Download Immunological Mechanisms and Natalizumab Treatment in Multiple Sclerosis Natalizumab Treatment in Multiple Sclerosis

Linköping University Medical Dissertations No. 1332
Johan Mellergård
Studies on lymphocytes, inflammatory markers and
magnetic resonance spectroscopy
Johan Mellergård Linköping 2012
Divisions of Neurology and Clinical Immunology
Department of Clinical and Experimental Medicine
Faculty of Health Sciences, Linköping University,
Sweden
Immunological Mechanisms and Natalizumab Treatment in Multiple Sclerosis
Immunological Mechanisms and
Natalizumab Treatment in Multiple Sclerosis
 Johan Mellergård, 2012
Paper I, III and IV have been reprinted
with permission of the respective copyright holders.
Cover: Cerebral MRI sagittal section showing MS
white matter lesions and atrophy (FLAIR protocol).
From Center for Medical Image Science and Visualization (CMIV), Linköping.
Printed by LiU-Tryck, Linköping, Sweden, 2012.
ISBN: 978-91-7519-787-6
ISSN: 0345-0082
To Sara,
Selma, Morris & Bea
For now we see only a reflection as in a mirror; then we shall see face to face.
Now I know in part; then I shall know fully, even as I am fully known.
1 Corinthians 13:12
Contents
Contents
Contents _________________________________________________ 5
Original publications _______________________________________ 8
Abstract ________________________________________________ 10
Abbreviations ___________________________________________ 11
1. Introduction ___________________________________________ 14
2. Background ___________________________________________ 16
2.1 Historical aspects of MS – the treatment revolution ________________16
2.2 Epidemiology and symptoms __________________________________16
2.3 Diagnosing multiple sclerosis _________________________________18
2.4 The immune system – introductory remarks ______________________19
2.5 Etiology and pathogenesis of multiple sclerosis ___________________19
2.6 Mechanisms of demyelination and neuroaxonal damage _____________21
2.7 Components of the immune response ___________________________22
2.8 Leukocyte adhesion and migration _____________________________28
2.9 Inflammation versus neurodegeneration _________________________29
2.10 Measuring diffuse pathology in MS ____________________________30
2.11 Disease-modifying treatments ________________________________31
2.12 Progressive multifocal leukoencephalopathy (PML) _______________33
3. Aims _________________________________________________ 34
4. Methods ______________________________________________ 36
4.1 Study subjects _____________________________________________36
4.2 Procedures ________________________________________________38
4.2.1 Disease scoring systems (paper I, II, III, IV) _____________________________ 38
4.2.2 Routine CSF analyses (paper I, II, III) __________________________________ 39
4.2.3 Multiplex bead assay (paper I, III) _____________________________________ 40
4.2.4 Flow cytometry (paper II, IV) ________________________________________ 41
4.2.5 Lymphocyte activation assay (paper II) _________________________________ 42
4.2.6 Markers of neurodegeneration in CSF (paper III) _________________________ 42
4.2.7 Magnetic resonance spectroscopy – MRS (paper III) ______________________ 43
4.2.8 Absolute quantification of MRS data (paper III) __________________________ 45
4.2.9 Real-time RT-PCR (paper IV) ________________________________________ 45
4.3 Statistical methods __________________________________________46
5
Contents
4.4 Ethical considerations _______________________________________46
5. Results and Discussion _________________________________ 48
5.1 Clinical outcome (paper I, II, III) _______________________________48
5.2 CSF and plasma variables (paper I, II, III) ________________________49
5.2.1 Routine CSF analyses (paper I, II, III) __________________________________ 49
5.2.2 Cytokines and chemokines in CSF and plasma – comparing multiple sclerosis with
non-inflammatory neurological diseases (paper I) _____________________________ 49
5.2.3 Cytokines and chemokines in CSF (paper I, III) __________________________ 51
5.2.4 Cytokines and chemokines in plasma (paper I) ___________________________ 52
5.3 Blood lymphocyte composition (paper II) ________________________53
Main lymphocyte populations _____________________________________________ 53
Lymphocyte subpopulations ______________________________________________ 55
5.4 Lymphocyte activation assay (paper II) __________________________58
5.5 Markers of neurodegeneration in CSF (paper III) __________________60
5.6 1H-MRS metabolite concentrations in NAWM (paper III) ___________61
5.7 Transcriptional characteristics of CD4+ T cells (paper IV) ___________63
6. Concluding remarks and future perspectives _______________ 66
7. Conclusions ___________________________________________ 68
8. Sammanfattning på svenska _____________________________ 70
9. Acknowledgements _____________________________________ 72
10. References ___________________________________________ 74
6
7
Original publications
Original publications
This thesis is based on the following four papers, in the text referred to by their Roman
numerals.
I
Natalizumab treatment in multiple sclerosis: marked decline of chemokines and
cytokines in cerebrospinal fluid.
Johan Mellergård, Måns Edström, Magnus Vrethem, Jan Ernerudh and Charlotte Dahle
Mult Scler. 2010 Feb;16(2):208-17
II
An increase in B cell and cytotoxic NK cell proportions and increased T cell
responsiveness in blood of natalizumab-treated multiple sclerosis patients.
Johan Mellergård, Måns Edström, Maria C. Jenmalm, Charlotte Dahle, Magnus
Vrethem, Jan Ernerudh
Submitted
III
Association between change in normal appearing white matter metabolites and
intrathecal inflammation in natalizumab-treated multiple sclerosis.
Johan Mellergård, Anders Tisell, Olof Dahlqvist Leinhard, Ida Blystad, Anne-Marie
Landtblom, Kaj Blennow, Bob Olsson, Charlotte Dahle, Jan Ernerudh, Peter Lundberg,
Magnus Vrethem
PLoS One. 2012 Sept 17;7(9):e44739
IV
Transcriptional characteristics of CD4+ T cells in multiple sclerosis: relative lack of
suppressive populations in blood.
Måns Edström, Johan Mellergård, Jenny Mjösberg, Maria C. Jenmalm, Magnus
Vrethem, Rayomand Press, Charlotte Dahle, Jan Ernerudh
Mult Scler. 2011 Jan;17(1):57-66
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9
Abstract
Abstract
Background: Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central
nervous system (CNS), and a frequent cause of neurological disability among young adults. In addition
to focal inflammatory demyelinated lesions, diffuse white matter pathology as well as a
neurodegenerative component with accumulating axonal damage and gliosis have been demonstrated
and contribute to MS disease characteristics. The inflammatory component is considered autoimmune
and mediated by auto-reactive T lymphocytes together with other cell populations of the immune system
and their respective products like cytokines and chemokines. Treatment with natalizumab, a monoclonal
antibody directed against the α4β1-integrin (VLA-4), reduces migration of potential disease-promoting
cells to the CNS. The efficacy of natalizumab in reducing relapses and MRI activity is evident, however
associated effects on the immune response and the neurodegenerative component in MS are not clear.
Methods: In total 72 MS patients were included, distributed among paper I-IV. We investigated effects
associated with one-year natalizumab treatment in 31 MS patients regarding cytokine and chemokine
levels in CSF and blood using multiplex bead assay analyses (paper I), as well as treatment effects on
blood lymphocyte composition in 40 patients using flow cytometry, including functional assays of
lymphocyte activation (paper II). Normal appearing white matter (NAWM) metabolite concentrations
were assessed with proton magnetic resonance spectroscopy (1H-MRS) in 27 MS patients before and
after one year of treatment (paper III). We also evaluated the balance between circulating T helper (Th)
subsets in 33 MS patients using gene expression analyses of the CD4+ T cell related transcription factors
in whole blood (paper IV).
Results: One-year natalizumab treatment was associated with a marked decline in pro-inflammatory
cytokines (IL-1β and IL-6) and chemokines (CXCL8, CXCL9, CXCL10 and CXCL11) intrathecally.
Circulating plasma levels of some cytokines (GM-CSF, TNF, IL-6 and IL-10) also decreased after
treatment. Natalizumab treatment was further associated with an increase in lymphocyte numbers of
major populations in blood (total lymphocytes, T cells, T helper cells, cytotoxic T cells, NK cells and B
cells). In addition, T cell responsiveness to recall antigens and mitogens was restored after treatment. As
to 1H-MRS metabolite concentrations in NAWM, no change in levels were detected post-to
pretreatment on a group level. However, correlation analyses between one-year change in metabolite
levels (total creatine and total choline) and levels of pro-inflammatory IL-1β and CXCL8 showed a
pattern of high magnitude correlation coefficients (r=0.43-0.67). Gene expression analyses
demonstrated a systemically reduced expression of transcription factors related to immunoregulatory T
cell populations (regulatory T cells and Th2) in relapsing MS compared with controls.
Conclusions: Our findings support that an important mode of action of natalizumab is reducing
lymphocyte extravasation, although cell-signalling effects through VLA-4 also may be operative.
Correlation analyses between changes 1H-MRS metabolite concentrations and inflammatory markers
possibly point towards an association between intrathecal inflammation and gliosis development in
NAWM. Finally, gene expression analyses indicate a systemic defect at the mRNA level in relapsing
MS, involving downregulation of beneficial CD4+ phenotypes.
10
Abbreviations
Abbreviations
ANOVA
APC
BBB
Breg
CD
cDNA
cMRI
Cr
CNS
CCL
CSF
CXCL
DC
EAE
EDSS
FOXP3
GA
GFAP
Gln
Glu
GM-CSF
HLA
1
H-MRS
IFN
Ig
IL
Lac
LFA-1
MAdCAM-1
MBP
MHC
MHC I
MHC II
mIns
MRI
mRNA
MS
MSIS-29
MSSS
NAA
NAAG
NAGM
NAWM
NFL
NK cell
NKT cell
OND
PBMC
11
analysis of variance
antigen presenting cell
blood brain barrier
regulatory B cell
cluster of differentiation
complementary deoxyribonucleic acid (DNA)
conventional magnetic resonance imaging
creatine
central nervous system
CC-chemokine ligand
cerebrospinal fluid
CXC-chemokine ligand
dendritic cell
experimental allergic (autoimmune) encephalomyelitis
expanded disability status scale
forkhead box P3
glatiramer acetate
glial fibrillary acidic protein
glutamine
glutamate
granulocyte-monocyte colony-stimulating factor
human leukocyte antigen
proton magnetic resonance spectroscopy
interferon
immunoglobulin
interleukin
lactate
leukocyte function-associated antigen 1
mucosal addressin cell adhesion molecule 1
myelin basic protein
major histocompatibility complex
major histocompatibility complex class I
major histocompatibility complex class II
myo-Inositol
magnetic resonance imaging
messenger ribonucleic acid
multiple sclerosis
multiple sclerosis impact scale 29
multiple sclerosis severity score
N-acetylaspartate
N-acetylaspartylglutamate
normal appearing grey matter
normal appearing white matter
neurofilament light protein
natural killer cell
natural killer cell with T cell properties
other neurological diseases
peripheral blood mononuclear cell
Abbreviations
PCR
PML
PNS
PPMS
P-tau
qMRI
RRMS
S1P
SDMT
SPMS
rRNA
tCho
tCr
TCR
TGF
Th
tGlx
tNA
TNF
Treg cell
T-tau
VCAM-1
VLA-4
polymerase chain reaction
progressive multifocal leukoencephalopathy
peripheral nervous system
primary progressive multiple sclerosis
phosphorylated tauprotein
quantitative magnetic resonance imaging
relapsing remitting multiple sclerosis
sphingosine-1-phosphate
symbol digit modalities test
secondary progressive multiple sclerosis
ribosomal ribonucleic acid
total choline
total creatine
T cell receptor
tumor growth factor
T helper
total glutamate + glutamine
total N-acetylaspartate
tumor necrosis factor
regulatory T cell
total tauprotein
vascular cell adhesion molecule 1
very late activation antigen 4
12
13
1. Introduction
1. Introduction
The human nervous system contains about 100 billion nerve cells, neurons, and principally
constitutes groups of neurons connected to each other, creating a network of nerve circuits with
astounding complexity. Apart from neurons, specialized to receive and transmit electric
impulses, the nervous system also consists of cells with supportive functions, the neuroglia
cells. The brain and spinal cord define the central nervous system (CNS) and the cranial and
spinal nerves with their ganglia the peripheral nervous system (PNS). A neuron consists of a
cell body with two types of extensions; the receiving dendrites with many branches in the
immediate neighborhood of the cell body, and one axon that conducts the nerve impulse away
from the cell body. Axons may be myelinated or unmyelinated depending on whether they are
surrounded by a sheath of isolating lipoproteins, the myelin, or not. Myelin is required for the
rapid propagation of action potentials along the axon. Parts in the CNS containing many
myelinated axons designate the white matter, whereas parts containing aggregations of neuron
cell bodies designate the gray matter (Heimer 1995).
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS. In MS,
the inflammatory response results in myelin lesions (demyelination) causing deteriorated nerve
impulse propagation (Bruck 2005). Although the hallmark of MS is focal demyelinated white
matter, the disease is also denoted by axonal damage and gliosis (Dutta and Trapp 2007).
Depending on the size and anatomical localization of the lesion, the clinical result is reduction
or loss of the corresponding neurological function. The onset of symptoms is often referred to
as a relapse. Within weeks the local inflammation attenuates and eventually the myelin may be
partially restored (remyelination) resulting in fully or partial regained neurological function
(Kotter, Stadelmann et al. 2011). With time there might be no fully recovery from relapsecaused neurological deficits and there is an accumulation of axonal damage causing increasing
neurological disability (Dutta and Trapp 2011). The MS disease now clinically changes
character into a progressive course, where inflammation as underlying pathomechanism seems
replaced by neurodegeneration.
This thesis discusses the immunological mechanisms and the inflammatory response in MS and
deals with the interplay between inflammation and neurodegeneration. The approach is by
mainly focusing on the immunomodulating treatment with natalizumab, highlighted from
different perspectives.
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15
2. Background
2. Background
2.1 Historical aspects of MS – the treatment revolution
The first description of MS pathology dates back to 1868, when Charcot documented the focal
lesions in the brain of a young woman who had suffered from nystagmus, intention tremor and
scanning speech. In his report, “Histologie de la sclérose en plaques”, he described the
characteristic plaques that still constitute the hallmark of MS (Compston 1988). Up to the last
decade of the 20th century there were no disease-modulating treatments available, only
symptomatic treatment could be offered. Life with MS was an evitable journey towards
increasing disability in various extensions. In the mid-90th interferon beta-1b was approved as
the first disease-modifying therapy for relapsing-remitting MS (RRMS). The approval was a
result of a randomized, double-blind, placebo-controlled trial in 1993 (Paty and Li 1993). That
event started a new era of MS history with the possibility to modify the disease course for
patients with relapsing MS. Ever since, the number of disease-modifying treatments has grown
and today (august 2012) there are five approved immunomodulators for the treatment of
relapsing MS (interferon beta-1a, interferon beta-1b, glatiramer acetate, natalizumab and
fingolimod), and the number will further increase. By using disease-modifying treatment early
in the disease course the accumulation of irreversible brain damage and subsequent
neurological disability may decrease. In the foreseeable future there is no cure for MS, but with
the increasing pallet of disease-modifying drugs, hopefully MS may become a disease with a far
better reputation than in the past.
2.2 Epidemiology and symptoms
The MS prevalence worldwide is unevenly distributed, in general varying from about 10/100
000 to 100/100 000 what is believed to depend on geographical and ethnical/genetic factors
(Rosati 2001). The highest prevalence is seen in northwestern Europe and in northern North
America and the lowest in Asia and Africa. Women are affected roughly twice as often as men.
Sweden has among the highest nationwide prevalence estimates of MS in the world with
189/100 000 (Ahlgren, Oden et al. 2012), which correspond to 600 new cases in Sweden each
year. Most patients are diagnosed with MS between the ages of 20-50 (incidence peak at 30
year) and MS is, after acquired disability from accidents, the most frequent cause of
neurological disability among young adults. The usual clinical presentation, occurring in
approximately 85% of patients, is a relapsing-remitting disease course with varying
neurological deficits fluctuating over time (Figure 1). Early features that predict a benign
disease course are complete remission of the first relapse, low or moderate initial relapse
frequency and dominance of afferent symptoms (Skoog, Runmarker et al. 2012). With time,
however, the inflammatory activity in the CNS seems to decline and the relapsing disease turns
into a progressive state distinguished by increasing axonal loss and deterioration of
neurological function without clinical relapses (secondary progressive MS, SPMS). For
16
2. Background
approximately 15% of patients the disease course is progressive from the start (primary
progressive MS, PPMS) (Miller and Leary 2007).
