INTERLEUKIN 6 RELEASE FROM t98g HUMAN GLIAL CELL LINE AS a PREDICTIVE MARKER FOR CHRONIC PAIN, AND THE CHARACTERIZATION OF SUBSTANCE(S) INVOLVED IN PAIN

INTERLEUKIN-6 RELEASE FROM T98G HUMAN
GLIAL CELL LINE AS A PREDICTIVE MARKER FOR
CHRONIC PAIN, AND THE CHARACTERIZATION OF
SUBSTANCE(S) INVOLVED IN CHRONIC PAIN

TAY SUAN ANNABEL
B.Sc. (Hons.), NUS

A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF ANAESTHESIA
NATIONAL UNIVERSITY OF SINGAPORE
2013


i


Acknowledgements
I would like to express my sincere gratitude to my supervisor A/P Low
Chian Ming, for his support, advice and constant encouragement throughout my
research career at the National University of Singapore. Throughout the course of
my project, I have not only learned many laboratory techniques, but also learned
many invaluable life skills that will benefit me for life. I would also like to
express my gratitude to Prof Shinro Tachibana, for his guidance and support
throughout the course of my project. Without his directions, this work would not
have come to fruition. My heartfelt thanks are also due to A/P Liu Hern Choon
Eugene and Prof Lee Tat Leang, for supporting my work constantly, for their
helpful advice when things did not go well, and for helping me procure the
valuable samples from National University Hospital for the project. Thank you for

your supervision, guidance and support throughout my study. My sincere thanks
also goes out to Prof Toshiaki Minami for helping me procure the precious
samples from Osaka Medical University Hospital for my research work.
I would also like to express my heartfelt thanks to Mdm Li Chunmei for
her constant support, advice and encouragement. Thank you for all your help with
my HPLC work and other technical support. My sincere thanks go out to Ms
Wang Anni for her assistance in western blot and for brightening up my days as a
researcher. Thank you both so much for all the fun and laughter you have brought
to my days in the laboratory. I am also very grateful to Ms Jeyapriya Raja
Sundaram for her help and guidance in primary astrocyte culture and
immunocytochemistry, and for her kind advice throughout my study. My sincere
ii


thanks are also due to Mdm Karen Ho Ban Shian and Mrs Mariam Mathew
for their administrative support and assistance.
Lastly, I am grateful to my family and friends for their understanding,
endless support and encouragement throughout the course of my research work.

iii


Table of Contents
Acknowledgements

ii

Summary

ix

List of Tables

xi

List of Figures

xii

List of Abbreviations

xiv

List of Publications

xvii

List of Conference Papers

xvii

Chapter 1

Introduction

1.1 Chronic Pain

2

1.1.1 Epidemiology of Chronic Pain

2

1.1.2 Pathophysiology of Chronic Pain

3

1.1.2.1 Role of Glial Cells in Chronic Pain

4

1.1.2.1.1 T98G Cell Line as an in vitro Astrocytic Model

7

1.1.3 Treatment Strategies for Chronic Pain and their Challenges

8

1.1.4 Conditions related to Chronic Pain

11

1.1.4.1 Post-Herpetic Neuralgia (PHN)

11

1.1.4.2 Osteoarthritis

12

1.2 Neurotransmitters/Neuromodulators in Chronic Pain
1.2.1 Small Molecule Neurotransmitters

14
14

1.2.1.1 Amino Acids

14

1.2.1.2 Prostaglandins

16

1.2.2 Neuropeptides

16

1.2.2.1 Substance P

17
iv


1.2.2.2 Nociceptin/orphanin FQ and Nocistatin
1.2.3 Pro-inflammatory Cytokines

17
18

1.2.3.1 Tumour Necrosis Factor-α (TNF-α)

19

1.2.3.2 Interleukin-1β (IL-1β)

21

1.2.3.3 Interleukin-6 (IL-6)

22

1.3 Importance of Cerebrospinal Fluid (CSF) in Chronic Pain Research
1.3.1 Biological Markers in the CSF
1.4 Aim and Scope of Study

