoproliferation and a minimal role in regulating cytokine production except for IL4. In

support of these findings, normal IL2 levels were observed under the same conditions as those

tested in splenic T cells isolated from JNK1-/- mice (Dong et al. 1998). Despite our findings,

the role of the JNK1 and JNK2 isoforms remains controversial. While Sabapathy et al. (2001)

demonstrated that T cells from JNK1-/- mice had reduced IL2 production, Dong et al. (1998)

reported that there was no effect on cytokine production under the same conditions. Similarly

while Sabapathy et al. (1999) observed a reduction in IL2 production by JNK2-/- splenic T

cells, Yang et al. (1998) reported normal cytokine levels from JNK2-/- spleen cells in response

to PMA/ionomycin. Similar to our data, this discrepancy may be due to the variation in

stimuli. In support of this, a previous study by Veiopoulou et al. (2004) demonstrated that the


172

regulation of IL2 production by another MAPK, p38 was dependent on the mode of

stimulation.

Thus we have not only demonstrated the similarity between human and mouse T cell cytokine

production with regard to the role of the JNK signalling pathway but also significantly

advanced our understanding by showing that JNK regulates Th1 and Th2 cytokine production

and that individual cytokines within these subsets may also be differentially regulated.

In the anti-CD3-anti-CD28 model, there was a substantial difference between SP600125 and

TAT-JIP153-163. While the peptide enhanced T cell proliferation in this model, the chemical

inhibitor suppressed the response. This may be explained by the kinase CK1, which was

solely inhibited by SP600125 and not TAT-JIP153-163 (Table 7.4). Importantly, CK1 exists

with NFAT in resting T cells and dissociates upon activation (Okamura et al. 2004). The CK1

phosphorylation motif is required for NFAT import which is critical for IL2 production and

proliferation (Okamura et al. 2004), thus this may be an explanation for the reduction in

lymphoproliferation and cytokine production observed.

Examining the role of JNK in antigen-induced T cell responses produced unexpected findings.

In the MLR, Tetanus Toxoid and Der p 2 experiments, JNK regulated Th1 but not Th2

cytokine production (Table 6.1). Based on the TAT-JIP153-172 peptide, JNK only regulated IL2

production in response to Tetanus Toxoid and IL2 and LT production in response to Der p 2.

Thus the results suggest that inhibition of JNK signalling may increase responses to antigens

by enhancing IL2 production and lymphoproliferation and decrease responses to allergens by

enhancing these responses. In addition, any approaches to targeting JNK in this manner could

promote autoimmunity.


173

Similarly targeting JNK in allergy may lead to a decrease in Th1 cytokines favouring the

balance towards Th2 cytokines and an increase in the allergic inflammatory response. At

present, there is increasing interest with regard to the role of Th1 cytokines in immune

responses of atopic individuals to allergens (Heaton et al. 2005). A recent study demonstrated

an association between atopy to inhalant allergens and a mixed Th1/Th2 immune response in

children (Heaton et al. 2005). Furthermore, this study highlighted that Th1 cytokines may be

responsible for the pathogenesis of allergic disease (Heaton et al. 2005).

7.5 Interaction between members of the MAPK family in T cell function

A summary of the roles of the ERK, p38 and JNK pathways in T cell proliferation and

cytokine production in response to PHA-PMA, CD3-CD28, Tetanus Toxoid and Der p 2 are

illustrated in Fig. 7.1, 7.2 and 7.3. Using the ERK pathway inhibitor, PD98059, we

demonstrated that the MAPK negatively regulates PHA-PMA-induced proliferation of human

T cells, while playing a positive regulatory role in response to anti-CD3-anti-CD28-induced

activation. This was also observed with regard to cytokine production although changes in the

PHA-PMA model did not reach significance. Nevertheless the data suggest that when T cells

are activated independently of the TCR, ERK reduces the immune response and engagement

of the TCR by antigens enhances the response. This is in agreement with previous work

which also demonstrated a concentration-dependent reduction in the proliferative response of

anti-CD3-PMA-induced human T cells pre-treated with PD98059 (Li et al. 1999a). Hence like

JNK, these data suggest that the mode of stimulation is critical in determining the role of the

ERK in T cell function.