Figure 1. The usual disease course in MS. Subclinical disease activity within the CNS is followed by a relapsingremitting disease course. With time there is an accumulation of disability and a gradual transition to the
progressive phase. In parallel with inflammation, neuroaxonal damage causes increasing cerebral atrophy.
Magnetic resonance imaging (MRI) is a valuable tool to assess disease activity and progression.
The clinical symptoms related to a relapse vary depending on where the demyelinated lesion is
located in the CNS and thus highly diverse signs and symptoms may arise. There is however a
high propensity for focal lesions to occur in the periventricular white matter, cerebellum and
brain stem (Fazekas, Barkhof et al. 1999). The first clinical presentation of MS is usually a
clinically isolated syndrome (CIS) (Miller, Chard et al. 2012). Principally there are so called
negative and positive symptoms. Negative symptoms are characterized by a reduction or loss of
function like motor weakness, reduced sensation ability, reduction or loss of vision and reduced
coordination. It is easy to comprehend that affected nerve conduction in demyelinated axons
may cause these symptoms. Positive symptoms on the other hand arise as a consequence of
ectopic and emphatic nerve transmission with symptoms like paresthesias, spasms and
paroxysmal pain (Raine 1997). In general optic neuritis, sensory disturbances and mild to
moderate paresis are common in the initial disease course, but with time ataxia, increased
muscle tonus with spasticity and genito-urinary as well as bowel dysfunction develop. Apart
from the described focal neurological deficits, cognitive dysfunction is common (Chiaravalloti
17
2. Background
and DeLuca 2008). Loss of energy, fatigue, is one common symptom associated with cognitive
dysfunction, although the mechanisms behind are elusive.
2.3 Diagnosing multiple sclerosis
The principal when diagnosing MS is to demonstrate dissemination of CNS lesions in time and
space as well as excluding alternative diagnosis (Miller, Weinshenker et al. 2008) (Miller,
Chard et al. 2012). A careful medical history and a thorough neurological examination form the
basis for further diagnostic considerations. Since 2001 the McDonald criteria are the foundation
for diagnosing MS (McDonald, Compston et al. 2001). Although MS may be diagnosed on
clinical grounds alone, the use of magnetic resonance imaging (MRI) is fundamental for an
accurate MS diagnosis. According to the latest revision of the McDonald criteria from 2010,
the use of MRI for demonstration of dissemination of lesions in time and space has been
emphasized, in some circumstances facilitating an MS diagnosis by a single MRI scan
(Polman, Reingold et al. 2011). The investigation of cerebrospinal fluid (CSF) with the
assessment of intrathecal production of immunoglobulins and oligoclonal bands (reflecting
intrathecal Ig production) can support the diagnosis but is not mandatory. However, when to
exclude an alternative diagnosis to MS, CSF analyses are crucial, although intrathecal Ig
production occurs in other inflammatory conditions. In the future, with the identification of
diagnostic and prognostic markers, the importance of CSF evaluations may increase.
18
2. Background
2.4 The immune system – introductory remarks
The immune system constitutes a complex network of different cells and molecules necessary
for our protection from various microbes, but also to fight cancer cells and promote healing.
Furthermore, the components of the immune system also participate in a wide range of other
biological processes from embryology to behavior, in close interplay with the endocrine and the
nervous system. Traditionally the immune system is divided into innate and adaptive responses
(Abbas A.K. 2012). Innate immunity is the first, constantly mobilized, line of defense against
infections, and involves epithelial barriers, phagocytic cells (neutrophils and macrophages),
natural killer (NK) cells, mast cells, dendritic cells, the complement system and many other
soluble mediators. Innate immunity uses “standardized” procedures to fight invading microbes,
utilizing structures that are common to groups of microbes, but cannot evolve highly specific
immune mechanisms and, importantly, has no memory function. On the contrary, adaptive
immune responses evolve slower (within days), use highly specific immune mechanisms to
combat various infections and have a memory function. The adaptive immune system consists
of lymphocytes, with a total estimated number of 4.6 x 1011 (Ganusov and De Boer 2007).
Lymphocytes are divided into T and B lymphocytes, commonly and here referred to as T and B
cells. During T cell maturation in the thymus, the T cell receptor (TCR) evolves and there is an
expression of either CD4 or CD8 co-receptor molecules, denoted as CD4+ and CD8+ T cells
respectively. CD4+ and CD8+ T cells have different capabilities that complement each other.
The development of antigen specific immunity is dependent on the TCR recognition of
antigens presented by antigen presenting cells (APC). The most efficient APC is the dendritic
cell, thus a member of innate immunity with a major role in initiating adaptive immunity. Also
macrophages and B cells act as APCs. The antigen is presented to the T cells by major
histocompatibility complex (MHC) molecules (in humans often referred to as HLA, human
leukocyte antigen) on the APC. MHC molecules are polymorphic glycoproteins that transport
antigens and display them on the cell-membrane. There are two main types of MHC molecules,
class I and class II. MHC class I molecules (MHC I) are constitutively expressed on all
nucleated cells and platelets, and present peptides from endogenously synthesized antigens to
CD8+ T cells. MHC class II molecules (MHC II) are expressed mainly on dendritic cells,
macrophages and B cells and present peptides from processed exogenous antigens to CD4+ T
cells thereby initiating CD4+ T cell immune responses (Abbas A.K. 2012).
2.5 Etiology and pathogenesis of multiple sclerosis
The precise etiology of MS is still unknown but a complex interplay between genetic
susceptibility and environmental factors is most likely (Sospedra and Martin 2005). The
strongest disease association is linked to human leukocyte antigen (HLA) class II genes (in
particular within the HLA-DRB1*15:01 haplotype), which increase the risk for disease about 34 times (International Multiple Sclerosis Genetics, Wellcome Trust Case Control et al. 2011).
MS is considered an autoimmune disease; i. e. the basis for the inflammatory lesions is an
immune response against self-antigens. Generally, this conclusion is based on what we learned
19
2. Background
from the animal model of MS, experimental allergic (or autoimmune) encephalomyelitis
(EAE). The general procedure of inducing EAE is to inject a myelin-derived protein in
conjunction with Freund´s adjuvant into a rodent. The rodent will then develop an immune
response involving myelin-specific T cells that migrate into the brain initiating an inflammatory
attack on myelin structures. This results in a disability and histopathology that share many
similarities with MS (Raine 1997). Since EAE could be transferred to naïve animals by in vitro
reactivated myelin-specific CD4+ T cells (adoptive transfer), it was assumed that MS too is a T
cell-mediated autoimmune disease (Zamvil and Steinman 1990). Still, substantial evidence
accumulated over many years confirms that the histopathological hallmark of MS is
inflammatory demyelination with axonal damage (Hemmer, Archelos et al. 2002). However,
the initial processes that trigger this inflammatory response are yet to be defined. In addition to
inflammation, a process of degeneration is crucial in MS pathology and it has even been
suggested that MS primarily is a neurodegenerative disease instead of an autoimmune disease.
Thus there are data suggesting that the first event in the pathogenesis of MS lesions is the loss
of oligodendrocytes independent of adaptive immunity mechanisms (Henderson, Barnett et al.
2009). On the contrary, among genes associated with MS, there is a significant
overrepresentation of genes with immunological relevance, in particular genes related to T cell
differentiation (International Multiple Sclerosis Genetics, Wellcome Trust Case Control et al.
2011), which supports the inflammatory theory.
Furthermore, although T and B cell populations are considered major contributors to
inflammation and tissue damage (Goverman 2009, Fletcher, Lalor et al. 2010, Disanto,
Morahan et al. 2012), there is increasing evidence that other mechanisms and factors may have
a major impact on disease development and progression (Sriram 2011).
Principally, the pathogenesis of MS involves (1) the activation and clonal expansion of autoreactive lymphocyte clones (T and B cells) in the peripheral lymph nodes and spleen or the
gastrointestinal (Berer, Mues et al. 2011) or respiratory tract (Odoardi, Sie et al. 2012), (2) the
migration and infiltration of these clones into the CNS and (3) the reactivation of these cells
within the CNS by resident immune cells presenting self-antigens (Figure 2). (1) Although still
uncertainties about what specific antigens constituting the target for auto-reactive cells, many
studies indicate the importance of myelin proteins (Goverman 2011). CNS antigens could
either be released to the periphery or there may be a cross-reaction to foreign antigens (OchoaReparaz, Mielcarz et al. 2011). (2) The subsequent homing of activated T and B cells to the
inflammatory region in CNS and migration through the blood-brain barrier (BBB) is dependent
on chemokine signalling and the interaction between cell surface molecules (as integrins) and
corresponding endothelial adhesion molecules (Rice, Hartung et al. 2005). (3) Once inside the
CNS, T and B cells are reactivated by neurons and glial cells as well as APCs like microglia,
through recognition of antigens presented on MHC class I and II molecules respectively
(Hemmer, Archelos et al. 2002). This interaction is also largely dependent on signalling
molecules like cytokines and chemokines. Furthermore, the magnitude of the inflammatory
response depends on the balance between auto-reactive T cells and immunosuppressive T cells.
With time, the inflammation may be self-generating and compartmentalized within the CNS
(Meinl, Krumbholz et al. 2008).
20
2. Background
Figure 2. Schematic view of mechanisms of lymphocyte migration and effector responses. Briefly, after activation
of CD4+ T cells in the periphery by antigen presenting cells (APC),migration through the blood-brain barrier
(BBB) into the CNS are dependent on the interaction between integrin VLA-4 and VCAM-1 and chemokine
gradient guidance. Within the CNS, reactivation of auto-reactive T cell clones by macrophages and microglia
initiates an inflammatory cascade, which includes the secretion of different cytokines, eventually leading to
demyelination and axonal damage. Pro-inflammatory (red) and immunoregulatory (green) cytokines and Th cell
subsets are also shown.
2.6 Mechanisms of demyelination and neuroaxonal damage
The formation of a demyelinated lesion in CNS involves the interplay between mechanisms of
both adaptive and innate immunity (Weiner 2009). As to adaptive immunity, activated CD4+ T
cells facilitate effector mechanisms of other immune cells by cell-to cell interactions and the
secretion of pro-inflammatory cytokines. A pro-inflammatory cytokine milieu activates
residence cells like microglia and astrocytes as well as recruits additional immune cells like
monocytes, mast cells, CD4+ and CD8+ T cells and B cells to the site of inflammation. CD8+ T
cells, can mediate direct cytotoxic effects to neurons and glial cells by the release of toxic
granules inducing apoptosis of the target cell. B cells, apart from acting as APCs and regulatory
cells can secrete cytokines and exert humoral responses, by producing autoantibodies that
specifically may target neuronal and glial cells.
Turning to innate immunity responses, these are main players in inflammation and
demyelination. As to cell populations, monocytes/macrophages, dendritic cells, NK cells, mast
21
2. Background
cells, NKT cells and γδ-T cells have all been assigned involvement in inflammation and tissue
damage (Gandhi, Laroni et al. 2010). As one prominent example, monocytes are differentiated
into tissue macrophages releasing pro-inflammatory cytokines and injurious reactive oxygen
intermediates and nitric oxide. Additionally, macrophages may also exert direct phagocytic
attacks on myelin sheaths. Also microglia, being resident cells of the macrophage lineage, are
involved in the process (Sriram 2011).
Soluble factors as matrix metalloproteinases and histamine further add to the breakdown of the
BBB and damage to neurons and glial cells (Larochelle, Alvarez et al. 2011). Disturbances in
glutamate metabolism among glia cells may also contribute to white matter damage (Matute,
Domercq et al. 2006).
2.7 Components of the immune response
This thesis investigates some of the immunological mechanisms underlying MS. Below is a
description of the main components assessed. As mentioned, MS pathogenesis has been linked
to the adaptive immune system by T cell-mediated immune regulation, involving both CD4+ T
helper cells and CD8+ T cytotoxic cells (McFarland and Martin 2007). However, the
pathogenetic picture has become far more diverse also including B lymphocytes and
lymphocytes of the innate immune system. Clearly, the distribution of different cell populations
is an important facet of MS pathology and regulation.
CD4+ T cells
CD4+ T cell directed immune responses can roughly be mediated by effector T cells as T helper
1 (Th1), Th2, Th17 and regulatory T cells (Treg cells). As to MS, Th1 and Th17 cells are
thought to represent auto-reactive disease-promoting T cell populations, whereas Th2 and Treg
cells seem to have protective abilities (Fletcher, Lalor et al. 2010, Kasper and Shoemaker
2010). Although attempts have been done, mainly in experimental models (Racke, Bonomo et
al. 1994, Ekerfelt, Dahle et al. 2001, Hallin, Mellergard et al. 2006), utilizing this concept
therapeutically in autoimmunity has been proven difficult. CD4+ Th1 cells have traditionally
been assigned a pivotal disease-promoting role in autoimmunity by the production of proinflammatory cytokine interferon γ (IFN-γ) (Petermann and Korn 2011), indeed underscored by
the worsening of MS during IFN-γ therapy (Panitch, Hirsch et al. 1987). The discovery of the
Th17 subset has changed this view since Th17 cells were shown absolutely essential for
initiating EAE (Langrish, Chen et al. 2005). Th17 cells produce the pro-inflammatory cytokine
IL-17 and elevated intracellular expression of IL-17 in peripheral blood mononuclear cells
(PBMC) has been demonstrated in patients with active MS (Durelli, Conti et al. 2009) and also
in CSF (Matusevicius, Kivisakk et al. 1999). Moreover, MS endothelial cells express high
levels of IL-17 receptors and IL-17 is shown to facilitate BBB permeability, altogether further
promoting CNS inflammation (Kebir, Kreymborg et al. 2007). Interestingly, it was recently
proposed that granulocyte-monocyte colony-stimulating factor (GM-CSF) was required to
22
2. Background
induce CNS pathology in EAE irrespective of a Th1- or a Th17-mediated disease mechanism
(Codarri, Gyulveszi et al. 2011).
Treg cells are distinguished by their high expression of the IL-2 receptor alpha chain (CD25)
and the transcriptor factor forkhead box p3 (FOXP3). Although FOXP3 is the signature marker
of Treg cells, CD4+ cells with a slightly lower CD4 expression (CD4dim) and high CD25
expression (CD25bright) are also shown to correspond well to FOXP3-high expressing cells
(Mjosberg, Svensson et al. 2009). Treg cells have been designated a central role for
maintenance of tolerance and for the protection against autoimmune diseases (Zozulya and
Wiendl 2008). Treg cells may prevent the development of EAE (Kohm, Carpentier et al. 2002)
and immunosuppressive function of Treg cells has been shown impaired in MS patients
compared to controls (Viglietta, Baecher-Allan et al. 2004, Haas, Hug et al. 2005).
The differentiation of a naive CD4+ T cell into an effector T cell is dependent on its recognition
of antigens presented by MHC II molecules on APCs. However, for the proper activation of the
T cell, co-stimulatory cell-to-cell interactions as well as soluble signals are also required. The
co-stimulatory signals from APCs are referred to as the second signal. A well-known example
is the binding of T cell surface molecule CD28 to B7-1 (CD80) and B7-2 (CD86) on APCs.
The soluble signals required refers to the cytokine milieu in which the activation takes place.
Furthermore, part of the T cell effector mechanisms involves secretion of specific cytokines
that orchestrate the subsequent immune response (Figure 3). For example, Th1 requires IL-12
for its differentiation and is characterized by secretion of IFN-γ, whereas Th2 requires IL-4 and
secretes IL-4, IL-5 and IL-13 (Farrar, Asnagli et al. 2002, Murphy and Reiner 2002). As to
Th17, differentiation is dependent on IL-23, transforming growth factor β (TGF-β) and IL-6
and effector responses are characterized by secretion of IL-17A, IL-17F, IL-21 and IL-22
(Miossec, Korn et al. 2009). Treg differentiation requires TGF-β and is denoted by the
secretion of IL-10 and TGF-β (Sakaguchi, Miyara et al. 2010). Intracellularly the development
of Th subpopulations is denoted by expression of lineage specific transcription factors as
Tbet/TBX21 (Th1) (Szabo, Kim et al. 2000), GATA3 (Th2) (Zheng and Flavell 1997), RORC
(Th17) (Ivanov, McKenzie et al. 2006) and FOXP3 (Treg) (Fontenot, Gavin et al. 2003)
(Figure 3).