Chapter 2

23
24
26

Release of Pro-inflammatory Cytokines in T98G Cells Upon
Exposure to CSF of PHN Patients

2.1 Objectives of Chapter

29

2.2 Materials and Methods

31

2.2.1 Materials

31

2.2.2 CSF Samples

32

2.2.3 Cell Culture

33

2.2.4 Measurement of TNF-α, IL-1β and IL-6 Release

34

2.2.5 Measurement of Dexamethasone in CSF

35

2.2.6 Statistical Analyses

36

2.3 Results

37

2.3.1 TNF-α, IL-1β and IL-6 Releasing Activity in T98G Cells Upon
Exposure to CSF of PHN Patients

37

2.3.2 Comparison of IL-6 Releasing Activity Between CSF of Different
PHN Treatment Groups

39

2.3.3 Effect of in vitro Steroid on IL-6 Releasing Activity
2.4 Discussion

41
44

v


Chapter 3

Comparison of IL-6 Releasing Activity between CSF of Chronic
Pain Patients and Pain-free Patients

3.1 Objectives of Chapter

49

3.2 Materials and Methods

50

3.2.1 Materials

50

3.2.2 CSF Samples

50

3.2.3 Culture of Primary Astrocytes

51

3.2.4 Immunocytochemistry

52

3.2.5 Measurement of IL-6 in T98G and Primary Astrocyte Cell Culture

53

3.2.6 Statistical Analyses

53

3.3 Results

54

3.3.1 Comparison between PHN Therapy Effective, Therapy Ineffective
and Control Group

54

3.3.2 Comparison between Osteoarthritis and Control Group

56

3.3.3 IL-6 Release from Primary Astrocytes Upon Exposure to CSF of
Chronic Pain Patients

57

3.4 Discussion

Chapter 4

59

Purification of Protein-like Compounds from CSF of
Chronic Pain Patients

4.1 Objectives of Chapter

64

4.2 Materials and Methods

65

4.2.1 Materials

65

4.2.2 CSF Samples

65

4.2.3 Preliminary Experiments on CSF

66

4.2.3.1 Separation of CSF by Molecular Weight
vi

66


4.2.3.2 Treatment of CSF with Pronase

66

4.2.3.3 Measurement of IL-6 in T98G Cell Culture

67

4.2.4 Separation of CSF into Different Fractions

67

4.2.4.1 CSF Fractionation using HPLC

68

4.2.4.2 Measurement of IL-6 in T98G Cell Culture

70

4.2.5 IL-6 Release from T98G Cells When Exposed to Lignocaine and
Albumin

70

4.2.5 Statistical Analyses

71

4.3 Results

72

4.3.1 CSF Fraction >10 kDa Molecular Weight Triggered IL-6 Release in
T98G Cells

72

4.3.2 Pronase Attenuated IL-6 Release in T98G Cells

74

4.3.3 Separation of CSF by HPLC

75

4.3.3.1 HPLC on Pooled CSF

75

4.3.3.2 HPLC on Pooled Active Fractions

77

4.3.4 Comparison between Chromatograms Obtained from HPLC of
Osteoarthritis CSF and Control CSF
4.3.5 Effects of Lignocaine and Albumin on IL-6 Release
4.4 Discussion