174

In agreement with these findings, the role of p38 is also stimulus-dependent. In PHA-PMA-

induced T cells, proliferation was substantially inhibited in the presence of the p38 pathway

inhibitor, SB203580. In anti-CD3-anti-CD28-stimulated T cells, however, a concentration-

dependent increase in the lymphoproliferative response was observed. A corresponding

reduction and increase in cytokine production was also observed in both the PHA-PMA and

anti-CD3-anti-CD28 models respectively. Thus the data suggest that in a pathogenic reaction

not involving the TCR, p38 acts to enhance the immune response and down-regulates the

response in antigen-driven T cell activation. This finding is supported by previously published

data which demonstrate that in the presence of SB203580, IL2 production is enhanced

following TCR-induced activation under suboptimal stimulation conditions (Kogkopoulou et

al. 2006).

Interestingly, the finding that p38 activation down-regulated TCR-induced

lymphoproliferation was in contrast to the findings of Koprak et al. (1999), who reported that

SB203580 had no affect on human T cell proliferation in response to anti-CD3-anti-CD28

antibodies. This may be due to the differences in T cell purification procedures. While we

used plastic plate adherence and nylon wool columns to purify T cells, Koprak et al. (1999)

used negative selection via antibody cocktails and anti-mouse Ig-coated glass bead columns.

Irrespective of whether T cells were stimulated via PHA-PMA or anti-CD3-anti-CD28

antibodies, simultaneous inhibition of ERK and JNK gave rise to a normal proliferative

response. This is not surprising since individually these pathways oppositely regulated

proliferation. In contrast, inhibition of both p38 and JNK in the PHA-PMA model, up-

regulated T cell proliferation. This therefore suggests that the response may be dominated by

the ERK signalling pathway.


175

However, the results were different for T cells stimulated via the TCR, using anti-CD3-anti-

CD28 antibodies. Inhibition of either two or three of the MAPK did not influence the

proliferative response, suggesting that the MAPK may influence one another. This is

supported by the finding that the interaction between ERK and p38 in human T cells regulates

the production of IL2 (Kogkopoulou et al. 2006). In our studies, examination of cytokine

production revealed a similar dominance by ERK, such that when all three MAPK were

inhibited, reduced cytokine production was observed, similar to the results obtained with the

inhibition of ERK alone. The present results suggest that the balance and timing of the

activation of the MAPK may be critical in the response achieved.


176

CD28

TCR/CD3

CD4

anti-CD28

p38

+

-

-

Proliferation,

IL2

JNK

ERK

p38

Proliferation,

IL4

T cell

anti-CD3

Proliferation,

IFNγ, LT, IL2CD28

TCR/CD3

CD4

anti-CD28

p38

+

-

-

Proliferation,

IL2

JNK

ERK

p38

Proliferation,

IL4

T cell

anti-CD3

Proliferation,

IFNγ, LT, IL2

A.

B.

Fig. 7.1. Summary of the role of the MAPK in human T cell function in response to

PHA-PMA (A) and anti-CD3-anti-CD28 antibodies (B).

CD2

TCR/CD3

CD4

CD28

PHA

PMA

p38

-

+

+

Proliferation

Proliferation,

IFNγ, LT, IL2, IL4

JNK

ERK

p38

Proliferation,

IFNγ, LT, IL2

T cell

CD2

TCR/CD3

CD4

CD28

PHA

PMA

p38

-

+

+

Proliferation

Proliferation,

IFNγ, LT, IL2, IL4

JNK

ERK

p38

Proliferation,

IFNγ, LT, IL2

T cell


177

Fig. 7.2. Summary of the role of the MAPK in human T cell function in response to

Tetanus Toxoid. N/A indicates that the pathways were not analysed.

CD28

TCR/CD3

CD4

p38-

N/A

JNK

ERK

p38

Proliferation,

IL2

T cell

N/A

MHC class II

B7

Tetanus Toxoid

peptide

APCLFA-1

CD2

CD45

ICAM-1

LFA-3

CD22

CD28

TCR/CD3

CD4

p38-

N/A

JNK

ERK

p38

Proliferation,

IL2

T cell

N/A

MHC class II

B7

Tetanus Toxoid

peptide

APCLFA-1

CD2

CD45

ICAM-1

LFA-3

CD22


178

Fig. 7.3. Summary of the role of the MAPK in human T cell function in response to Der

p 2 allergen. N/A indicates that the pathways were not analysed.