CD8+ T cells
CD8+ T cells are major effector cells of the adaptive immune system. Consistent with this, it is
reported that MS lesions show a predominance of CD8+ T cells and that axonal damage within
lesions correlate best with the number of CD8+ T cells (Kuhlmann, Lingfeld et al. 2002, Friese
and Fugger 2009). Thus, even though CD4+ T cells are considered to initiate or direct the
immune attack on myelin, CD8+ T cells are crucial for continuous demyelination and
subsequent axonal damage. Effector mechanisms of activated CD8+ T cells include the
secretion of IFN-γ and tumor necrosis factor (TNF) and direct toxic properties to myelin and
axons by perforin and granzymes (Kaech and Wherry 2007).
23
2. Background
Figure 3. Schematic view of CD4+ T cell lineages and corresponding cytokines. Different cytokines drive T helper
(Th) cells into different subsets that express different lineage-specific transcription factors. Different Th subsets
secrete different sets of cytokines. Note that the inducing cytokines are mainly secreted by other cells, in particular
antigen presenting cells (APC). Also note that the Treg cells depicted here are induced Treg cells, while natural
Treg cells are produced in thymus.
B cells
The importance of B cells in the pathophysiology of MS has become indisputable in recent
years. The background for this include the findings of clonally expanded B cells producing
immunoglobulins with overlapping repertoire within CSF and brain lesions (Obermeier, Lovato
et al. 2011), large numbers of plasma cells in subacute and chronic MS plaques (Henderson,
Barnett et al. 2009) and the therapeutic effect of B cell depletion (Bar-Or, Calabresi et al.
2008). In addition, some EAE models are mainly dependent on antibodies (Pollinger,
Krishnamoorthy et al. 2009). The presence of antibody production in terms of CSF oligoclonal
bands is demonstrated in more than 95% of MS patients and thus constitutes a typical finding in
MS (Freedman, Thompson et al. 2005). Furthermore, CSF levels of B cell marker cytokine
CXCL13, is shown to correlate both with clinical disease activity and progression as well as
with paraclinical measures as CSF total leukocyte and B cell counts, intrathecal IgG synthesis,
markers of demyelination and MRI activity (Khademi, Kockum et al. 2011) (Disanto, Morahan
et al. 2012). Apart from antibody-dependent effector mechanisms of B cells, this lymphocyte
population can also be divided into subsets with divergent functions including proinflammatory or regulatory subsets. Memory B cells (CD19+CD27+) are reported to be
24
2. Background
distinguished from naïve B cells (CD19+ CD27-) by the secretion of pro-inflammatory
cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) upon stimulation, whereas naïve
B cells secrete immunoregulatory IL-10 (Duddy, Niino et al. 2007). Reduction in IL-10
producing B cells (Breg) in MS was accompanied by a reduced naïve/memory Breg ratio
during relapse but not in remission (Knippenberg, Peelen et al. 2011). Breg cells have, despite
their name, no association with Treg cells with regard to differentiation, action and expression
of FOXP3. Still, Breg cells constitute a heterogeneous population with different definitions,
one being CD25 expression. CD19+CD25high Breg cells have been shown to suppress CD4+ T
cell proliferation and enhance Treg properties (Kessel, Haj et al. 2012), as well as suggested a
regulatory role in vasculitis (Eriksson, Sandell et al. 2010).
Natural killer cells
Natural killer (NK) cells are lymphocytes of the innate immune system, having both cytotoxic
and regulatory functions (Gandhi, Laroni et al. 2010).(Berzins, Smyth et al. 2011, Kaur,
Trowsdale et al. 2012). They constitute a unique cell population considering their ability to
direct lyse tumor cells and virus-infected cells without a preceding sensitization. The regulatory
functions are primarily executed by the CD3- CD56bright subset secreting cytokines as IL-10
(Poli, Michel et al. 2009). Based on different expression of chemokine receptors and adhesion
molecules, cytotoxic NK cells (CD56dim) and regulatory NK cells (CD56bright) have different
migration preferences with CD56dim migrating to inflammatory sites while CD56bright
preferentially home to secondary lymphoid organs (Campbell, Qin et al. 2001). Interestingly,
NK cells are shown to mediate differentiation of dendritic cells, thereby shaping the subsequent
immune response (Moretta, Marcenaro et al. 2008). In that respect, NK cells are a striking
example of the close interplay between innate and adaptive immunity. NK regulatory subset
(CD56bright) is proposed having a beneficial effect in MS since there is an increase in their
numbers on immunomodulatory treatment (Bielekova, Catalfamo et al. 2006, Saraste, Irjala et
al. 2007) and in ameliorated MS during pregnancy (Airas, Saraste et al. 2008). Additionally, a
reduction in NK cell function in the periphery was demonstrated to correlate with the onset of
clinical relapse in MS patients (Kastrukoff, Morgan et al. 1998, Kastrukoff, Lau et al. 2003).
Cell surface markers of activation and co-stimulation
Activation of a T cell (upon ligand recognition of the TCR) triggers intracellular signalling
pathways that cause activation of gene transcripts and eventually the production of
corresponding proteins. Proteins synthesized may then be expressed on the cell-surface and
used as a measure of T cell activation. Based on the time elapse from TCR stimulation to
expression on the cell surface, activation markers may be referred to as early or late markers of
activation.
CD69 is a membrane bound receptor that acts as a co-stimulatory molecule. It is a marker of
very early activation of T cells since it is detectable within 1-2 hours of antigen ligation of the
TCR (Ziegler, Ramsdell et al. 1994). It facilitates the activation and proliferation of other T
cells and is also suggested to have a downregulatory role in immune responses (Sancho, Gomez
et al. 2005). CD69 expression could be transient but in an environment of pro-inflammatory
25
2. Background
cytokines, i. e. at the inflammatory site, CD69 expression is prolonged (Sancho, Gomez et al.
2005). CD25 is part of the IL-2 receptor (α-chain) and is expressed within 24 hours after TCR
stimulation (Caruso, Licenziati et al. 1997). Signalling through the IL-2 receptor is crucial for
the activation, differentiation and expansion of effector T cells (Liao, Lin et al. 2011). HLADR, a MHC class II cell-membrane receptor molecule, is expressed after about 48 hours and
thus constitutes a marker of late T cell activation (Caruso, Licenziati et al. 1997).
OX40 (CD134) and its ligand OX40L (CD252) represent a co-stimulatory signalling system
that promotes effector T cell expansion and survival (Croft, So et al. 2009). OX40L is not
constitutively expressed but induced within hours of T cell activation. OX40/OX40L
interactions also modulate cytokine production from NK cells, NKT cells and APCs and have
also been shown important for the development of EAE (Weinberg, Bourdette et al. 1996).
Moreover, co-stimulation by T cell OX40L was suggested to deviate a Th1 response towards a
Th2 response under certain circumstances (Mendel and Shevach 2006).
Cytokines
Cytokines comprise a large group of small signalling proteins secreted by all kinds of cells of
the immune system as well as by several non-immune cells. They constitute the key mediators
of communication between immune cells and thus regulate and shape all immunological
pathways. Cytokine effects are characterized by pleiotrophism and redundancy, the former
refers to the ability of a specific cytokine to exert different effects on different cell types, the
latter implicates that multiple cytokines may have the same or similar functional effects (Akdis,
Burgler et al. 2011) (Table 1). However, as already described, different CD4+ T cells are
denoted by their secretion of specific cytokines. Accordingly, the balance between proinflammatory and regulatory cytokines has a major influence on the initiation and development
of MS (Sospedra and Martin 2005). As to IL-1 (IL-1α and IL-1β), it is an important regulator of
homeostasis in CNS under physiologic conditions, but levels are rapidly increased in response
to injury (Basu, Krady et al. 2004). It is suggested that IL-1 is at the top of the cytokine
signalling cascade by acting as an upstream signal for other pro-inflammatory cytokines and
mediators (Basu, Krady et al. 2004).
26
2. Background
Table 1. Description of cytokines and chemokine CXCL8 included in paper I and III. (Commins, Borish et al.
2010, Akdis, Burgler et al. 2011, Ernerudh 2012).
Cytokine
IL-1β
Main producers
Monocytes/macrophages,
neutrophils, glia cells,
keratinocytes and numerous other
cells.
IL-2
Activated CD4+ and CD8+ T
cells.
IL-4
Th2 cells, basophils, eosinophils,
mast cells and NKT cells.
IL-5
Th2 cells, eosinophils, mast cells,
NK cells and NKT cells.
IL-6
Monocytes/macrophages,
endothelial cells, T cells, B cells
and numerous other cells.
IL-10
Monocytes/macrophages,
dendritic cells, T cells and B
cells.
TNF
Mononcytes/macrophages, T
cells, B cells, NK cells,
endothelial cells, mast cells and
neutrophils.
IFN-γ
Th1 cells, cytotoxic T cells and
NK cells.
GM-CSF
T cells, fibroblasts, epithelial
cells, endothelial cells,
monocytes/macrophages, mast
cells, eosinophils and neutrophils.
CXCL8 (IL-8)
Monocytes/macrophages,
endothelial and epithelial cells, T
cells, granulocytes and numerous
other cells.
27
Important functions
Pro-inflammatory, induction of
adaptive immunity
Facilitates proliferation,
differentiation, and function of
both innate and adaptive immune
system cells, in particular T cells.
Upregulation of adhesion
molecules like integrins and
selectins. Endogenous pyrogen.
Regulation of adaptive
immunity
Proliferation of effector T and B
cells, development of Treg cells,
differentiation and proliferation of
NK cells and growth factor for B
cells.
Induction and regulation of
adaptive immunity
Induction of Th2 differentiation,
IgE class switch and upregulation
of MHC II expression on B cells.
Regulation of adaptive
immunity Facilitates chemotactic
activity and adhesion capacity of
eosinophils.
Pro-inflammatory, induction of
adaptive immunity
T cell activation and
differentiation, induction of Th17
differentiation, B cell
differentiation and inducer of
acute phase proteins.
Immunosuppressive
Downregulation of MHC II and
co-stimulatory molecules on
APCs, inhibition of production of
pro-inflammatory cytokines.
Pro-inflammatory
Induces anti-tumor immunity,
facilitates granulocyte function
and chemotaxis and promotes
vascular leakage.
Induction and regulation of
adaptive immunity
Induces cell-mediated immunity
by stimulating antigen
presentation and cytokine
production, promotes macrophage
mobilization and function.
Growth and differentiation
Supports maturation of dendritic
cells, neutrophils and
macrophages, stimulates platelet
and erythrocyte production,
activation of granulocytes and
monocytes.
Chemoattractant
Potent chemoattractant for
neutrophils, NK cells, T cells and
mobilization of hematopoietic
stem cells.
Important cell targets
T cells, endothelial cells
and epithelial cells.
CD4+ and CD8+
T cells, NK cells and B
cells.
T cells and B cells.
Eosinophils, basophils, and
mast cells.
Hepatocytes, leukocytes, T
cells, B cells and
hematopoietic cells.
Monocytes/macrophages T
cells, B cells, NK cells,
mast cells, dendritic cells
and granulocytes.
Endothelial cells and
granulocytes.
Monocytes/macrophages,
dendritic cells, NK cells,
granulocytes, endothelial
cells, T cells and B cells.
Eosinophils, neutrophils
dendritic cells,
monocytes/macrophages,
platelets and erythrocytes.
Neutrophils, T cells, NK
cells, basophils, eosinophils
and endothelial cells.
2. Background
Chemokines
Chemokines are described as “chemotactic cytokines” and constitue a large protein family
responsible for the trafficking of leukocytes to sites of inflammation in a concentrationdependent fashion (Commins, Borish et al. 2010). Chemokines are also involved in several
other processes including the regulation of leukocyte maturation (Baggiolini 1998, Ubogu,
Cossoy et al. 2006, Commins, Borish et al. 2010). Under normal conditions the CNS is
believed to be a zone of relative immune privilege, which means a relative lack of immune
surveillance. This implicates that although there are many immune competent cells in the CNS
like microglia, there are few patrolling immune cells recruited from the circulation. To
establish a local immune response, the recruitment of T cells to the site is therefore crucial and
likely a cornerstone in MS pahogenesis. Accordingly, CXCL8 (previously denoted IL-8) as
well as CXCL9 and CXCL10, the latter two denoted as Th1-associated chemokines, have all
shown to be elevated in CSF during relapse (Sorensen, Tani et al. 1999, Bartosik-Psujek and
Stelmasiak 2005). In contrast, CCL17 and CCL22 are associated with Th2 immune responses,
and are highly expressed in Th2-related diseases like airway hypersensitivity and atopic
dermatitis (Fujisawa, Fujisawa et al. 2002, Yamashita and Kuroda 2002, Hijnen, De BruinWeller et al. 2004).
2.8 Leukocyte adhesion and migration
The recruitment of pro-inflammatory cells to the CNS is dependent on the sequential
interactions of different adhesion and signalling molecules on leukocytes and endothelial cells
lining the BBB. The process can principally be divided into tethering, rolling, activation and
arrest followed by diapedesis (Luster, Alon et al. 2005). Initial tethering and subsequent rolling
of cells along the endothelium is mediated by E-, P-, and L-selectins or α4-integrins. E- and Pselectins are expressed by the endothelium and interacts with glycoproteins, glycolipids, Lselectins and α4-integrins expressed on the leukocyte. If the rolling leukocyte at this step
encounters additional signals from chemoattractive molecules like chemokines, there is an
activation of intracellular signalling pathways through G-protein–coupled receptors causing the
rapid activation of integrins. Integrins are heterodimeric leukocyte surface proteins composed
of an α- and β-popypeptide chain. The most important integrins for leukocyte trafficking
belong to the β2 subfamily (leukocyte function-associated antigen 1, LFA-1) and the α4integrins α4β1 (very late activation antigen 4, VLA-4) and α4β7 (Luster, Alon et al. 2005). The
integrins α4β1 and α4β7 bind to their corresponding molecules on endothelial cells, vascular
cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1
(MAdCAM-1), respectively. The activation of a leukocyte by chemoattractants thus
upregulates its expression of these integrins, promoting the arrest and firm adhesion of the
leukocyte to the endothelium. In the context of CNS inflammation, the ensuing diapedesis into
CNS tissue is facilitated by inflammation-caused local impairment of the BBB. This
impairment of the BBB implicates an increase in permeability with alteration of tight junction
structures altogether promoting the influx of pro-inflammatory leukocytes (Alvarez, Cayrol et
al. 2011).
28
2. Background
Inflammatory signals like cytokines and chemokines have a major role in cell-adhesion and
migration processes since they induce expression of selectins and integrins on leukocytes and
endothelial cells. To exemplify, VCAM-1 is normally not detectable in the brain parenchyma,
however upon stimulation with IFN-γ, IL-1β and TNF, astrocytes express VCAM-1
(Rosenman, Shrikant et al. 1995) and VCAM-1 can also be expressed by glial cells near sites of
MS lesions (Cannella and Raine 1995). Furthermore, peripheral lymphocyte expression of
adhesion molecules like VLA-4 and LFA-1 was associated with the development of MS lesions
as measured by T2 lesion load on MRI (Eikelenboom, Killestein et al. 2005).
2.9 Inflammation versus neurodegeneration
MS is traditionally considered an inflammatory disease with attacks on the myelin sheath by
auto-reactive T cells causing demyelination and axonal damage. Evidence for the association
between demyelination and axonal damage has come from histopathological studies showing
that transected axons are common in MS lesions and that the density of transected axons
correlated with the inflammatory activity in the lesion (Trapp, Peterson et al. 1998). Although
patients use to recover clinically from demyelinating exacerbations, there is shown to be
irreversible subclinical axonal damage also at early stages of the disease (Dutta and Trapp
2007). It is presumed that remyelination in combination with CNS plasticity accounts for the
ability to mask this progressive axonal damage early in the disease course. However, when the
accumulating axonal loss eventually reaches a threshold for the ability of the brain to
compensate, the result is irreversible neurologic disability. Clinically this point is characterized
by the transition to SPMS. MS treatment strategy has been focusing on the decrease of
inflammation and thereby indirectly also possibly interfering with the subsequent
neurodegenerative process. All available disease-modulating therapies for MS today are
immunomodulators and there is no specific treatment once the patient has reached the
secondary progressive phase of the disease. Consequently, in a recent systematic review of
treatment with IFNβ in SPMS, the conclusion was that treatment did not delay permanent
disability, although relapse risk was reduced (La Mantia, Vacchi et al. 2012).
However, the view that focal demyelinating inflammation drives the neurodegenerative process
in MS has been challenged by evidence that axonal damage, reflected in brain atrophy
progression, may occur independently of focal inflammatory lesions (Miller, Barkhof et al.
2002). In particular by means of new magnetic resonance imaging (MRI) techniques, several
studies have shown that the disease process in MS is ongoing rather than episodic, and have
emphasized the importance of neuronal and axonal pathology (De Stefano, Bartolozzi et al.