Chapter 5

80
81
83

Signaling Mechanism of Chronic Pain in the T98G Cell System

5.1 Objectives of Chapter

89

5.2 Materials and Methods

90

5.2.1 Materials

90

5.2.2 CSF Samples

90

5.2.3 Cell Culture and NF-κB Inhibition

91

vii


5.2.4 Cell Fractionation and Cell Lysis

91

5.2.5 Western Blot

93

5.2.6 Statistical Analyses

93

5.3 Results

95

5.3.1 Inhibition of NF-κB led to Reduced IL-6 Release in T98G Cells

95

5.3.2 NF-κB Activation in T98G Cells Upon Exposure to CSF of
Chronic Pain Patients

97

5.4 Discussion

Chapter 6

99

Conclusion and Future Directions

6.1 Conclusion

104

6.2 Limitations of Study

106

6.3 Future Directions

107

References

109

viii


Summary
Besides neurons, the central nervous system (CNS) consists of glial cells,
which are mainly microglia and astrocytes. Chronic pain is classically viewed as
being mediated solely by neurons, but there is mounting evidence that glial cells
also play a part. Glial cells, responding to stimulation by neurotransmitters and
peptides, are activated and release pain-enhancing substances like proinflammatory cytokines. These cytokines have been shown to play a role in
enhancing pain by their actions in the spinal cord.
This research work focuses firstly on investigating pro-inflammatory
cytokine release in a cell culture system as a potential marker for chronic pain.
Two conditions related to chronic pain were studied: post-herpetic neuralgia
(PHN) and osteroarthritis. Cerebrospinal fluid (CSF) from patients suffering from
either condition was used to trigger astrocytic cell line T98G cultures, and
subsequent pro-inflammatory cytokine release was measured by enzyme-linked
immunosorbent assay (ELISA). IL-6 release in the chronic pain patient groups
was found to be significantly higher compared to pain-free controls, as well as in
the PHN patient group whose steroid treatment was ineffective compared to those
whose treatment was effective. CSF samples collected before steroid treatment

also triggered higher IL-6 release than after treatment samples. These in vitro tests
provide an objective evaluation on the extent of chronic pain as well as the
efficacy of steroid therapy.

ix


Secondly, we attempted to separate and isolate pain-related substances in
chronic pain patients’ CSF, making use of the in vitro T98G cell system
established earlier. The CSF was separated by a two-step high performance liquid
chromatography (HPLC) technique, and the fractions that triggered IL-6 release
in T98G cells were isolated. We narrowed down to three peaks that could trigger
IL-6 release and these peaks would be subjected to future mass spectrometry
analysis to identify the protein-like substances in the CSF, which could be
potential markers for chronic pain. Lastly, we attempted to elucidate the IL-6
signaling pathway in this in vitro model of chronic pain. NF-κB inhibition studies
and western blot analysis confirmed that NF-κB acts upstream of IL-6 in this
T98G cell system.
In conclusion, we have established an effective in vitro assay system to
quantify chronic pain utilizing CSF and have taken the first steps in isolating
pain-related protein substances in the CSF of chronic pain patients. In an attempt
to better understand the complex mechanisms, we hope to contribute to the
management of unrelenting chronic pain.

x


List of Tables
Table 1-1

Common pharmacological treatments for chronic pain

10

Table 2-1

Patient data (PHN)

36

Table 3-1

Patient data (Control, PHN and Osteoarthritis)

53

xi


List of Figures
Figure 1-1

Schematic diagram of how chronic pain is transmitted

Figure 2-1

TNF-α, IL-1β and IL-6 release in T98G cells upon addition
of PHN CSF

37

Figure 2-2

Comparisons in IL-6 release between before and after
treatment groups, and between therapy effective and
ineffective groups

39

Figure 2-3

IL-6 release in T98G cells upon addition of PHN CSF and
methylprednisolone

41

Figure 2-4

Dexamethasone analyses in CSF of PHN patients

42

Figure 3-1

Comparisons in IL-6 release between control, PHN therapy
effective and PHN therapy ineffective groups

54

Figure 3-2

Comparison in IL-6 release between control and
osteoarthritis groups

55

Figure 3-3

Immunocytochemical analysis of primary astrocytes

56

Figure 3-4

IL-6 release in primary astrocytes upon addition of
PHN CSF

57

Figure 4-1

IL-6 release in T98G cells upon addition of pooled
osteoarthritis CSF of < 10 kDa molecular weight CSF and
pooled osteoarthritis CSF of > 10 kDa molecular weight

71

Figure 4-2

IL-6 release in T98G cells upon addition of pooled

osteoarthritis CSF pre-treated with pronase

72

Figure 4-3

HPLC of pooled osteoarthritis CSF and IL-6 releasing
activity of the peaks

73

Figure 4-4

HPLC of pooled active fractions from previous HPLC
run, and IL-6 releasing activity of the peaks

76

Figure 4-5

Chromatograms obtained from HPLC of (A) pooled
osteoarthritis CSF and (B) pooled pain-free control CSF