CD28

TCR/CD3

CD4

p38+

N/A

JNK

ERK

p38

Proliferation,

IL2, LT

T cell

N/A

MHC class II

B7

Der p 2

peptide

APCLFA-1

CD2

CD45

ICAM-1

LFA-3

CD22

CD28

TCR/CD3

CD4

p38+

N/A

JNK

ERK

p38

Proliferation,

IL2, LT

T cell

N/A

MHC class II

B7

Der p 2

peptide

APCLFA-1

CD2

CD45

ICAM-1

LFA-3

CD22


179

7.6 The relationship between Th1, Th2, Th17 and Tregs

In examining the cytokine patterns associated with T cell function, we have primarily focused

on the role of JNK in the Th1 and Th2 responses. The former being represented by IFNγ, LT

and IL2 and the latter by IL4 and IL10. However, it is also evident that other CD4+ Th cells

regulate the immune response, namely Th17 and Treg (Zhu et al. 2008). These cells are

distinguished by their own unique set of cytokines (Zhu et al. 2008). Most likely reactions in

response to bacteria, fungi, viruses, parasites, auto-antigens and allergens involve these

subsets of T cells in various ways. Thus the interpretation of our results is limited by the fact

that these subpopulations were not extensively studied.

However, on the basis of the results with IL10, which is also produced by iTreg cells (Zhu et

al. 2008), the JNK signalling pathway does not appear to play an important role in the

regulation of these cell responses, particularly involving TCR engagement. Interestingly

however, in the PHA-PMA response, JNK activation did promote IL10 production,

suggesting that microbial reactions in response to PA for example, may regulate these cells

through the JNK signalling pathway. Interestingly, ERK and p38 did not play a role in

regulating IL4 and IL10 cytokine production. Their influence on responses mediated through

TCR or non-TCR pathways were through the promotion of the Th1 cytokines and therefore

based on the IL10 results it is unlikely that they regulate Treg responses.

While this study has not investigated Treg and Th17 subpopulations, it is likely that the

MAPK have the greatest influence on the Th1 subset. The most convincing influence by

MAPK inhibition was observed in Th1 cytokine production. Thus overall the JNK signalling

pathway may play an important role in depressing chronic cell-mediated and allergic

inflammation by regulating Th1 cytokine production.


180

7.7 Infection and immunity, allergy and autoimmunity

The data are consistent with the proposal that MAPK play a role in immune responses to

microbial pathogens, allergens and auto-antigens in regulating CD4+ Th cells (Belkaid 2008;

Zhu et al. 2008). These cells express unique cytokines which induce specific functions. In

agreement with studies in the mouse, the results provide evidence that MAPK regulate

cytokine production. The data demonstrate that MAPK primarily regulate Th1 cytokine

production, namely IFNγ, IL2 and LT. This is important in the immune response to microbial

pathogens and autoimmunity. The JNK and p38 pathways promote Th1 cytokine production

when interactions occur independently of the TCR. This may be relevant to some microbial

pathogens such as PA which can stimulate T cells in this manner.

Engagement of the TCR, mimicked by the addition of anti-CD3-anti-CD28 antibodies,

promotes Th1 cytokine production via ERK (Table 5.2). Thus polyclonal activation of T cells

is likely to induce the ERK signalling pathway for the production of IFNγ, LT and IL2 (Table

5.2). In comparison, activation of p38 and JNK primarily result in the down-regulation of IL2

production and not surprisingly T cell proliferation (Table 5.1 and 5.2). This may cause T cell

proliferation to become self-regulated, depending on the degree of MAPK stimulation, which

may change during the course of an infection.

Although JNK was the only signalling molecule explored in the antigenic models, our

findings demonstrate the unique regulation of Th1 cytokine production by JNK with minimal

influence on IL4 and IL10 production in the presence of APC (Table 4.1). This was

demonstrated in the MLR and in lymphocyte cultures stimulated with antigen (Tetanus

Toxoid) and allergen (Der p 2). In all cases, the JNK signalling pathway regulated the

production of Th1 but not Th2 cytokines (Table 4.1). However, the type of antigen greatly


181

influenced the role of JNK. In the MLR and Tetanus Toxoid models, JNK activation down-

regulated both the proliferative response and Th1 cytokine production. This may play an

important role in controlling pathogenesis due to parasitic infection and autoimmunity.