2005) (Filippi, Rocca et al. 2012). Wallerian degeneration has been suggested to partly explain
the discrepancies between axonal damage and focal inflammation by providing a mechanism
for axonal loss in non-lesional white matter distal to the lesion. This notion is also consistent
with studies on axonal projections in corpus callosum (Evangelou, Konz et al. 2000) and the
corticospinal tract (Simon, Kinkel et al. 2000). However, the relation between inflammation
and neurodegeneration in MS is unclear and yet to be defined.
29
2. Background
Figure 4. Cerebral MRI (FLAIR protocol) showing a sagittal (left) and axial (right) section with periventricular
white matter lesions and cortical atrophy in MS (from CMIV).
2.10 Measuring diffuse pathology in MS
MRI is indisputable one of the most important paraclinical tools not only when diagnosing MS
but also in assessing disease progression and evaluating treatment effects (Figure 4). MRI in
combination with pathological studies has indeed expanded the way we understand this disase,
i. e. from a focal demyelinating disease of white matter to a disease with both focal and diffuse
pathology and involving both white and grey matter (Filippi, Rocca et al. 2012). Still,
conventional MRI (cMRI) only provides limited information about the degree of inflammation
and remyelination as well as data about diffuse white and grey matter pathology (Zivadinov,
Stosic et al. 2008). In this perspective novel MRI-techniques like proton magnetic resonance
spectroscopy (1H-MRS) and quantitative MRI (qMRI) can give valuable additional
information. 1H-MRS is a technique that measures signals originating from protons in organic
molecules in contrast to signals derived from protons in water (cMRI technique), thereby
allowing for non-invasive quantification of specific metabolites in cerebral tissues (Matthews,
Francis et al. 1991, Sajja, Wolinsky et al. 2009). 1H-MRS has provided data about pathologic
findings in what appears to be normal white (NAWM) and gray (NAGM) matter on cMRI (He,
Inglese et al. 2005). In 1H-MRS specific neurometabolites are represented by different
resonance peaks like N-acetylaspartate (NAA), creatine (Cr), myo-Inositol (mIns), choline
(Cho) and glutamate (Glu) (Sajja, Wolinsky et al. 2009). NAA, an aminoacid produced and
localized primarily in neurons, is considered to be a marker of axonal integrity and neuronal
viability. The resonance from Cr derives from creatine and phosphocreatine, which are
important in the energy metabolism. Since Cr is more abundant in glia cells (astrocytes and
oligodendrocytes) compared to neurons, elevated levels are an indicator of gliosis. mIns is also
30
2. Background
considered a glia cell marker whereas Cho indicates myelin turnover thus reflecting the process
of de- and remyelination. Glutamate is the principal excitatory neurotransmitter of the CNS but
has also been ascribed neurotoxic properties when at excessive amounts (Matute, Domercq et
al. 2006).
As a complement to 1H-MRS data on neurodegeneration in MS, levels of neurofilament light
protein (NFL) and glial fibrillary protein (GFAP) in CSF might be used (Giovannoni 2006).
NFL is, together with neurofilament intermediate chain and neurofilament heavy chain, the
major component of axonal cytoskeleton proteins. Axonal damage causes the release of
neurofilament proteins in the extracellular fluid and may accordingly be assessed in CSF
(Teunissen and Khalil 2012). CSF levels of NFL have been shown elevated in MS patients, in
particular during acute relapses (Malmestrom, Haghighi et al. 2003, Norgren, Sundstrom et al.
2004, Teunissen, Iacobaeus et al. 2009), and in patients with CIS (Teunissen, Iacobaeus et al.
2009). NFL levels in CSF were significantly reduced in MS patients by treatment with
natalizumab (Gunnarsson, Malmestrom et al. 2010). GFAP constitutes the major intermediate
filament cytoskeletal protein of astrocytes and is considered a marker of astrocytes and
astrogliosis (Middeldorp and Hol 2011). GFAP was found in a large MS plaque with fibrous
astrocytes and demyelinated axons (Eng, Vanderhaeghen et al. 1971), and is increasingly
expressed during CNS degeneration and aging. GFAP levels in CSF have been shown to
correlate with disability and disease progression in MS (Malmestrom, Haghighi et al. 2003,
Axelsson, Malmestrom et al. 2011).
2.11 Disease-modifying treatments
All approved disease-modifying therapies for relapsing MS are immunomodulators. The firstline treatment consists of interferon-β (IFNβ-1a or IFNβ-1b) and glatiramer acetate.
IFNβ is given as a subcutaneous injection every two or three days or as an intramuscular
injection once a week. IFNβ treatment has been shown to reduce relapse rate with about 30%
and reduce disease activity as measured by a reduction in new MRI lesions compared to
placebo (1995, Jacobs, Cookfair et al. 1996, 1998). The precise effector mechanism is not fully
defined, although inhibition of auto-reactive T cells, induction of Treg cells and interference
with leukocyte migration and cytokine modulation has been suggested (Dhib-Jalbut and Marks
2010).
Glatiramer acetate (GA) is given once a day as a subcutaneous injection. GA treatment was
shown to reduce relapse rate by 29% (Johnson, Brooks et al. 1995). As for IFNβ, the effector
mechanisms of GA are uncertain, however studies on EAE suggest that GA may generate
suppressor cells, inducing tolerance, expanding Treg populations and alter APC functions
(Racke, Lovett-Racke et al. 2010).
The second-line treatment for MS includes natalizumab and fingolimod. Fingolimod (FTY720)
is the first oral disease-modifying treatment available for relapsing MS and is a sphingosine-131
2. Background
phosphate (S1P) receptor agonist. S1P signalling mediate leukocyte trafficking in lymph nodes
and treatment with S1P has been shown to reversibly sequester naïve and activated central
memory T cells in the lymph nodes, whereas effector memory T cells remain in the peripheral
circulation (Mehling, Brinkmann et al. 2008). Treatment with fingolimod has been shown to
reduce relapse rate with 54% compared to placebo, and a significant decline in MRI lesions has
also been demonstrated (Kappos, Radue et al. 2010).
Natalizumab, which is of particular interest in this thesis, is a humanized monoclonal antibody
directed against the α4-chain (CD49d) of VLA-4 (α4β1) and α4β7. Humanized antibodies have
major advantages, since they are less immunogenic, which increases the in vivo life time and
allows for repeated administrations. Natalizumab antibodies are of the IgG4 subclass, which
facilitates their persistence in the circulation and eliminates the risk of complement activation
(Rice, Hartung et al. 2005). The α4-integrin is expressed on different types of leukocytes as T
and B cells, monocytes, eosinophilic and basophilic granulocytes, albeit neutrophil
granulocytes are excepted. The α4-chain heterodimerizes with β1 and β7 subunits and thereby
form a functional molecule (Abbas A.K. 2012). VLA-4 (α4β1) adheres toVCAM-1 on vascular
endothelium and α4β7 interacts with MAdCAM present on gut endothelium. The effect of
natalizumab in Crohn´s disease is considered mediated by the blocking effect on α4β7
(Guagnozzi and Caprilli 2008).
Evidence for the crucial role of α4-integrins in leukocute activation, migration and
differentiation originate from both in vitro and animal studies (Lobb and Hemler 1994). The
first study to demonstrate prevention of EAE by treatment with α4β1-antibodies came 1992
(Yednock, Cannon et al. 1992). Natalizumab-binding to VLA-4 blocks the attachment of
leukocytes to brain endothelial cells thereby preventing their entrance into the CNS. In a
randomized placebo-controlled trial, natalizumab reduced the relapse rate with about 68% over
one year and simultaneously reduced the accumulation of new or enlarging hyperintense
lesions as measured by T2-weighted MRI (Polman, O'Connor et al. 2006). Thus, natalizumab is
proven more efficient than the first-line treatments with IFNβ and GA. Apart from reduced
entrance of inflammatory cells to the CNS, other immunological effects are likely to occur that
may contribute to treatment effects. Principally, effects of α4-integrin chain antibody blockade
could be referred either to the mere blocking of VLA-4 adhesion to its ligands or to
natalizumab acting agonistically as a ligand on the VLA-4 complex (Gonzalez-Amaro,
Mittelbrunn et al. 2005). The latter could generate intracellular signals that may affect the
future behavior of the target cell. It is assumed that the major part of in vivo treatment effects of
α4-integrin chain blockade are related to the former, i. e. reduced extravasation of proinflammatory leukocytes. This is also indirectly supported by studies showing an increase in
the expression of pro-inflammatory lymphocytes in the peripheral circulation (Kivisakk, Healy
et al. 2009, Frisullo, Iorio et al. 2011) and data showing reduced leukocyte counts in CSF
(Stuve, Marra et al. 2006, Khademi, Bornsen et al. 2009) and cerebral perivascular spaces (del
Pilar Martin, Cravens et al. 2008) after natalizumab treatment. However, there are many
reasons to suspect additional effects of α4-integrin chain blockade apart from interference with
cell trafficking. To exemplify, VLA-4 is involved in the development and differentiation of
many cell types and participates in the induction of the immune response by facilitating APC
and T cell adhesion during antigen presentation (Gonzalez-Amaro, Mittelbrunn et al. 2005).
32
2. Background
Accordingly, ligand-binding to VLA-4 is shown to be a co-stimulatory signal for T cells (Sato,
Tachibana et al. 1995, Niino, Bodner et al. 2006) and VLA-4 expression is up-regulated during
maturation of dendritic cells (Puig-Kroger, Sanz-Rodriguez et al. 2000). Further evidence of
additional effects of VLA-4 blockade is alteration in gene expressions of peripheral blood cells
(Lindberg, Achtnichts et al. 2008) and changes in PBMC population composition and
activation (Skarica, Eckstein et al. 2011) in natalizumab treated MS patients, as well as the
intriguing finding that treatment with a small α4-integrin inhibitor (CDP323) in a phase II study
of relapsing MS failed to show efficacy (Harrer, Wipfler et al. 2011).
2.12 Progressive multifocal leukoencephalopathy (PML)
Exploring the full range of natalizumab treatment effects has become particularly important
considering the association to development of progressive multifocal leukoencephalopathy
(PML). PML is an opportunistic brain infection caused by JC polyomavirus (White and Khalili
2011). PML causes diverse neurological symptoms depending on the localization and extent of
the infection. Visual deficits, cognitive impairment and motor weakness have been reported,
and the disease course may be fatal (Tan and Koralnik 2010). In a survey study of cases of
PML associated to natalizumab treatment and reported up to February 2012, there were 212
confirmed cases of PML among 99,571 patients (2.1 cases per 1000 patients) (Bloomgren,
Richman et al. 2012). The risk of developing PML was related to positive status with respect to
anti-JC virus antibodies, prior immunosuppressive treatment and increased duration of
natalizumab treatment (Bloomgren, Richman et al. 2012). Considering the proposed action of
natalizumab on cell trafficking, it is suggested that impaired immune surveillance in the CNS
may explain the increased risk of PML (Stuve, Marra et al. 2006). Still, additional effects of
VLA-4 blockade may contribute to the risk of PML, emphasizing the importance for further
studies on these mechanisms.
33
3. Aims
3. Aims
The general aim of this thesis was to elucidate immunological mechanisms present in
multiple sclerosis and the immunomodulatory effects of natalizumab treatment, focusing
on lymphocytes, inflammatory markers and 1H-MRS.
The specific aims of each paper were:
I
To assess the effects of one-year natalizumab treatment on cytokine and chemokine
protein levels in plasma and cerebrospinal fluid of MS patients.
II
To explore changes in blood lymphocyte composition and functional T cell responses in
MS patients after one-year natalizumab treatment.
III
To detect changes in NAWM metabolite concentrations in MS patients after one-year
natalizumab treatment, and to assess whether changes were associated with intrathecal
inflammation or neurodegeneration as measured by biomarkers in the CSF.
IV
To evaluate the balance between circulating Th cell subsets in MS patients using the
expression of the CD4+ T cell related transcription factors in whole blood.
34
35
4. Methods
4. Methods
4.1 Study subjects
All patients in this thesis were included consecutively from the Department of Neurology at the
University Hospital of Linköping, Sweden. All were diagnosed with MS according to the
McDonald criteria from 2001 (McDonald, Compston et al. 2001). In total 72 patients were
included. Table 2 shows the distribution of patients in paper I-IV. The study design in paper I,
II and III was observational and not interventional, since there was no randomization to a
prospectively followed placebo-treated group. When treated with natalizumab, patients were
given intravenously infusions (300 mg) once a month.
Paper I
Natalizumab treatment was initiated in 31 patients, 18 men and 13 women (median age 35
years, range 23-50), because of unsatisfactory effect or intolerable adverse effects on treatment
with first-line treatments. Patients were diagnosed as either RRMS (n=23) or RRMS with
secondary progression (RRMS/SP) (n=8). RRMS/SP was defined as a clinically progressive
MS though with presumed inflammatory activity because of superimposed relapses and/or
disease progression measured by MRI. Median EDSS at inclusion was 3.5 (range 0-8). Within
the last three months before inclusion 26 patients were on immunomodulatory treatment (17
IFN-β, 6 GA, 2 mitoxantrone, 1 IvIg) and 5 patients were not on any treatment. CSF from 10
patients with non-inflammatory neurological diseases was used as a control for baseline CSF
analyses, and blood from 15 healthy donors was used as control for baseline blood analyses.
Paper II
Natalizumab treatment was initiated in 40 patients, 22 men and 18 women (median age 37
years, range 22-62), with active MS. Initiation of treatment was based on clinical and MRI
parameters, implicating an active relapsing disease. EDSS at inclusion was 2.5 (range 0-7).
Within the last three months before inclusion 31 patients were on immunomodulatory treatment
(26 IFN-β, 4 GA, 1 IvIg) and 9 patients were not.
Paper III
Natalizumab treatment was initiated in 27 patients, 14 men and 13 women (median age 40
years, range 22-62), with active MS. Initiation of treatment was based on clinical and MRI
parameters, implicating an active relapsing disease. EDSS at inclusion was 2.5 (range 0-7).
Within the last three months before inclusion 24 patients were on immunomodulatory treatment
(20 IFN-β, 4 GA) and 3 patients were not. For baseline comparisons of 1H-MRS variables, 20
healthy volunteers (median age 48 years, range 27-72) were used as controls.
36
4. Methods
Paper IV
33 patients, 18 men and 15 women (median age 35 years, 25th-75th percentile 30-43), with
RRMS were included. Median EDSS at inclusion was 2.0 and within the last three months
before inclusion 12 patients were on treatment with IFN-β and 23 patients were untreated. 20
age-and sex-matched healthy volunteers (median age 46 years, range 21-62) were used as
controls. All participants (patients and controls) were checked for ongoing infection and/or
other inflammatory conditions at the time of inclusion in order to minimize potential
confounding factors. None of the participants were excluded on these grounds.
Table 2. Distribution of patients in paper I-IV. Total no. of patients=72.
Year of birth
1987
1986
1983
1983
1982
1982
1982
1981
1980
1980
1979
1979
1979
1978
1978
1978
1976
1976
1976
1975
1975
1975
1975
1974
1974
1974
1974
1973
1973
1973
1973
1973
1972
1971
1971
1970
Sex
f
f
f
f
m
m
m
m
f
f
f
f
m
m
m
m
m
m
f
m
m
f
f
m
m
f
f
m
f
f
m
f
m
m
f
f
I
II
x
x
III
IV
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Year of birth
1970
1970
1970
1968
1968
1967
1967
1967
1967
1967
1967
1966
1966
1965
1965
1965
1965
1965
1965
1964
1964
1963
1963
1963
1963
1962
1962
1961
1960
1959
1959
1958
1957
1955
1945
1937
Sex
f
f
f
f
m
f
f
f
f
m
m
f
m
f
m
f
f
f
f
m
m
m
f
f
m
m
f
m
f
m
f
f
f
f
m
f
I
x
x
x
II
x
x
x
x
x
III
x
IV
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Year of birth and sex are given for each patient. m=male, f=female, x mark participation in respective study.
37
x
x
x
x
4. Methods
4.2 Procedures
4.2.1 Disease scoring systems (paper I, II, III, IV)
Medical records and the Swedish MS registry were used in order to evaluate disease course and
earlier immunomodulatory treatments. In paper I-III, all patients were examined by a
neurologist (CD or MV) or resident in neurology (JM) at inclusion and after one year of
treatment for the definition of Expanded Disability Status Scale (EDSS) score and subsequent
Multiple Sclerosis Severity Score (MSSS). In paper IV, EDSS was defined once (CD, MV or
JM). For paired comparisons the examination was carried out by the same clinician pre and
post one-year treatment (paper I, II and III).