78

Figure 4-6

IL-6 release in T98G cells upon addition of lignocaine

79

Figure 4-7

IL-6 release in T98G cells upon addition of albumin

80

Figure 5-1

Effect of NF-κB inhibitor Bay 11-7082 on IL-6 release
in T98G cells upon addition of osteoarthritis CSF

92

xii

6


Figure 5-2

Effect of NF-κB inhibitor Bay 11-7082 on IL-6 release
in T98G cells upon addition of control CSF

93

Figure 5-3

Western blot demonstrating NF-κB activation

95

Figure 5-4

Possible signaling pathway of IL-6 release in T98G cells

99

xiii


List of Abbreviations
ATCC

American Type Culture Collection

AUFS

Absorbance Units Full Scale

CER-1

Cytoplasmic Fractionation Reagent

CCI

Chronic Constriction Model

CNS

Central Nervous System

COX

Cyclooxygenase

CRPS

Complex Regional Pain Syndrome

CSF

Cerebrospinal Fluid

DAPI

4',6-diamidino-2-phenylindole

DMEM

Dulbecco's Modified Eagle Medium

DRG

Dorsal Root Ganglia

DSRB

Domain Specific Review Board

DTT

Dithiothreitol

EBSS

Earle's Balanced Salt Solution

ECM

Extra-cellular Matrix

ELISA

Enzyme-linked Immunosorbent Assay

EMEM

Eagle’s Minimum Essential Medium

FBS

Fetal Bovine Serum

GABA

γ-amino-butyric Acid

GFAP

Glial Fibrillary Acidic Protein

HPLC

High Performance Liquid Chromatography

HRP

Horseradish Peroxidase
xiv


IL-1

Interleukin-1

IL-1β

Interleukin-1β

IL-6

Interleukin-6

IL-8

Interleukin-8

IL-10

Interleukin-10

IL-18

Interleukin-18

i.t.

Intrathecal

L-PGDS

Lipocalin-type Prostaglandin D Synthase

LPS

Lipopolysaccharides

MALDI-TOF Matrix-assisted Laser Desorption/ionization–time-of-flight
NER-1

Nuclear Fractionation Reagent

NF-κB

Nuclear Factor-Kappa-B

NK1

Neurokinin 1

NMDA

N-methyl-D-aspartic Acid

N/OFQ

Nociceptin/orphanin FQ

NSAIDs

Non-steroidal Anti-inflammatory Drugs

NST

Nocistatin

PBS

Phosphate Buffered Saline

PBST

PBS with 0.1% v/v Tween-20

PGE2

Prostaglandin E2

PHN

Post-herpetic Neuralgia

PKA

Protein Kinase A

PKC

Protein Kinase C

xv


PMSF

Phenylmethylsulfonyl Fluoride

PNS

Peripheral Nervous System

ppN/OFQ

Prepronociceptin

PVDF

Polyvinylidene Difluoride

SEM

Standard Error of Mean

sIL-6R

Soluble IL-6 Receptor

SSNRIs

Selective Serotonin and Norepinephrine Reuptake Inhibitors

TCAs

Tricyclic Antidepressants

TFA

Trifluoroacetic Acid

TNF-α

Tumour Necrosis Factor-α

VAS

Visual Analogue Scale

WHO

World Health Organization

xvi


List of Publications
A.S. Tay, E.H. Liu, T.L. Lee, S. Miyazaki, W. Nishimura, T. Minami, C.-M. Low,
S. Tachibana, Cerebrospinal fluid of postherpetic neuralgia patients induced
interleukin-6 release in human glial cell-line T98G. (Manuscript submitted)