Therefore, JNK may not be the appropriate target in the treatment of autoimmune disease as

this may worsen the condition. In the presence of Der p 2, JNK activation up-regulated Th1

cytokine production. This may be important in regulating allergic inflammation and reactions

due to helminths, usually involving eosinophil, mast cell and basophil responses. The role of

ERK and p38 remains unknown under these conditions and will need to be further examined

in future investigations.

At this stage, the basis for the different roles of JNK in antigen versus allergen responses is

unclear and further investigation is required to appropriately interpret the result. The Th1

responses require the transcription factors, T-bet and STAT4 while GATA3 and STAT5 are

required for Th2 cytokine production (Zhu et al. 2008). It is likely that the relationship

between the JNK signalling pathway and these transcription factors may be the key to these

differences.

7.8 Concluding remarks

This study has demonstrated that the JNK pathway is important in the regulation of human T

cell proliferation and cytokine production. In an immune response whereby the TCR is

bypassed, i.e. in response to microbial pathogens, JNK activation may up-regulate

proliferation and Th1 cytokine production. The opposite result was observed when JNK is

activated via the TCR, i.e in response to antigen. With this case, JNK activation down-

regulates T cell proliferation and Th1 cytokine production again with minimal affect on Th2


182

cytokine patterns. The exception to this case occurs in atopic individuals in response to

allergen. Under these conditions, JNK activation up-regulates T cell proliferation and Th1

cytokine production. Therefore JNK may be important in the regulation of both autoimmunity

and allergy.

The MAPK may interact with one another for the regulation of T cell proliferation and

cytokine production. Inhibition of the p38 and JNK pathways, reduced T cell proliferation and

cytokine production, however, this response was suppressed by the inhibition of the ERK

pathway under non-TCR stimulatory conditions. The results were different in TCR-stimulated

T cells as inhibition of p38 and JNK did not affect T cell proliferation or cytokine production,

however, inhibiting all three MAPK caused a reduction in Th1 cytokine production,

confirming the dominance of the ERK signalling pathway. While this area of study remains

incomplete it is evident that the findings are likely to initiate new approaches in how we

assess the role of signalling molecules in biological responses.

The limitations of this study included the fact that we did not investigate the role of JNK in

Treg or Th17 cells and therefore while we can draw some conclusions about Treg cells from

our data on IL10, to determine if JNK plays a role in Th17 cells, IL17 production needs to be

analysed. In addition, the studies which compared the roles of ERK, JNK and p38 in T cell

function were only performed with the TAT-JIP153-163 peptide and therefore to confirm these

results, experiments may need to be repeated with the more selective TAT-JIP153-172 peptide.

To validate the results we obtained in the Der p 2 experiments, PBMC from atopic individuals

may need to be examined in response to Tetanus Toxoid. This would therefore establish that

the results obtained in response to allergen were specific for atopic individuals. Future

experiments may involve the examination of cytokine production at the mRNA level to

determine if JNK specifically affects gene transcription, however in accordance with previous


183

studies (Dumont et al. 1998; Koprak et al. 1999; Kogkopoulou et al. 2006), we would expect

the results to be similar to that obtained at the protein level. It may also be interesting to

determine the role central memory plays in the Tetanus Toxoid and Der p 2 responses. The

observed differences in lymphoproliferation and cytokine production resulting from JNK

inhibition may be due to the targeting of different cell types (central memory versus

effectors). Finally, further investigations may also involve skewed Th1 and Th2 cells in order

to determine if the TAT-JIP153-172 peptide inhibits initial cytokine transcription and later

polarisation of the cells. These experiments would require a longer culture period (i.e. 7 days)

as this is necessary for polarisation.

Analysis of the JNK pathway in future studies, should involve the TAT-JIP153-172 peptide in

preference to the shorter peptide and SP600125. However, the use of RNA interference also

provides another approach to investigate the role of JNK. Clearly, this method needs further

investigation especially with regard to primary cells and options such as electroporation and

nucleofection may prove more effective than those used in this study.

Overall this study demonstrated that the JNK signalling pathway plays an important role in

human T cell proliferation and cytokine production. In doing so, JNK may not only suppress

chronic inflammatory disease but also control allergic inflammation through the regulation of

Th1 cytokine production.


184

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