EDSS is widely used as a measure of disability in MS and dates back to 1955, but was revised
to present form 1983 (Kurtzke 1983). It is composed of eight Functional Systems (FS) and
allows neurologists to assign a Functional System Score (FSS) in each of these. The eight FS
are pyramidal function, cerebellar function, brainstem function, sensory function, bowel and
bladder function, visual function, higher cerebral function and other deficits. Other deficits can
include paroxysmal symptoms. The sum of all FSS is then referred to the EDSS scale to define
an EDSS score (Table 3). EDSS 0-4.5 refers to fully ambulatory patients and requires a
thorough neurological examination, whereas EDSS 5.0-9.0 are defined as the impairment to
ambulation.
MSSS was introduced in 2005 (Roxburgh, Seaman et al. 2005). It combines disability, as
defined by EDSS, with disease duration and thus is a measure of the disease progression rate.
The Multiple Sclerosis Impact Scale 29 (MSIS-29) was introduced in 2001 and consists of a
questionnaire about the patients’ view on the physical and psychological impact of MS within
the last two weeks (Hobart, Lamping et al. 2001). There are 12 questions related to physical
impairment and 17 related to psychological impairment. Each question constitutes a statement
and can be answered with four alternatives rendering different scores (1=not at all, 2=a little,
3=moderately, 4=quite a bit, 5=extremely). The sum of the questionnaire-score constitutes the
MSIS-29 score. MSIS-29 questionnaires were distributed to patients in connection to
natalizumab infusions.
Symbol Digit Modalities Test (SDMT) is a quick screening test for cognitive dysfunction
(Smith 1991). The procedure is that the patient, by using a reference key, has 90 seconds to pair
specific numbers with a given geometric figure. Answers can be oral or written. SDMT was
conducted by a nurse in connection to natalizumab infusions.
38
4. Methods
Table 3. The Kurtzke Expanded Disability Status Scale with description of EDSS scores.
0.0 Normal neurological examination.
1.0 No disability, minimal signs in one FS.
1.5 No disability, minimal signs in more than one FS.
2.0 Minimal disability in one FS.
2.5 Mild disability in one FS or minimal disability in two FS.
3.0 Moderate disability in one FS, or mild disability in three or four FS. Fully ambulatory.
3.5 Fully ambulatory but with moderate disability in one FS and more than minimal disability in
several others.
4.0 Fully ambulatory without aid, self-sufficient, up and about some 12 hours a day despite
relatively severe disability; able to walk without aid or rest some 500 meters.
4.5 Fully ambulatory without aid, up and about much of the day, able to work a full day, may
otherwise have some limitation of full activity or require minimal assistance; characterized by
relatively severe disability; able to walk without aid or rest some 300 meters.
5.0 Ambulatory without aid or rest for about 200 meters; disability severe enough to impair full
daily activities (work a full day without special provisions).
5.5 Ambulatory without aid or rest for about 100 meters; disability severe enough to preclude full
daily activities.
6.0 Intermittent or unilateral constant assistance (cane, crutch, brace) required to walk about 100
meters with or without resting.
6.5 Constant bilateral assistance (canes, crutches, braces) required to walk about 20 meters without
resting.
7.0 Unable to walk beyond approximately five meters even with aid, essentially restricted to
wheelchair; wheels self in standard wheelchair and transfers alone; up and about in wheelchair
some 12 hours a day.
7.5 Unable to take more than a few steps; restricted to wheelchair; may need aid in transfer; wheels
self but cannot carry on in standard wheelchair a full day; may require motorized wheelchair.
8.0 Essentially restricted to bed or chair or perambulated in wheelchair, but may be out of bed itself
much of the day; retains many self-care functions; generally has effective use of arms.
8.5 Essentially restricted to bed much of day; has some effective use of arms retains some self-care
functions.
9.0 Confined to bed; can still communicate and eat.
9.5 Totally helpless bed patient; unable to communicate effectively or eat/swallow.
10.0 Death due to MS.
4.2.2 Routine CSF analyses (paper I, II, III)
Analyses of CSF total leukocyte counts, IgG index, albumin ratio and the investigation of
oligoclonal bands were performed as routine analyses at the Department of Clinical Chemistry
at the University Hospital of Linköping. IgG index is a measure of intrathecal immunoglobulin
synthesis and constitutes, together with the presence of oligoclonal bands, the CSF hallmarks of
an intrathecal inflammatory process as in MS. IgG index compensates for levels of IgG in
serum and effects on the BBB (Figure 5 a). Albumin ratio is a measure of BBB integrity
(Figure 5 b). An increase in albumin ratio is a non-specific sign of damage to the BBB and
39
4. Methods
could thus be caused by inflammatory processes as well as by infections, tumors, stroke and
trauma.
a: IgG index = [CSF-IgG (mg/l) / serum-IgG (g/l)] / [CSF-albumin (mg/l) / serum-albumin (g/l)]
b: Albumin ratio = [CSF-albumin (mg/l) / serum-albumin (g/l)]
Figure 5 a-b. How to calculate IgG index and albumin ratio.
4.2.3 Multiplex bead assay (paper I, III)
For assessment of cytokine (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-10, IFN-γ, TNF
and GM-CSF) concentrations in plasma and CSF, an ultra-sensitive 10-plex kit was used in
accordance with the instructions provided (Invitrogen Cytokine Ultrasensitive Human 10plex Panel). For assessment of chemokine (CXCL9, CKCL10, CXCL11, CCLL17 and CCL22)
concentrations in plasma and CSF, an in-house assay was used as described in paper I. Both the
assessment of cytokines and chemokines included the use of a multiplex bead assay (Figure 6).
Briefly, polystyrene microspheres (beads) are internally dyed with two different fluorochromes
in combinations, creating up to 100 different microspheres, all with a unique spectral signature
(Vignali 2000). A capture antibody to the analyte of interest is then coupled to the microsphere,
thereby obtaining a link between a specific analyte (in this thesis a cytokine or chemokine) and
a spectral-specific bead. Several bead sets may be mixed, enabling the assessment of many
analytes in a single sample. The beadmix with different captured analytes is then incubated
with detection antibodies labeled with a reporter fluorochrome. Using flow cytometry, each
bead is then characterized one at a time with respect to bead type and fluorescence intensity.
The mean fluorescence intensity (MFI) is proportional to the amount of bound analyte, and by
using a standard curve of defined concentrations of the analyte, the concentration of each
analyte in the sample could be determined.
40
4. Methods
Figure 6. Principle of a multiplex bead assay.
4.2.4 Flow cytometry (paper II, IV)
Flow cytometry makes it possible to detect, sort and count different cell populations on the
basis of their properties related to size, granularity and characteristic cell-surface proteins. The
cells are first incubated with fluorescent-labeled monoclonal antibodies against the cell-surface
(or intra-cellular) molecules of interest. In the flow cytometer, labeled cells then passes one at a
time through a laser beam causing scattering of the light and excitation of the attached
fluorochrome, if any, to a higher energy state. The excitation of the fluorochrome causes
emission of a certain wavelength that then can be detected. According to the specific scattering
profile of each cell, its volume or size (forward scatter, FSC) and inner complexity including
cytoplasmatic granules (side scatter, SSC), can be determined. In addition, based on the
fluorescence emission characteristics of each cell the expressions of cell-surface molecules of
interest are assessed.
Briefly, whole blood was drawn from EDTA tubes. The cells were labeled with antibodies for
15 min at room temperature in the dark. Antibodies used were (paper II) anti-CD3, anti-CD45,
anti-CD4, anti-CD8, anti-CD16/56, anti-CD19, anti-CD28, anti-CD56, anti-CD57, anti-HLADR, anti-CD69, anti-CD25, anti-OX40L, anti-CD5 and anti-CD27 and (paper IV) anti-CD4,
anti-CD3, anti-CD25. All antibodies were purchased from BD Biosciences (San José, CA,
USA). For analysis of absolute cell numbers (cells/μl) in lymphocyte populations, TruecountTM
41
4. Methods
tubes (BD Biosciences) were used. Removal of erythrocytes was done using FACS Lysing
Solution (BD Biosciences) followed by another 15 min incubation in the dark. Cells were
collected and analyzed using the FACS Canto II system (BD Biosciences). The initial
lymphocyte gate was based on FSC and SSC properties. The principle for gating was then to
define discrete populations or to use negative populations to define cut-off for continuously
expressed markers as described in paper II.
4.2.5 Lymphocyte activation assay (paper II)
The Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole
blood (FASCIA) (Svahn, Linde et al. 2003) was used with some modifications to evaluate
lymphocyte function. This method is based on determination of induced lymphoblast numbers
after in vitro stimulation. Briefly, diluted peripheral whole blood cultures were stimulated with
influenza antigen 1:1000 (Vaxigrip; Sanofi Pasteur, Solna, Sweden), PPD 10 µg/mL (SSI,
Copenhagen, Denmark), CMV antigen 0.125 µg (BD Biosciences), tetanus toxin 5.7 Lf/mL
(SSI), phytohaemagglutinin (PHA) 5 µg/mL (Sigma Aldrich), pokeweed mitogen (PWM) 10
µg/mL (Sigma Aldrich) or myelin basic protein (MBP) 100 µg/mL (Sigma Aldrich) diluted in
cell-culturing media. Culturing ensued for seven days at 37°C with 5% CO2, after which cells
were harvested and labeled with anti-CD3, anti-CD4, anti-CD8, and anti-CD108. After
labeling, erythrocytes were lysed by incubating cells with 0.8% NH4Cl solution for 15 min at
room temperature in the dark. Collection and analysis of data were performed using the FACS
Canto II system. TruecountTM tubes were used separately for assessment of absolute cell
numbers. Negative controls, cultured without antigen, were set up separately.
Initially, a lymphoblast gate was set based on FSC and SSC properties. A negative gate, gating
non-activated lymphocytes, was set using the unstimulated cultures, and this gate was used to
distinguish activated from resting lymphocytes. The cells were then further gated to CD3+CD4+
and CD3+CD8+ gates, respectively. From these populations, CD108+ cells were selected as a
marker of cytotoxic T cells. The number of cells in the lymphoblast gate was compared
between patients for the respective population and stimulus. For each stimulus, the mean
number of lymphoblasts in unstimulated cultures was subtracted, thereby compensating for
baseline activation of cells.
4.2.6 Markers of neurodegeneration in CSF (paper III)
Assessment of CSF myelin basic protein (MBP), total tau (T-tau), tau phosphorylated at
threonine 181 (P-tau), neurofilament light protein (NFL) and glial fibrillary acidic protein
(GFAP) was performed at the Clinical Neurochemistry Laboratory at The Sahlgrenska
Academy, Gothenburg, Sweden. Briefly, MBP was analyzed using a sandwich ELISA (Active
MBP ELISA, Diagnostic Systems Laboratories Inc., Webster, TX, USA). T-tau and P-tau were
determined using the Luminex xMAP technology and the INNOBIA AlzBio3 kit (Innogenetics
Zwijndrecht, Belgium) as described previously (Olsson, Vanderstichele et al. 2005). NFL and
42
4. Methods
GFAP were analyzed using ELISA methods described previously (Rosengren, Wikkelso et al.
1994, Rosengren, Karlsson et al. 1996).
4.2.7 Magnetic resonance spectroscopy – MRS (paper III)
All body tissues have a high water content and thus also protons (H+ ions). If a magnetic field
is applied to a tissue, the containing protons will align to the direction of this field. In the static
magnetic field of a MRI scanner, radio frequency currencies are systematically applied to
create electromagnetic fields that alter the alignment of the protons. When the electromagnetic
field is changed, the protons will return to the equilibrium state (relaxation), causing the
emission of a radio frequency signal that can be detected by the scanner. Since protons in
different tissues and under different pathophysiological conditions have different relaxation
properties, these variations are used to create high-resolution images of the investigated tissue.
Proton MRS (1H-MRS) is based on MRI methodology and measures signals originating from
protons in organic molecules in contrast to signals derived from protons in water as in
conventional MRI (cMRI). It is an important complement to cMRI because of the ability to
assess the chemical situation of tissues in vivo, as measured by the concentration of specific
metabolites. Accordingly, the use of MRS for the investigation of cerebral tissues in MS is
feasible (Sajja, Wolinsky et al. 2009). In clinic routine MRS is mainly used to differentiate
between inflammatory and malignant focal pathology. Metabolite data is collected from a
volume (voxel) defined by cMRI (Figure 7 A). The results are presented as a MRS-spectrum
where each resonance peak represents a specific metabolite (Figure 7 B).
All MRS examinations as well as the calculation and evaluation of spectral data were
performed by Anders Tisell at the Center for Medical Image Science and Visualization
(CMIV), Linköping. Briefly, examinations were performed using an Achieva 1.5 T MR scanner
(Philips, Best, The Netherlands), and an eight channels SENSE head coil. A T1 weighted axial,
and a FLAIR sagittal, and a T2 weighted coronal image volumes were acquired for placing the
MRS voxels. For quantification of R2 (1/T2) an axial ‘Multi Echo GRAdient echo and Spin
Echo’ (ME-GRASE) pulse sequence was used to acquire data of the tissue water signal. MRS
data was acquired using ‘Point RESolved Spectroscopy’ (PRESS), with an echo time of 30 ms
and a repetition time of 3.0 s, 128 water suppressed spectra were averaged. In addition, eight
spectra without water suppression were obtained as an internal reference standard. The MRS
voxel was placed in periventricular NAWM (Figure 7 A). Lesions were avoided by freely
rotating the voxel and adjusting the size (ranging 2-3 ml). The healthy controls (HC) were
similarly examined using two MRS voxels (PRESS TR 30 ms, TR 3.0 s), which were placed
bilaterally in parietal white matter.
43
4. Methods
Figure 7. 1H-MRS
examination with description
of metabolites (from
Mellergård, Tisell et al,
2012).
A. Typical placement of a
spectral voxel in normal
appearing white matter.
B. Typical 1H-MR spectrum
with LCModel fit and residual
of a natalizumab-treated MS
patient (male, 30 years old).
Spectral assignments: 1, CrCH2-; 2, mIns; 3, Cho (CH3)3;
4, Cr (CH3); 5, NAA; 6, Gln
γ/β; 7, Glu γ/β; 8, NAAGmethyl; 9, NAA-methyl; 10,
Lac CH3.
C. Description of 1H-MRS
metabolites, for reference see
Dahlqvist O. (Dahlqvist
2010).
44
4. Methods
4.2.8 Absolute quantification of MRS data (paper III)
MRS results are often expressed as ratios, with the most frequent denominator creatine (Cr),
assuming that Cr levels are stable over time. However, this is not a recommended procedure as
it has been shown that Cr is neither unaffected nor stable (Mader, Roser et al. 2000, Li, Wang
et al. 2003). In paper III we therefore utilized absolute quantitative 1H-MRS to measure
metabolite concentrations by using the internal water signal as an internal reference. The
metabolite concentrations are thus estimated as absolute ‘aqueous fraction concentrations’ (mM
aq.). The MRS spectra were analysed using LCModel (S. Provencher, Canada) (Provencher
1993), and the metabolites total creatine (tCr), total choline (tCho), myo-Inositol (mIns), total
N-acetylaspartate (tNA), the sum of glutamate and glutamine (tGlx) and lactate (Lac) (Figure 7
C) were analyzed.
Since we only intended to measure NAWM, evaluation of possible plaque contamination in
each voxel was done. The volume of plaque contamination in each MRS voxel was estimated
by segmenting all plaques in the proximity of the MRS voxel. The segmentations were
performed on T2w image by a neuroradiologist using MEVISLAB (MeVis Medical Solutions
AG, Bremen, Germany). Of all investigated MRS voxels, only one contained more than 2 % of
plaque. Statistical tests were therefore redone excluding this potential outlier, however, the
observed significant correlations presented were then still valid.
4.2.9 Real-time RT-PCR (paper IV)
Gene expression analyses were performed using real-time Taqman reverse transcriptase (RT)PCR. Real-time Taqman RT-PCR enables the amplification and quantification of mRNA in the
same procedure. Principally, a reporter dye (fluorescent) and a quencher dye are attached to a
target probe that is complementary to the specific mRNA sequence of interest. During the PCR
procedure the probe hybridizes with the cDNA template. The subsequent extension of template
by Taqman polymerase results in the cleavage and release of both the reporter and quencher
dye causing an increase in fluorescent intensity. Hence, the amplification plot reflects the
increasing amount of reporter dye during amplification and is directly related to the formation
of the PCR product of interest.