List of Conference Papers
A.S. Tay, C. Li, A. Wang, T.L. Lee, S. Tachibana, C.-M.Low, Cerebrospinal fluid
from post-herpetic neuralgia Japanese patients triggers interleukin-6 release in
glioblastoma cells. 2nd Singapore-Duke Anaesthesia Update (Singapore, 2010)
Poster presentation
A.S. Tay, C. Li, A. Wang, E.H. Liu, T.L. Lee, S. Chiang, S. Fujiwara, W.
Nishimura, T. Minami, C.-M. Low, S. Tachibana, Cerebrospinal fluid from postherpetic neuralgia Japanese patients triggers interleukin-6 release in glioblastoma
cells, J. Neurochem. 115 Supplement 1 (2010). 10th Biennial Meeting of the
Asian-Pacific Society of Neurochemistry (Phuket, Thailand, 2010) Poster
presentation
A.S. Tay, E.H. Liu, T.L. Lee, S. Miyazaki, W. Nishimura, T. Minami, C.-M. Low,
S. Tachibana, Interleukin-6 release from human glial cell line T98G as a
predictive marker on the effectiveness of steroid therapy in reducing neuropathic
pain of post-herpetic neuralgia patients. Yong Loo Lin School of Medicine 2nd
Annual Graduate Scientific Congress (Singapore, 2012) Oral presentation

xvii


Chapter 1

Introduction

1


1.1 Chronic Pain
The International Association for the Study of Pain defines chronic pain as
pain that persists for at least 3 months. Chronic pain is normally triggered by
injury or disease, which damages the nervous system in such a way that it is
unable to restore its normal physiological functions to homeostatic levels. It can
persist for months or years, long after the original injury has healed (Gao and Ji,
2010). Chronic pain is heterogeneous, and multiple molecular and cellular
mechanisms act in combination within the peripheral nervous system (PNS) and
central nervous system (CNS) to produce the chronic pain (Scholz and Woolf,
2002).

1.1.1 Epidemiology of Chronic Pain
Chronic pain has become a major healthcare challenge as it interferes with
normal daily life. The World Health Organization (WHO) approximates that one
in five people worldwide experiences chronic pain. In a large-scale survey
involving 16 countries, 19% of respondents over 18 years old had suffered pain
for more than 6 months. 61% of these chronic pain sufferers were unable to work
outside the home, and 19% had lost their jobs. 40% of them had insufficient pain
management and only 2% were seeing a pain specialist (Breivik et al., 2006).
A study carried out in Singapore in 2009 showed that the prevalence of
chronic pain was 8.7%. Chronic pain afflicted women at a higher rate than men,
and the prevalence increased sharply beyond 65 years of age. Although a
2

seemingly lower prevalence of chronic pain was seen in this study, it remains a
healthcare problem of high importance, due to a rapidly ageing population in
many developed countries including Singapore, and is likely to consume an
increasingly large amount of healthcare resources within the next few years (Yeo
and Tay, 2009).

1.1.2 Pathophysiology of Chronic Pain
Chronic pain can be classified as either nociceptive or neuropathic.
Nociceptive pain is derived from mechanical, chemical or thermal irritation to the
peripheral sensory nerves, and is typically well-localized. Neuropathic pain, on
the other hand, is pain initiated by damage to or a lesion of the nervous system,
and is characterized by poor localization (Goucke, 2003).
Chronic pain results from the development of neural plasticity in the PNS
and CNS. It was traditionally believed that only neurons and their neural circuits
were responsible for pain development and maintenance. Hence current
therapeutics for chronic pain focuses on neuronal targets, which include Nmethyl-D-aspartic acid (NMDA) receptor antagonists and opioid analgesics. Such
therapies provide transient pain relief and do not resolve the underlying
pathological processes that lead to chronic pain. Hence, studies on non-neuronal
cells, in particularly glial cells in chronic pain conditions, have increased
tremendously in recent years.

3


1.1.2.1 Role of Glial Cells in Chronic Pain
Recent studies and review articles have highlighted a communication
cross-talk that exists between the immune system and the nervous system (Scholz
and Woolf, 2007). Neurons and glial cells in the nervous system have close
interactions with each other on a cellular and molecular level. Injury to peripheral
nerves triggers an inflammatory response in peripheral glia and immune cells. The