For the purpose of acquiring a situation with as much resemblance to the in vivo situation as
possible, whole blood was used to investigate gene expression. Briefly, total RNA was
extracted from whole blood PAXgene tubes (PreAnalytix, GmbH, Hombrechtikon,
Switzerland) and then converted to complementary DNA (cDNA) using a random hexamer
method (High Capacity cDNA Synthesis Kit, Applied Biosystems, Foster City, CA, USA).
Before cDNA synthesis, all samples were diluted to a fixed concentration to standardize the
performance of the real-time RT-PCR. mRNA expression was quantified using the Applied
Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). Amplification of cDNA
was performed with the TaqMan Universal PCR Master Mix (2X), No AmpErase UNG
(Applied Biosystems). For quantification of cDNA a five-point serially four-fold diluted
45
4. Methods
standard curve was developed from PBMC cultures stimulated with phytohaemagglutinin
(PHA). The mRNA expression of T helper cell transcription factor TBX21 (Th1), GATA3
(Th2), RORC (Th17), FOXP3 (Treg) and EBI3 was standardized to 18S (human rRNA), and
all results expressed as ratio in relation to 18S-expression. Primers and probes for TBX21,
RORC and Ebi3 expression were analyzed with Taqman® Gene Expression assays (Applied
Biosystems), while GATA3, FOXP3 and 18S probes were constructed in-house using Primer
Express 3.0 (Applied Biosystems).
4.3 Statistical methods
Statistical analyses on variables with assumed non-normal distribution were performed using
non-parametric methods. Such variables included clinical data (clinical scoring systems), CSF
routine analyses (cell count, IgG index and albumin ratio), levels of cytokines, chemokines,
markers of neurodegeneration and gene expression of transcription factors. In contrast, flow
cytometry and 1H-MRS data were analyzed using paired and independent samples t test.
Comparisons between several groups (lymphocyte activation assay data in MS patients and
controls and different treatment at baseline 1H-MRS) were performed with ANOVA and
Tukey´s post-hoc test (paper II and III). Parametric bi-variate correlation analyses (Pearson)
were used to evaluate flow cytometry data in relation to clinical data (paper II) and nonparametric bi-variate correlation analyses (Spearman) were used to evaluate 1H-MRS data in
relation to CSF biomarkers (paper III). A p-value of <0.05 was considered statistically
significant, except for flow cytometry data in paper II, where p<0.01 was considered
statistically significant and p<0.05 was considered a tendency.
4.4 Ethical considerations
All studies included in this thesis were approved by The Regional Ethics Committee in
Linköping. Written (paper I and III) or informed (paper II and IV) consent was obtained from
all patients.
46
47
5. Results and Discussion
5. Results and Discussion
5.1 Clinical outcome (paper I, II, III)
The studies were observational, thus the purpose was not to evaluate clinical effects of
treatment. The studies were open and treatment was initiated on clinical indications.
Nevertheless, it was mandatory to carefully record the clinical outcome including tests as
described in the Methods section. One-year treatment with natalizumab was associated with a
clinical improvement as measured both by a reduction in relapse rate and improvement of
clinical scoring systems. Annualized relapse rates at follow up were 0.35 (paper I), 0.10 (paper
II) and 0.15 (paper III) which is in line with earlier results from a randomized placebocontrolled trial with a reduction of relapse to 0.26 after one year of natalizumab treatment
(Polman, O'Connor et al. 2006). EDSS was stable at follow-up or improved though not
significantly in all studies. MSSS was assessed in paper II and III and declined significantly
upon treatment as did MSIS-29 (Table 4 a-b).
Table 4 a-b. Clinical scoring system outcome in paper II and III.
a: Paper II, n=40
EDSS
MSSS
MSIS-29
physical
psychological
SDMT
Baseline
2.5 (0-7.0)
3.82 (0.19-8.55)
Follow-up
2.5 (0-8.0)
3.20 (0.17-9.20)
p
0.08
<0.0005
2.18 (1.00-4.75)
2.11 (1.00-4.56)
48 (5-66)
1.40 (1.00-4.20) a
1.44 (1.00-4.56) a
50 (11-65) b
<0.0005
<0.0005
0.03
b: Paper III, n=27
EDSS
MSSS
MSIS-29
physical
psychological
SDMT
Baseline
2.5 (0-7)
3.9 (0.2-8.9)
Follow-up
2.0 (0-6.5)
3.1 (0.2-7.9)
p
0.007
<0.0005
2.2 (1.0-3.7)
2.1 (1.0-4.2)
49 (5-69)
1.5 (1.0-3.6)
1.6 (1.0-3.8)
53 (3-74)
<0.0005
<0.0005
0.03
Median values are given and range within brackets. p refers to Wilcoxon signed
rank test comparing baseline and follow-up. a n=37 and b n=38 because of lack of
follow-up data. EDSS=Expanded Disability Status Scale, MSSS=Multiple
Sclerosis Severity Score, MSIS-29=Multiple Sclerosis Impact Scale 29,
SDMT=Symbol Digit Modalities Test.
48
5. Results and Discussion
5.2 CSF and plasma variables (paper I, II, III)
5.2.1 Routine CSF analyses (paper I, II, III)
There were significant declines in CSF total white blood cell counts and IgG index after one
year of natalizumab treatment (Table 5). This result is in accordance with the proposed main
mode of action of natalizumab; the reduced migration of leukocytes into the CNS, and have
been demonstrated earlier (Stuve, Marra et al. 2006, del Pilar Martin, Cravens et al. 2008,
Khademi, Bornsen et al. 2009). In contrast, albumin ratios did not change significantly post- to
pre-treatment, which may reflect that damage to the BBB is irreversible or take long time to
recover from.
Table 5. CSF total white blood cell (wbc) count and IgG index changes after one year of natalizumab treatment.
CSF
wbc count (x106 cells/l)
IgG index
Paper
I
II
III
I
II
III
Baseline
2.5 (0.0-31.0)
2.6 (0.2-28.0)a
2.2 (0.2-31.0)
0.97 (0.56-4.1)
0.92 (0.48-3.0)a
0.83 (0.48-4.06)
Follow-up
0.9 (0.1-3.6)
1.1 (0.0-4.0)b
1.0 (0.0-3.6)
0.83 (0.49-3.8)
0.77 (0.45-2.4)b
0.75 (0.45-3.84)
p
<0.0005
<0.0005
0.002
<0.0005
<0.0005
0.001
Median values are given and range within brackets. p refers to Wilcoxon signed rank test comparing baseline and
follow-up. n=31 (paper I), n=25 (paper III), a n=38, b n=36.
5.2.2 Cytokines and chemokines in CSF and plasma – comparing multiple
sclerosis with non-inflammatory neurological diseases (paper I)
CSF levels of IL-1β, IL-8 (hereafter referred to as CXCL8), CXCL10, CXCL11 and CCL22,
were shown to be significantly higher in natalizumab-naive MS patients compared with patients
with non-inflammatory other neurological diseases (OND), whereas levels of GM-CSF, IFN-γ,
TNF, IL-2, IL-4, IL-5, IL-10 and CCL17 were too low to be detected (Table 6). In plasma,
levels of IFN-γ, IL-2, IL-5 and CXCL8 were significantly higher in natalizumb-naive MS
patients compared with healthy blood donors (Table 6).
Although levels of several cytokines proved to be low and therefore difficult to detect, still
indications of an intrathecal inflammation in MS was present in terms of consistent and
significant higher levels of chemokines (CXCL8, CXCL10, CXCL11, CCL22) and IL-1β in
CSF from MS patients compared with OND patients. Of note, levels of IL-1β were higher
intrathecally than in plasma, possibly reflecting its important role in hypothalamic activity.
49
5. Results and Discussion
Table 6. Cytokine and chemokine concentrations (pg/ml) in plasma (top) and CSF (bottom) in healthy blood
donors and patients with other neurological diseases (OND) compared with natalizumab-naïve MS patients
(paper I).
No. of MS
patients with
measurable
levels
MS (n=31)
No. of healthy
blood donors
with
measurable
levels
Healthy blood
donors (n=15)
IL-1β
IL-2
IL-4
IL-5
IL-6
IL-8 (CXCL8)
IL-10
TNF-α
IFN-γ
GM-CSF
31/31
31/31
31/31
31/31
31/31
31/31
31/31
31/31
31/31
31/31
0.2 (0.2-0.5)
172.5 (117.6-194.4) *
77.4 (51.4-136.5)
18.1 (11.8-23.1) *
15.9 (7.4-27.3)
39.7 (23.3-46.3) *
19.2 (9.1-27.4)
67.6 (48.8-81.8)
88.3 (58.1-98.4) *
57.4 (30.6-90.4)
15/15
15/15
15/15
15/15
15/15
15/15
15/15
15/15
15/15
15/15
0.2 (0.2-0.4)
123.4 (72.9-150.1)
95.4 (43.0-112.0)
12.8 (6.3-19.0)
22.8 (7.7-27.5)
30.3 (17.2-35.0)
15.2 (8.3-27.1)
56.9 (31.4.70.0)
51.3 (37.7-78.0)
62.1 (26.1-108.5)
CXCL9
CXCL10
CXCL11
CCL17
CCL22
31/31
31/31
31/31
31/31
31/31
79.5 (58.5-135.0)
49.6 (35.0-80.3)
48.3 (34.6-92.4)
22.3 (15.9-41.5)
196.8 (149.5-317.2)
15/15
15/15
15/15
15/15
15/15
84.6 (41.3-144.1)
44.8 (33.0-67.5)
48.7 (35.0-96.1)
36.6 (21.2-74.6)
192.2 (153.8-281.8)
No.of MS
patients with
measurable
levels
MS (n=31)
No. of OND
patients with
measurable
levels
OND (n=10)
IL-1β
IL-2
IL-4
IL-5
IL-6
IL-8 (CXCL8)
IL-10
TNF-α
IFN-γ
GM-CSF
29/30 a
0/30
0/30
29/30
28/30
29/30
24/30
26/30
0/30
8/30
4.4 (3.7-5.3) **
0.6 (0.6-0.6)
1.1 (0.7-1.9)
83.8 (70.2-111.3) **
0.2 (0.2-0.2)
0.3 (0.3-0.3)
0.4 (0.4-0.4)
9/9 b
0/9
0/9
9/9
7/9
9/9
0/9
5/9
0/9
0/9
2.7 (2.6-3.8)
0.6 (0.6-0.6)
1.4 (0.3-2.8)
51.1 (43.3-71.3)
0.3 (0.3-0.3)
-
CXCL9
CXCL10
CXCL11
CCL17
CCL22
31/31
31/31
31/31
31/31
31/31
50.1 (37.3-73.6)
188.3 (146.4-294.1) ***
5.4 (4.7-7.2) *
1.0 (1.0-1.0)
21.4 (12.8-35.3) ***
10/10
10/10
10/10
10/10
10/10
38.3 (28.8-55.6)
87.1 (74.2-118.9)
4.4 (3.2-4.9)
1.0 (1.0-1.0)
8.8 (6.1-9.7)
Plasma
Cytokines
Chemokines
CSF
Cytokines
Chemokines
Median values are given and interquartile range within brackets. Values in bold represent significant differences
compared to healthy blood donors (plasma) or patients with OND (CSF). a n=30, while analyse was not done in
one patient, b n= 9 while analyse was not done in one patient, * p<0.05 compared with healthy blood donors, **
p<0.01 compared with OND patients, *** p<0.001 compared with OND patients.
50
5. Results and Discussion
5.2.3 Cytokines and chemokines in CSF (paper I, III)
There was a significant decline in cytokine (IL-1β, IL6) and chemokine (CXCL8, CXCL9,
CXCL10, CXCL11, CCL22) levels in CSF after one-year natalizumab treatment as reported in
paper I (Figure 8). Considering that CSF levels of IL-1β, CXCL8, CXCL10, CXCL11 and
CCL22 were significantly higher in MS patients pre-treatment compared to OND patients, it is
notable that after treatment with natalizumab, levels of CXCL11 and CCL22 were reduced to
levels similar to OND patients (data not shown). In paper III the significant decline in CSF
cytokine (IL-1β) and chemokines (CXCL10, CXCL11, CCL22) after one year of natalizumab
treatment was confirmed, also when 12 patients were excluded from the cytokine and
chemokine analyses because of their former inclusion in paper I.
Figure 8. CSF levels of cytokines and chemokines pre-and post-treatment (paper I). Median values are given and
whiskers mark the interquartile range. * p<0.05, ** p<0.01, *** p<0.001. Please note the broken y-axis.
We interpret the decline in CSF cytokine and chemokine levels as a reflection of the decrease
in intrathecal inflammation. It seems logic that a decline in CSF leukocyte numbers would lead
to a subsequent decrease in cell-derived mediators like cytokines and chemokines intrathecally.
However, the biological and clinical significance of these changes are uncertain.
51
5. Results and Discussion
5.2.4 Cytokines and chemokines in plasma (paper I)
Interestingly, the decline in intrathecal levels of pro-inflammatory cytokines and chemokines
was not accompanied by an increase in plasma levels of these pro-inflammatory mediators, as
would have been expected. Instead, levels of IL-6, IL-10, GM-CSF, IL-6 and TNF decreased
significantly after one-year natalizumab treatment. This result is partially in contrast to
previous gene expression data, where mRNA expression of IFN-γ and TNF in PBMC were
shown to increase after natalizumab treatment. However, the expression of IL-23, IL-17A, IL17F, IL-13, IL-10, IL-4, and TGF-β was unchanged (Khademi, Bornsen et al. 2009). There is
however limited data on natalizumab treatment effects on cytokine and chemokine protein
levels in plasma. In one report on protein levels in plasma assessed by flow cytometry, IL-1β,
IL-2 and IL-5 were significant elevated after treatment for 9-12 months, whereas IL-10 and
IFN-γ did not change (Ramos-Cejudo, Oreja-Guevara et al. 2011). IL-6, GM-CSF and TNF
were not assessed in this study. To conclude, our findings of a decrease in plasma levels of IL6, IL-10, GM-CSF, IL-6 and TNF are difficult to interpret. However, the fact that there is no
consistent finding in the literature of elevated plasma protein levels of pro-inflammatory
cytokines and chemokines in natalizumab treated patients, as would have been expected,
indicate that natalizumab has other effects apart from reducing leukocyte extravasation.
52
5. Results and Discussion
5.3 Blood lymphocyte composition (paper II)
Main lymphocyte populations
There was a significant increase in absolute numbers of all investigated main lymphocyte
populations in peripheral blood after one year of natalizumab treatment (Table 7). This increase
was most marked for NK cells (195% increase compared with baseline) and B cells (105%
increase compared with baseline). As to changes in proportions within the lymphocyte
population, the fractions of NK cells and CD8+ T cells increased significantly, whereas CD3+ T
cells and CD4+ T cells decreased significantly (Table 7).
Table 7. Changes in main lymphocyte populations in blood before (baseline) and after one-year (follow-up)
treatment with natalizumab.
Number (cells/μl)
Percentage of parent population (%)
baseline
follow-up
change
p
baseline
follow-up
change
p
Lymphocytes
1989 ± 630
3889 ±1163
+96 %
<0.0005
27.7 ± 6.8
40.3 ± 7.0
+45 %
<0.0005
T cells
1501 ± 554
2591 ± 915
+73 %
<0.0005
75.0 ± 7.0
66.1 ± 6.7
-12 %
<0.0005
CD4+
896 ± 285
1435 ± 397
+60 %
<0.0005
61.3 ± 9.7
56.6 ± 7.7
-8 %
<0.0005
CD8+
502 ± 316
975 ± 599
+94 %
<0.0005
31.8 ± 8.4
35.9 ± 8.6
+13 %
<0.0005
NK cells
277 ± 145
816 ± 248
+195 %
<0.0005
14.3 ± 6.6
21.4 ± 5.2
+50 %
<0.0005
B cells
258 ± 182
528 ± 296
+105 %
<0.0005
12.5 ± 6.4
13.2 ± 5.1
+6 %
0.3
Mean ± SD, n=40 except for CD4+ where n=38, p values refers to paired samples t test comparing number of cells
and percentage of parent population, respectively, at baseline and follow-up. Change refers to difference between
baseline and follow-up mean given in % of baseline values.
53
5. Results and Discussion
An overview of blood lymphocyte population compositions before and after one-year
natalizumab treatment is given in figure 9 a-d.
Figure 9 a-d. Overview of lymphocyte populations in patients before and after one year of natalizumab treatment.
a-b: Distribution of lymphocytes (% of total leukocytes) and CD3 + T cells (% of total lymphocytes). Comparisons
are pairwise. Bars denote mean values. c-d: Relative distribution of discrete lymphocyte subpopulations before (c)
and after (d) natalizumab treatment.
The increase in circulating numbers of lymphocyte populations associated with natalizumab
treatment is in accordance with the proposed mode of action of this therapy. However, since
VLA-4 is expressed on many different lymphocyte populations and its main ligand VCAM-1 is
ubiquitously expressed on activated endothelium throughout the body, the overall effect of
VLA-4 antagonism on leukocyte populations measured in peripheral blood is not only a result
of reduced leukocyte migration across the BBB but also across endothelium in many peripheral
tissues. Furthermore, considering the low numbers of leukocytes intrathecally compared with
numbers in the peripheral compartment, it is unlikely that reduced migration to the CNS may
account for the total increase in circulating leukocytes during natalizumab treatment.
Accordingly, it is also found that natalizumab mobilizes hematopoietic progenitor cells out of
the bone marrow (Bonig, Wundes et al. 2008, Zohren, Toutzaris et al. 2008).
54
5. Results and Discussion
It is further reported that the magnitude of VLA-4 expression varies between different
lymphocyte populations, e. g. being higher in B cells than T cells, and higher in CD8+ T cells
than CD4+ T cells (Niino, Bodner et al. 2006). In addition, different lymphocyte subsets seem
to respond to natalizumab treatment in various magnitudes. It has recently been reported that
the amount of natalizumab binding to CD3- NK cells is higher than the amount binding to
CD19+ B cells and CD3+ T cells in a descendant scale (Harrer, Pilz et al. 2012). This diversity
in binding preference of natalizumab among different lymphocyte subsets is well in line with
our observation of the highest increase in the number of NK cells after treatment (195 %)
followed by B cells (105 %) and T cells (CD3+) (73 %). The overall effect of these differences
(expression of VLA-4 and binding preference of natalizumab), regarding migration capacity
and function for different lymphocyte subsets, is however uncertain.
Lymphocyte subpopulations
NK cells
Among CD3-CD56+ NK cells, a significant decrease in percentage of CD3-CD56bright NK cells,
was accompanied by a significant increase in CD3-CD56dim NK cells (Figure 10 a-b). There
was a tendency to a decrease in early activated CD3-CD56+CD69+ NK cells (Figure 10 c).
Based on different expression of chemokine receptors and adhesion molecules, cytotoxic NK
cells (CD56dim) and regulatory NK cells (CD56bright) have different migration preferences with
CD56dim migrating to inflammatory sites while CD56bright preferentially home to secondary
lymphoid organs (Campbell, Qin et al. 2001), where they can exert an immunoregulatory
function by interacting with dendritic cells (Gandhi, Laroni et al. 2010). Expansion of the
CD56bright NK cell subset in blood has been reported in MS patients treated with interferon-beta
or daclizumab, in contrast to what we observed during natalizumab treatment (monoclonal
antibody against CD25, a part of the IL-2 receptor) (Bielekova, Catalfamo et al. 2006, Saraste,
Irjala et al. 2007). Our finding of an increase in the fraction of circulating cytotoxic NK cells,
may suggest that these cells are relatively more affected by a decrease in extravasation caused
by natalizumab than regulatory NK cells. This interpretation seems logic considering the
cytotoxic NK cell preference for homing to inflammatory sites with endothelial upregulation of
VCAM-1, compared to regulatory NK cells that preferentially home for secondary lymphoid
organs, with a possible less expression of VCAM-1.
T cells
Regarding activation of T cells, there was a significant decrease in the proportion of activated
CD4+ cells when using CD4+CD25+ T cells among CD4+ cells as marker of activation (Figure
10 d). ). However, there was a tendency to an increase in CD4+CD69+ cells, representing early
activated Th cells (Figure 10 e), with similar findings for CD8+HLA-DR+ cells, representing
late activated cytotoxic T cells (Figure 10 f). Fractions of both CD4+OX40L+ cells and
CD8+OX40L+ cells, representing activated immunomodulatory T cells, decreased significantly
on treatment (Figure 10 g-h). There was a tendency to a decrease in CD4dimCD25bright Treg
55
5. Results and Discussion
cells after treatment (Figure 10 i). The population of CD8+CD28-CD57+ T cells, representing
senescent cytotoxic T cells, decreased significantly (Figure 10 j).
No significant change in the Treg (CD4dimCD25bright) population was demonstrated, a result in
line with earlier observations and the demonstrated low expression of VLA-4 on Treg cells
(Stenner, Waschbisch et al. 2008). The changes in expression status of activation markers and
co-stimulatory molecules were to some extent inconsistent; both a decrease (significant in
fractions of CD4+CD25+, CD4+OX40L+, CD8+OX40L+, CD8+CD28-CD57+ T cells and a
tendency in fractions of CD3-CD56+CD69+ NK cells) as well as a tendency to an increase (in
fractions of CD4+CD69+ and CD8+HLA-DR+ T cells) were observed. The interpretation of
these findings is challenging. The OX40-OX40L interaction has been ascribed an important
role in facilitating survival and clonal expansion of effector and memory T cells, as well as
regulating T cell-mediated cytokine production (Croft, So et al. 2009) and possibly promoting
Th2 immune responses (Ohshima, Yang et al. 1998). Our finding of a significant decline in
proportions of both CD4+OX40L+ and CD8+OX40L+ cells after natalizumab treatment,
possibly suggests an attenuation of effector T cell responses in the periphery. Since OX40OX40L interaction also favors a Th2 immune response, our result may also have implications
on the peripheral Th1 –Th2 balance. On the contrary, the observed tendency of an increase in
fractions of early activated Th cells (CD4+CD69+) and late activated cytotoxic T cells
(CD8+HLA-DR+) and the restored T cell responsiveness in the functional assays after treatment
(as described below) may indicate a regained responsiveness of T cells in the periphery. A
recovered T cell responsiveness after treatment is also in line with the observed significant
decrease in CD8+CD28-CD57+ T cells, since the loss of CD28 and gain of CD57 expression is
considered a sign of chronic activation and a marker for immune senescence (Strioga,
Pasukoniene et al. 2011).
Whether these observations implicate direct effects of natalizuamb on cell-surface markers of
activation and co-stimulation, or constitute indirect effects, demands further investigation.
B cells
The proportion of CD19+CD27+ cells, representing memory B cells, increased significantly on
treatment as well as the fraction of CD19+CD25+ cells, possibly representing regulatory B cells
(Figure 10 k-l). The increase in the fraction of memory B cells was significantly higher than the
increase in regulatory B cells (p=0.005).
The increase in absolute numbers of B cells after treatment are in line with earlier studies
showing an increase in circulating B cells during natalizumab treatment (Krumbholz, Meinl et
al. 2008, Haas, Bekeredjian-Ding et al. 2011, Planas, Jelcic et al. 2012). Within the B cell
population, the fractions of both memory CD19+CD27+ B cells and possibly regulatory
CD19+CD25+ B cells increased significantly after treatment. This is in accordance to recent
reports showing an increase in memory B cells while the population of naïve B cells decreased
(Haas, Bekeredjian-Ding et al. 2011, Planas, Jelcic et al. 2012). The latter finding was
suggested to depend on differences in α4-integrin expression between these two B cell subsets.
Nevertheless, the marked increase in circulating B cells during natalizumab treatment is a
56
5. Results and Discussion
consistent finding throughout many studies and further yields this lymphocyte population a
probably essential role in treatment effects and side effects.
Figure 10 a-l. Phenotypic characteristics of blood lymphocyte subpopulations in MS patients before and after one
year of natalizumab treatment (pre and post, respectively). p<0.01 is considered statistically significant. All
comparisons are pairwise. Bars show mean value, whiskers denote SD.
57
5. Results and Discussion
5.4 Lymphocyte activation assay (paper II)
The number of activated lymphocytes (lymphoblast-transformed cells) after antigen- or
mitogen stimulation was assessed at baseline and compared with the response after one year of
natalizumab treatment. The results from these assays are given in figure 11 a-f. As controls, we
also analyzed blood cells of healthy individuals, showing a significantly stronger response
compared with pre-treatment levels of patients regarding influenza antigen-induced CD4+ and
CD4+CD108+ Th cell responses (Figure 12). However, post-treatment levels of patients were in
the same range as those for controls.
Figure 11 a-f. Response towards antigens in different T cell populations. Pairwise comparison between patients
before and after one year of natalizumab treatment. PPD=purified protein derivative, PWM=pokeweed mitogen,
CMV=cytomegalovirus, n=37 in both groups.
58
5. Results and Discussion
Figure 12. Response towards antigens in healthy controls and patients before and after one year of natalizumab
treatment. For visualization purposes, data is normalized to the average of the healthy controls for the respective
antigen. Analyses performed with one-way ANOVA with Tukey’s post hoc test. *: p<0.05, comparison between
controls and pre-treatment patients. ¶: p<0.05, comparison between pre- and post-treatment patients. n=23 for
controls, n=37 for both pre- and post-treatment groups. Bars represent mean values, whiskers mark SEM.
The overall pattern of the lymphocyte activation assays was a consistently higher response
post-treatment compared with pre-treatment, although not always statistically significant. Since
the assay determines the number of responding cells, this observation is in accordance with a
reduction in immune cell migration into tissues by VLA-4 blockade and thus expectedly an
increase in the number of reactive memory T cells in the circulation. In addition, when
comparing healthy individuals to MS patients post-treatment, comparable levels of reactivity
were observed, although pre-treatment MS patients overall showed lower reactivity than
healthy controls. Speculatively this may be explained by the notion that in a neuroinflammatory
setting, the immune-competent T cells are residing at the inflammatory focus, i.e. the CNS.
Normally, T cells are constantly migrating between peripheral lymphoid tissues, blood and
other tissues. In MS, this migratory pattern could be disturbed by trapping of effector T cells to
the focus of inflammation, however, during natalizumab treatment these cells can remain in the
circulation.
59
5. Results and Discussion
5.5 Markers of neurodegeneration in CSF (paper III)
There was a significant decrease in CSF levels of MBP (from 1.08 to 0.97 ng/ml, median
values, p<0.0005) and NFL (from 509 to 289 ng/l, median values, p<0.0005) after one year of
natalizumab treatment. GFAP levels increased (from 314 to 382 ng/l, median values, p=0.004),
whereas T-tau and P-tau were unchanged. Reduced CSF levels of MBP are in accordance with
a decline in demyelination activity (Cohen, Brooks et al. 1980) because of reduced intrathecal
inflammation, and the decrease in levels of NFL is interpreted as the consequence of a general
decline in neuroaxonal damage associated with natalizumab treatment (Norgren, Sundstrom et
al. 2004). The increase in levels of GFAP may indicate a process of increasing astrogliosis
(Malmestrom, Haghighi et al. 2003, Middeldorp and Hol 2011). However, in a multicenter
Swedish cohort of 99 MS patients, including nine patients from the present study (paper III),
there was no increase in GFAP levels during natalizumab treatment (Gunnarsson, Malmestrom
et al. 2010). Thus, the increase in GFAP should be interpreted with caution. No change in T-tau
and P-tau levels was observed. The accumulation of Tau-proteins in neurons and glia cells and
elevated levels in CSF are typical signs of the neurodegenerative process in Alzheimer´s
disease. Although elevated levels of tau-proteins in CSF among MS patients have been
reported, this is not a consistent finding (Teunissen, Dijkstra et al. 2005).
60
5. Results and Discussion
5.6 1H-MRS metabolite concentrations in NAWM (paper III)
Pre-treatment 1H-MRS metabolite concentration levels in NAWM of MS patients differed
significantly from levels in healthy controls regarding tNA, tCho, mIns and tGlx, but no
significant group level changes in metabolites were observed after one year of natalizumab
treatment (Table 8).
Table 8. 1H-MRS metabolite concentrations at baseline and at follow-up after one year of natalizumab treatment
(presented as units of mM aq).
tNA
tCr
tCho
mIns
tGlx
Lac
HC
baseline
(n=20)
mean
SD
13.35
6.82
2.59
5.95
10.56
0.29
0.82
0.38
0.31
0.94
1.60
0.27
pa
0.001
0.5
0.02
0.004
0.003
0.2
MS
baseline (n=27)
MS
follow-up (n=27)
mean
SD
mean
SD
12.20
6.91
2.83
7.08
12.42
0.45
1.40
0.55
0.34
1.60
2.47
0.52
11.95
6.93
2.83
6.91
12.33
0.55
1.25
0.46
0.26
1.16
2.11
0.62
change during
treatment
mean
CI
difference
-0.25
-0.72, 0.22
0.02
-0.25, 0.29
0.00
-0.11, 0.10
-0.17
-0.70, 0.36
-0.09
-1.26, 1.09
0.10
-0.21, 0.41
pb
0.3
0.9
0.9
0.5
0.9
0.5
To the left the mean and standard deviation (SD) of metabolite concentrations among healthy controls (HC). p a
refers to significance level with independent samples t test comparing HC with MS patients at baseline. Significant
differences were still valid when adjusting for age using logistic regression analyses. To the right the mean and
the standard deviation (SD) of metabolite concentrations among MS patients are presented at baseline and at
follow-up. At the far right the mean difference in metabolite concentrations post-to pretreatment are presented
with a 95% confidence interval (CI). p b refers to significance level with paired samples t test comparing levels of
metabolites in MS patients post-to pretreatment.
Since we aimed to explore possible associations between on one hand changes in 1H-MRS
metabolite levels, reflecting the development of disease pathology, and on the other hand and
the intrathecal status of inflammation and neurodegeneration, we performed correlation
analyses between one-year change in H-MRS metabolite levels versus baseline and follow-up
levels of markers for inflammation and neurodegeneration. The rationale for this procedure was
that although there were no observed overall changes in 1H-MRS metabolite levels at one-year
follow-up, this finding does not imply that there was a zero change in metabolite concentrations
in all patients. We thus wanted to explore if individual variations in 1H-MRS metabolite change
could be associated with individual levels of CSF markers of inflammation and
neurodegeneration. By using correlation analyses we also could make use of the broad
investigational approach (examinig both markers of inflammation and neurodegeneration as
well as 1H-MRI data) and the longitudinal design of the study.
Interestingly, we found a consistent pattern of high magnitude correlation coefficients
indicating an association between change in specific 1H-MRS metabolite concentrations in
NAWM and the inflammatory markers IL-1β and CXCL8 (Table 9 a). In specific, intrathecal
levels of IL-1β and CXCL8, the latter previously known as IL-8, correlated positively at
61
5. Results and Discussion
baseline and at follow-up to change in levels of tCr (IL-1β and CXCL8) and tCho (IL-1β and
for CXCL8 at follow-up only) over one year (Table 9 b). These results suggest that patients,
despite treatment, being characterized by high levels of inflammatory markers at follow-up,
experienced an increase in density of glia cells and also increased membrane turnover in the
MRS voxel during the one-year follow-up time. Inversely, elevated levels of inflammatory
markers at baseline could possibly be a prognostic factor for the development of increased
glia cell density and membrane turnover. This finding may be speculatively interpreted as an
involvement of subclinical persistent inflammation in MS diffuse white matter pathology.
Table 9 a-b. Correlation analyses map (bivariate non-parametric) between change in 1H-MRS metabolite
concentrations during one-year versus levels of CSF markers at baseline and follow-up.
a: Map of all correlation coefficients.
1
H-MRS
change Inflammation
CXCL8
IL-1β
*
* **
* **
Th1
IL-6
TNF
CXCL10
CXCL11
Th2
Neurodegeneration
CCL22
NFL
GFAP
T-tau
P-tau
MBP
tNA
tCr
tCho
**
**
*
mIns
tGlx
Lac
The magnitude of all correlation coefficients (r) is shown. ** r > 0.5, * r = 0.4-0.5, no asterisk r < 0.4. CSF
markers are subdivided into markers of inflammation, chemokines representing T helper cells 1 (Th1) and 2 (Th2)
and neurodegeneration.
b: Detail of correlation analyses map showing one-year change in metabolite concentrations versus baseline
(left column for each analyte) and follow-up (right column for each analyte) levels of CXCL8 and IL-1β.
1
H-MRS
change
tNA
tCr
tCho
mIns
tGlx
Lac
CXCL8 (n=24)
r
p
r
p
r
p
r
p
r
p
r
p
baseline
0.01
1.0
0.48
0.02
0.38
0.1
0.21
0.3
-0.26
0.2
0.14
0.5
follow-up
-0.04
0.9
0.67
<0.0005
0.56
<0.01
0.09
0.7
-0.14
0.5
-0.004
1.0
IL-1β (n=24)
baseline
-0.02
0.9
0.49
0.02
0.43
0.03
0.30
0.2
-0.25
0.2
0.09
0.7
follow-up
-0.15
0.5
0.52
0.01
0.52
0.01
0.10
0.6
-0.25
0.2
-0.04
0.8
High magnitude correlation coefficients in bold. r=correlation coefficient, p=significance level.