vital role of inflammation in the development of chronic pain is demonstrated in a
study whereby injection of pro-inflammatory agents such as carrageenan or
complete Freund’s adjuvant around the sciatic nerve induced mechanical
allodynia in animal models (Sorkin and Schafers, 2007). The peripheral
inflammatory response then sets off a series of downstream reactions through the
release of neurotransmitters or neuromodulators from primary afferents. These
substances in turn lead to activation of glial cells that are in close proximity to the
afferent neuron terminals (Vallejo et al., 2010).
Glial cells, forming 70% of the total cell population in the brain and spinal
cord, have long been viewed as performing mainly neuronal housekeeping and
support functions, providing insulation and protection to the neurons. They do not
have axons, and hence they have been perceived to have no role in nerve signal
transmission. However, this view has slowly been changing. Pathological pain is
classically viewed as being mediated solely by neurons, but there is mounting
evidence that glial cells also play a part in exaggerated pain states created by
inflammation and neuropathy (Hashizume et al., 2000; Watkins and Maier, 2002).
Astrocytes, in particular, play an important role in pain processing. They are the
4


most abundant cells in the CNS (constituting 40-50% of all glial cells) in terms of
number and volume, forming networks with themselves via gap junctions and
making very close contacts with neuronal synapses and blood vessels (Halassa et
al., 2007). Astrocytes express receptors for numerous neurotransmitters,
neuroactive substances and amino acids, and hence provide support and
nourishment for neurons as well as regulate the external chemical environment
during synaptic transmission (Verkhratsky and Steinhauser, 2000; Hanson and
Ronnback, 2004).
In normal physiological conditions, glial cells are in a quiescent state. In
most cases of early glial response to injury or disease conditions, activation of

microglia occurs first. Activated microglia display a change in surface markers
and membrane proteins, and this triggers the production and release of painenhancing substances such as reactive oxygen species, excitatory amino acids,
nitric oxide, prostaglandins and pro-inflammatory cytokines (Wieseler-Frank,
Maier and Watkins, 2004). This subsequently leads to activation and proliferation
of astrocytes, correlating with the release of even more pain enhancing substances.
These substances modulate pain processing by influencing either presynaptic
release of neurotransmitters and/or postsynaptic excitability, leading to
persistence in hypersensitivity and chronic pain (Guo et al., 2007). Figure 1-1
shows the schematic diagram of chronic pain transmission.

5


Figure 1-1 Schematic diagram of how chronic pain is transmitted. After
receiving a pain stimulus, peripheral neurons transmit signals to the primary
afferent neuron, causing the release of neurotransmitters and neuromodulators that
activate the receptors on microglia and astrocytes. The glial cells are activated and
release pro-inflammatory cytokines and other pain enhancing substances. This
affects the presynaptic release of neurotransmitters, postsynaptic excitability, as
well as a self-propagating mechanism of enhanced production of pain enhancing
substances by the glial cells.

6


Astrocytic reaction is more persistent than microglial reaction after nerve
injury, lasting more than 150 days after nerve injury. Activation of astrocytes
results in prolongation of the pain state, and is accompanied by a reduction in
microglial activity over time (Tanga et al., 2004). Thus, microglia are involved in
the early development of chronic pain, while astrocytes function in sustaining the

pain (Vallejo et al., 2010). An interesting finding was that nerve injury induces an
increase in interleukin-18 (IL-18) and IL-18 receptors in activated microglia and
astrocytes respectively, suggesting an interaction between microglia and
astrocytes in the physiology of chronic pain (Miyoshi et al., 2008).
However in some cases, astrocytes are activated without microglial
activation, and are shown to be sufficient on their own to produce chronic pain.
Davies et al. (2008) showed that transplantation of astrocytes derived from glialrestricted precursor cells triggered the onset of mechanical allodynia and thermal
hyperalgesia after spinal cord injury in rats. Hald et al. (2009) also demonstrated
that development of hypersensitivity and activation of astrocytes occurred without
microglial activation in chronic pain mouse models.

1.1.2.1.1 T98G Cell Line as an in vitro Astrocytic Model
The cell line used in this project is the T98G human glial cell line. T98G
cell line is a derivative of glioblastoma and is of astrocytic origin. T98G cells
have the transformed characteristics of immortality and anchorage independence.
However at the same time, they act like normal human cells in that they become

7