62
5. Results and Discussion
The correlation analyses included several variables and when considering multiple
comparisons, each with a significance test, the problem with mass significance is true. Our
solution to this problem was not focusing on repeated significance testing of isolated
correlations one by one. Instead, the magnitude of the correlation coefficients and their pattern
was the main focus. The magnitude of the correlation coefficient reflects the strength of the
association between two variables, irrespective of whether this coefficient happens to be
“significant” or not in a null hypothesis testing. Furthermore, and most important, data revealed
a pattern of high magnitude correlation coefficients in a consistent direction that was relevant
and interesting.
5.7 Transcriptional characteristics of CD4+ T cells (paper IV)
Paper IV is a cross-sectional, descriptive study on the balance of circulating CD4+ T cell
populations in blood of 33 patients with relapsing MS. The different T cell populations were
defined by expression of their respective associated transcription factor. The main finding was
a significantly diminished expression of Treg-associated FOXP3, Th2-associated GATA3 and
immunoregulatory EBI3 in RRMS compared with healthy blood donors (Figure 13 A-E).
Similar findings were present also when recognizing the possible confounding factor of IFN-β
treatment. In contrast, transcription factors associated with proposed pro-inflammatory and
disease-promoting T cell subsets Th1 (TBX21) and Th17 (RORC) did not differ compared with
controls. On the basis of the reciprocal relation between TBX21 and GATA3 (Ouyang,
Ranganath et al. 1998) as well as RORC and FOXP3 (Ichiyama, Yoshida et al. 2008), ratios of
TBX21/GATA3 and RORC/FOXP3 expression were also calculated. An increase in
RORC/FOXP3 ratios among MS patients was then found (Figure 14).
63
5. Results and Discussion
Figure 13 A-E. Comparison of transcription factor expression fold change in patients with MS and healthy
controls (HC). All values represent ratios between transcription factor and 18S expression. Bars represent median
values and interquartile range and whiskers mark the 10 th and 90th percentiles. rrMS=relapsing-remitting MS
(n=33), HC=healthy controls (n=20), * p<0.05, ** p<0.01.
64
5. Results and Discussion
Figure 14. Transcription factor ratios in MS. Bars represent median values and whiskers mark the interquartile
range (25th and 75th percentiles). rrMS=relapsing-remitting MS (n=33), HC=healthy controls (n=20),
***p<0.005.
We interpret these findings as an indication of defects at the mRNA level systemically,
involving downregulation of beneficial CD4+ phenotypes in RRMS. These results stress the
importance of assessing the balance between pro-inflammatory and regulatory subsets when
exploring disease mechanisms. Notably, data was based on whole blood analyses and
accordingly we cannot argue that myelin-specific cells were measured. Nevertheless, our
findings may suggest that a defect in the relative immune suppression capacity of CD4+ T cells
in the periphery could contribute to Th-mediated pathogenic mechanisms.
65
6. Concluding remarks and future perspectives
6. Concluding remarks and future perspectives
This thesis discusses different aspects of the immune response and its consequences in the
context of neuroinflammation and neurodegeneration in MS. We assessed both soluble
mediators as cytokines and chemokines in plasma and CSF, different lymphocyte populations
in blood, diffuse white matter pathology using 1H-MRS as well as expression of intracellular
transcription factors. Although one main aim of the thesis was to elucidate immunological
mechanisms and consequences of natalizumab treatment, we did not compare natalizumabinduced changes with what would have been the natural course, that is without natalizumab
treatment. For obvious reasons, a placebo group cannot be used and therefore the study design
was to compare baseline status with status after one year of treatment. Consequently we could
not, and did not aim at evaluating clinical effects of treatment. However, we still wanted to
describe the clinical setting being a basic description of the material.
Our results regarding clinical outcome after one-year natalizumab treatment are well in line
with previous reports and confirm the efficacy of VLA-4 blockade. As to the effects on
lymphocyte populations in the circulation, changes in the major populations (T cells, B cells,
NK cells) may be explained by their expression levels of VLA-4, thus reflecting the effect of
natalizumab on cell trafficking. In addition, the reduction of cytokine and chemokine levels
intrathecally, also tallies with a decline in leukocyte extravasation due to VLA-4 blockade.
However, the differential effects on lymphocyte subpopulations, including cell-surface
molecules of co-stimulation and immunomodulation, as well as the intriguing divergent
findings of plasma levels of cytokines and chemokines, seem hard to explain solely by effects
on cell trafficking. Hence, additional effects of natalizumab involving cell signalling are likely.
Knowing these effects may help to understand the mechanisms of adverse reactions as the
development of PML.
Although there is a reduction in intrathecal inflammation and immune surveillance during
natalizumab treatment, the overall systemic increase in lymphocyte populations (especially
considering cytotoxic NK cells and memory B cells) indicate a preserved or even an increase in
the ability for immune responses systemically. However, since natalizumab also reduces
leukocyte migration into peripheral tissues, immune surveillance and immune responses at
other sites like the gut and lungs may be insufficient. This may have to be accounted for in
treatment considerations.
Apart from the changes in soluble mediators and lymphocyte populations associated with
natalizumab treatment, we also addressed diffuse white matter pathology in MS. The distinct
reduction in inflammatory markers (total leukocyte cell counts, IgG index, cytokines and
chemokines) and markers for demyelination and neuroaxonal damage (MBP and NFL)
intrathecally after one year, constituted the rationale to explore if these variables were
associated with NAWM changes as measured by 1 H-MRS metabolite concentrations. The
observation of a possible relation between intrathecal subclinical inflammation and
development of presumably degenerative processes in NAWM, as measured by 1H-MRS , may
66
6. Concluding remarks and future perspectives
be speculative and needs further confirmation, yet it may have important implications for the
understanding of diffuse pathology in MS and how to prevent it.
In the future, it would be interesting to evaluate the effects of natalizumab treatment on the
balance of circulating CD4+ T cell populations as defined by the expression of transcription
factors (in analogy to paper IV). This would hint at possible mechanisms of natalizumab
treatment that goes beyond the effect on cell trafficking. Such mechanisms could also be
successfully studied in an in vitro setting. Furthermore, additional functional studies on
lymphocytes, comparing pre-to post-treatment characteristics, should be done. One relevant
study would be to find ways to identify responders and non-responders (as to clinical effects of
natalizumab) by means of in vitro assessment of lymphocyte responsiveness. Another study of
high relevance is to evaluate 1H-MRS metabolite concentrations after three years of
natalizumab treatment.
67
7. Conclusions
7. Conclusions
Paper I
Natalizumab treatment was associated with a global decline in cytokine and chemokine levels
at a protein level. This finding was most pronounced in CSF, in line with the reduced migration
of cells into the CNS, whereas reduction also in plasma levels indicates other possible
mechanisms of natalizumab treatment.
Paper II
Natalizumab treatment was associated with a pronounced effect on the lymphocyte
composition in blood with an increase in numbers of all investigated major populations, most
marked for NK cells and B cells. The overall pattern of the functional T cell responses was a
consistently higher response post-treatment compared to pre-treatment, although not always
statistically significant. Changes in the major populations (T cells, B cells and NK cells) may
be explained by their expression levels of VLA-4, thus reflecting the effect of natalizumab on
cell trafficking, but the differential effects on subsets of these populations, indicate additional
effects of natalizumab involving cell-signalling.
Paper III
The levels of 1H-MRS metabolite concentrations were unchanged after one year of natalizumab
treatment on a group level. However, despite a clinical improvement and a global decrease in
levels of inflammatory markers in CSF during treatment, high levels of pro-inflammatory
CXCL8 and IL-1β were associated with an increase in 1H-MRS metabolites indicative of
continued gliosis development and membrane turnover in NAWM.
Paper IV
Gene expression analyses of transcriptional factors indicated systemic defects at the mRNA
level in RRMS, involving downregulation of beneficial CD4+ phenotypes. This relative lack of
suppression might play a role in disease development by permitting the activation of harmful T
cell populations.
68
69
8. Sammanfattning på svenska
8. Sammanfattning på svenska
Bakgrund: Multipel skleros (MS) är en kronisk sjukdom i det centrala nervsystemet (CNS) där
nervtrådarnas skyddshölje, myelin, angrips av en inflammatorisk process. MS utgör, näst efter olyckor,
den vanligaste orsaken till neurologisk funktionsnedsättning hos unga vuxna. Förutom lokala skador på
grund av inflammation har MS också visat sig medföra en diffust utbredd skadeprocess i hjärnan som på
sikt innebär en tilltagande funktionsnedsättning och minskad hjärnvolym (atrofi). Huruvida denna
process är en självständig del av MS-sjukdomen eller beror på inflammation är oklart. Den
inflammatoriska komponenten vid MS anses orsakad av immunsystemets T-lymfocyter, även kallade Tceller (en typ av vita blodkroppar), i samarbete med andra immunceller och deras producerade
substanser som cytokiner och kemokiner. Natalizumab, den hittills mest effektiva behandlingen mot
MS, utgörs av så kallade antikroppar (sökmolekyler) som binder och blockerar molekylen α4β1-integrin
(VLA-4) på cellytan. Genom denna blockering kan bland annat T-celler inte ta sig in till hjärnan
eftersom antikropparna hindrar deras bindning till kärlväggen. Natalizumab minskar på så sätt
inflammationen i hjärnan och därmed minskar också skovfrekvensen hos patienten. Däremot är det
okänt om natalizumab också påverkar andra processer i hjärnan och likaså om blockering av VLA-4
molekylen leder till andra förändringar utöver effekten på celltrafik till hjärnan.
Metoder: Totalt inkluderades 72 stycken MS-patienter, fördelade på 4 olika delarbeten. Vi undersökte
effekterna av ett års natalizumab-behandling hos 31 MS-patienter vad gäller koncentrationer av
inflammationsmediatorer, cytokiner och kemokiner, i ryggvätska (cerebrospinalvätska, CSV) och blod
med hjälp av multiplexteknik (arbete I). Vidare undersöktes sammansättningen av immunceller i blod
hos 40 patienter med hjälp av flödescytometri före och efter ett års behandling (arbete II). Diffusa
sjukdomsförändringar i till synes normal vit hjärnvävnad (NAWM) bedömdes genom att mäta
koncentrationen av olika ämnen i NAWM med hjälp av proton-magnetresonansspektroskopi (1H-MRS)
hos 27 patienter före och efter behandling (arbete III). Slutligen analyserades balansen mellan olika
hjälpar-T-celler i blod hos 33 MS-patienter med hjälp av molekylärbiologisk metod (arbete IV).
Resultat: Ett års behandling med natalizumab ledde till en tydlig minskning av
inflammationsmediatorer i ryggvätska, det vill säga både av cytokiner (IL-1β och IL-6) och kemokiner
(CXCL8, CXCL9, CXCL10 och CXCL11). Nivåer av några cytokiner i blod minskade också (GMCSF, TNF, IL-6 och IL-10). Behandlingen ledde dessutom till en ökning av det totala antalet lymfocyter
i blod inklusive undergrupper av lymfocyter (hjälpar-T-celler, mördar-T-celler, NK-celler och B-celler).
En normalisering av T-cellernas förmåga att reagera mot virus och på ämnen för celltillväxt påvisades
också. Vi kunde däremot inte med 1H-MRS på gruppnivå påvisa någon skillnad före jämfört med efter
ett års behandling i sammansättning av ämnen i till synes normal vit hjärnvävnad. Vi fann vidare ett
samband mellan förändring i de 1H-MRS-påvisade ämnena kreatin och kolin under ett års behandling
och nivåer av inflammationsmediatorerna IL-1β och CXCL8. Analys av balansen mellan olika hjälparT-celler visade tecken på en minskad andel gynnsamma hjälpar-T-celler i blod hos MS-patienter jämfört
med hos friska kontrollpersoner.
Slutsatser: Våra fynd stöder att behandling med natalizumab påverkar celltrafiken mellan blodbanan
och annan vävnad. Samtidigt tycks behandlingen också påverka undergrupper av immunceller, vilket
också kan bidra till behandlingsresultatet. Undersökningen av inflammationsmediatorer i ryggvätska i
kombination med 1H-MRS påvisade ett möjligt samband mellan inflammationsprocesser och tilltagande
diffus hjärnskada. Avslutningsvis tycks det finnas en ofördelaktig sammansättning mellan olika hjälparT-celler i blod hos MS-patienter jämfört med friska kontrollpersoner. Denna obalans skulle kunna bidra
till utveckling av MS och en korrigering av obalansen kan möjligen utgöra en framtida
behandlingsstrategi.
70
71
9. Acknowledgements
9. Acknowledgements
I wish to express my sincere gratitude to all who made this thesis possible, especially I would
like to thank:
Magnus Vrethem, chief tutor and co-author, for great support and encouragement during these
years. As head of the MS-team you have been invaluable for the progress of this thesis. A
special thanks also for always generously sharing your knowledge and vast experience in MS
clinics.
Jan Ernerudh, co-tutor and co-author, for sharing your impressive knowledge in the field of
immunology and for invaluable support and guidance. Thanks for always taking the time for
discussions and giving essential comments on manuscripts.
Charlotte Dahle, co-tutor and co-author, for your never-ending enthusiasm and encouragement
throughout this project, as well as for inspiring discussions on topics beyond immunology.
Måns Edström, co-author, for truly appreciated collaboration both in the laboratory and during
the preparation of manuscripts. Thanks for always answering my labquestions and sharing my
interest for syncopated beats!
Anders Tisell, co-author at CMIV, for great collaboration during the work with the MRS
study.
Peter Lundberg, Olof Dahlqvist Leinhard, Anne-Marie Landtblom, Ida Blystad, Kaj
Blennow and Bob Olsson, co-authors, for stimulating collaboration during the MRS study.
Maria Jenmalm and Jenny Mjösberg, co-authors, for sharing your knowledge and skills as to
immunological methods and for valuable comments on manuscripts.
Rayomand Press, co-author, for nice collaboration during the work with the transcription
study.
Petra Cassel, co-worker at AIR, for helping me out with the laboratory work and for your
generous attitude.
Jan-Edvin Olsson, professor emeritus, for all support and encouragement.
Mats Andersson, Anne-Marie Landtblom, Nil Dizdar and Patrick Vigren for providing me
with the opportunity to combine clinical work and research during their time as Heads of the
Department of Neurology.
To members of the MS-team, Helene Lundberg, Gunn Johansson, Britt Morberg, Anne
Wickström, Marielle Kagre, Annette Sverker, Katja Asplund, Eva Nilsson, Liz
Myhrinder, Annica Insulander, Elisabeth Fransson, Martin Hemmingsson, Daniel Ulrici,
Iréne Håkansson and Yumin Link for all practical help and for sharing everyday work with
our MS patients.
72
9. Acknowledgements
Per Hultman, my first research supervisor at Medical School, for introducing me to research
and autoimmunity, in this case mercury-induced autoimmunity in rodents. I will never forget
the shape of green-fluorescent antinucleolar antibodies in the dark!
Christina Ekerfelt, tutor during my year at the Biomedical Research School, for
encouragement and introduction to MS immunology including “immune deviation in vitro”.
Olof Danielsson, former clinical tutor, for introducing me to the clinical skills of neurology
and for your good example of devotion to the subject of neurology.
Mats Fredrikson, for statistical advice on paper III.
The staffs at AIR and Clinical Immunology, for handling of samples and performing flow
cytometry analyses, respectively.
All highly appreciated colleagues and co-workers at the Department of Neurology for their
support and understanding. A special thanks to the staff at neurology ward 73, for taking care
of patients during natalizumab infusions.
To all patients who generously participated in all studies, without you this thesis would never
have been written.
All dear friends, without you, life would be truly empty!
A special help and encouragement when working on this thesis I got from Sara and Johan and
from Charlotte and Mattias.
My parents-in-law, Margaretha and Rodney, for great generosity and practical help whenever
needed.
My parents, Aina and Beo, for endless love and support from the very start and throughout the
years. My sisters, Camilla, Martina and Hanna and my brother Joseph and their families for
love and friendship.
My beloved Selma, Morris and Bea; you fill me with tremendous joy and gratitude!
My beloved Sara for all your love, immense support and for being a true life companion.
Words fail me!
This work was supported by grants from The National Research Council (VR/NT), The
Swedish Society of Medicine, The Swedish Association of Persons with Neurological
Disabilities, Linköping Society of Medicine, The University Hospital of Linköping, The
County Council of Östergötland and Linköping University. The project was further supported
by an unrestricted grant and award from Biogen Idec Sweden.
73
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